Anne-Nathalie Volkoff

Kenneth Vernick

Yannick Outreman

Samuel Alizon

Remy Froissart

Christophe Boete

Mylene Ogliastro

Anne-Sophie Gosselin-Grenet

Merci Beaucoup

Fabienne Justy

Sandra Unal

Patrick Ma- Koundou

Philippe Clair

Thomas Cantinelli

Frédérique Cerqueira

Mine Altinli
email: mine.altinli@umontpellier.fr

Filiz Gunay

Bulent Alten

Mylene Weill

Mathieu Sicard
email: mathieu.sicard@umontpellier.fr

Wolbachia diversity and cytoplasmic incompatibility patterns in Culex pipiens populations in Turkey

Keywords: Wolbachia, Culex pipiens, Cytoplasmic incompatibility, Turkey, Vector control

Culex pipiens -Wolbachia -Densovirus Tripartite Interactions

Virus discovery has long depended on the observation of outbreaks, and the research to understand their pathogenicity depended on their culture. The advances of sequencing technology and molecular biology techniques allowed researchers to discover the ubiquity and the diversity of viruses. Despite these advances, virus discovery remains mainly focused on vertebrate viruses. Furthermore, even the known diversity of these viruses is poorly studied in term of virus ecology. Arthropods with their ancient evolutionary history represent 80% of animal diversity on our planet. The ubiquity of endosymbiosis in arthropods and their ability to support (sometimes multiple) symbionts suggest that their interactions with their viruses might be of symbiotic nature, intimate long-term associations with outcomes ranging from mutualistic to antagonistic. In this context, this PhD dissertation focuses on a mosquito densovirus, Culex pipiens densovirus (CpDV), to investigate their prevalence and diversity in natural populations of Culex pipiens mosquitoes and their interactions with endosymbiotic bacteria Wolbachia in a natural host-bacteria-virus system (Cx. pipiens -Wolbachia -CpDV) both in natural populations and laboratory colonies. We reveal a high prevalence and diversity of CpDV in worldwide Cx. pipiens populations. Moreover, CpDV persistently infects laboratory lines and are vertically transmitted along with their hosts' endosymbiont Wolbachia. We reveal high CpDV prevalence and diversity in Turkey and Tunisia and further focus on these two countries to investigate Wolbachia-CpDV interactions in natural populations. In Tunisia, in a narrow contact zone where two genetically distinct Wolbachia groups coexist, different Wolbachia groups influences CpDV prevalence and diversity. Moreover, we show a positive correlation between Wolbachia density and CpDV density. Overall, our results suggests that CpDV-Cx.pipiens -Wolbachia system is a good model system to study the ecological interactions between these partners and their evolution together both in the nature and in laboratory conditions. Densoviruses with their diversity and long-term evolution with their hosts, and their easily manipulated small genomes, are good models to study complex host-virus-bacteria interactions as well as virus ecology and evolution in a symbiotic context.

1. 4.2.1 Brevidensoviruses ............................................................................................... 9 1.4.2.2 Ambidensoviruses .............................................................................................. 12 1.4.

1.1! Symbiosis

All multicellular organisms evolved in a microorganism rich environment and interacted with them through the course of their evolution. Hence these interactions shaped the evolution of the both partners and continues to do so. While the fact that acquisition of the mitochondrion leading to the formation of the first eukaryotic cells originated from a symbiotic event was discussed for a long time, the important impacts of microbes on all multicellular organisms were not properly appreciated until recently (Sagan, 1967;McFall-Ngai, 2015). For most of the scientific past, microbial research mainly focused on the pathogenic interactions where microorganisms caused diseases especially in humans or other economically important organisms. Associations with non-pathogenic microorganisms was thought to be rare and limited to some specific organisms such as lichens or corals (McFall-Ngai, 2015). This general view started to change in the late 1970s with the use of sequencebased analysis of phylogenetic relationships for the first time, using 16S ribosomal RNA sequences (Woese and Fox, 1977). These new molecular methods coupled with the rapid progress of sequencing technologies i) enlightened the shared evolutionary history between the three "domains" of life ii) led scientists to appreciate the ubiquity and the real diversity of bacteria and archea, as well as the diversity of the interactions and interdependencies they have with their hosts (Woese and Fox, 1977;Woese, 2004;Gilbert et al., 2012;Pace et al., 2012;McFall-Ngai et al., 2013). Symbiosis term first used by a German botanist to describe the living together of fungi and algae as lichens, is today used to describe the countless forms of long term intimate relationships between organisms, that can range from mutualism to parasitism (Relman, 2008;Combes et al., 2018).

Long term intimate symbiotic associations have been described in more than half of the animal phyla so far, especially in arthropods which represents, about 80 % of animal diversity (McFall-Ngai, 2015). While exceptionally rare in vertebrates, in arthropods long-term intimate endosymbioses, i.e.

where microorganisms colonize inside of the body wall and sometimes even in host cells, are also found widespread (Douglas, 2015(Douglas, , 2016)).

As the appreciation of the symbiotic interactions grew, their classification in terms of mutualistic versus parasitic also became more and more difficult. Even though some clearly mutualistic symbionts might exist, for most of the symbionts the nature of their interactions with their hosts are not so clear. Moreover even when they are seen as mutualistic, their interactions with their hosts are context dependent and can change with the ecological context (Keeling and McCutcheon, 2017). As such, many microorganisms causing chronic infections are expected to have both mutualistic and pathogenic properties and they can evolve rapidly into benefiting or harming their hosts (Moran, 2006). Transmission modes of the symbionts have been demonstrated to affect the nature of these interactions: vertically transmitted, from parent to offspring, are more likely to evolve towards mutualism as their fitness is related to their host fitness, while horizontally transmitted symbionts are expected to evolve towards antagonism (Cressler et al., 2016). In nature most of the symbionts exhibit both of these transmission modes, called mixedmode transmission (Ebert, 2013).

Symbiotic relationships can also vary in that they are obligate or facultative (Moran, 2007). Many vertically transmitted symbionts are required for their hosts' normal development and kept in a specialized organ called bacteriome. For instance Buchnera aphidicola, obligate symbionts of aphids, are unculturable and bacteria-free aphids grow poorly and produce few or no offspring (Douglas, 1998). On the other hand facultative symbionts are not restricted to one tissue in the host (Dobson et al., 1999;Fukatsu et al., 2000).

The spread of facultative symbionts in the host populations can be selectively favoured either due to their ability to confer fitness benefits to their hosts (e.g. protection against natural enemies, heat tolerance), or alternatively manipulate their host reproduction (Stouthamer et al., 1999;Werren et al., 2008;Engelstädter and Hurst, 2009). Reproductive manipulation seem to have evolved separately in phylogenetically diverse insect symbionts, such as Arsenophonus nasoniae, Spiroplasma, Rickettsia species (e.g. Wolbachia) and can include many different strategies such as male killing, cytoplasmic incompatibility, feminization, and parthenogenesis that increases the infected females in the host population (Engelstädter and Hurst, 2009).

Without a doubt, symbionts and their hosts influence each other's phenotypes. This influence can also be observed in their genome evolution.

Symbiosis between obligate bacteriome-associated symbionts and their hosts are usually very old where partners co-evolved and co-diversified, as seen in Buchnera -Aphid symbiosis (Clark et al., 2000). Interestingly their genomes were found to be more static, showing a lack of recombination, phages or mobile elements and rearrangements and usually gone through a gene reduction (Moran et al., 2008). An extreme example for this has been observed in cicadas (McCutcheon and Moran, 2012). Cicadas carry an obligate nutritional symbiont, Candidatus Hodgkinia cicadicola, which has a genome size about 145kb which is thirty times smaller than the genome of a free living bacteria such as E. coli (McCutcheon and Moran, 2012). Contrarily facultative symbionts, like Wolbachia, do not show a clear pattern of co-diversification with their hosts, as they are able to be horizontally transmitted, and their genomes are more dynamic with rearrangements, recombination, phages and mobile elements (Moran et al., 2008). Additionally such extreme genome-size reduction has not been demonstrated for facultative symbionts (Moran et al., 2008).

1.2! Viruses as endosymbionts

Similar to bacteria, viruses have been seen only as causative agents of diseases, since the start of virology with the discovery of Tobacco Mosaic virus in 1886 (Mayer et al., 1942) until recently. While the sequencing of the 16S ribosomal RNA gene revolutionized the way we see other microbes, study of viruses -lacking a similar common phylogenetic marker -mainly relied on the observation of the disease outbreaks and ability to culture these viruses in cell lines until almost three decades after.

Starting with the first study of marine viral communities in 2002, the number of published metaviromes grew significantly during the last decade and with this our understanding of the diversity of viruses, their ecology and the wide-range of environmental conditions they exist in (Breitbart et al., 2002;Breitbart and Rohwer, 2005;Edwards and Rohwer, 2005;Rosario and Breitbart, 2011). This knowledge also highlighted that most viruses do not cause any detectable damages to their hosts contrarily of what is thought (Roossinck and Bazán, 2017).

In fact, viruses can better be described as endosymbionts, given their intimate relationships with their hosts. Similar to bacterial symbionts their interactions with their hosts can be anywhere from mutualism to antagonism on the symbiotic continuum (Roossinck, 2015a). While antagonistic relationships are the most studied for viruses, mutualistic viruses have also been reported notably in plants (Roossinck, 2015b). For instance, several plant viruses have been demonstrated to confer drought and cold tolerance to their hosts (Xu et al., 2008), highlighting the huge impact that viruses can have on their host's ecology.

Studying viruses as endosymbionts, trying to understand ecological context of host-virus interactions rather than only focusing on pathogenic interactions, can improve our understanding of virushost interactions. Indeed, viruses can be good models to study the evolution of symbiotic relationships (Roossinck and Bazán, 2017). As the virus-host interactions are usually fast evolving and dynamic, study of their evolution and population dynamics can also give insights on how mutualism or antagonism evolve (Roossinck, 2011).

The studies considering viruses in this broad context are so far mainly focused on plant viruses while there are not many studies on arthropod viruses. Given their ancient evolutionary history, diversity and the fact that they are able to support endosymbionts (sometimes multiple endosymbionts), arthropods are really likely candidates to observe long-term intimate symbiotic relationships with viruses.

1.3! Symbiont-symbiont interactions

Another change in paradigm that came with the advances in the sequencing techniques was the understanding that the diseases were not caused by only one micro-organism (i.e. "the" pathogen) but rather driven or influenced by the interactions between microorganisms (including bacteria, protists, fungi and viruses) (Vayssier-Taussat et al., 2014). Even in the cases of more studied bacterial symbiosis, interactions between symbionts have been poorly studied and research mostly focused on binary host-symbiont interactions (Douglas, 2016). The study of the complex interactions of microbial communities, called microbiota, so far mainly focused on vertebrates (mainly human and mice) and especially on human gut microbiota (Clemente et al., 2012) or on arthropods of medical or economic importance such as mosquitoes and aphids (Morin et al., 1999;Gottlieb et al., 2010;Weiss and Aksoy, 2011;Su et al., 2013;Johnson, 2015a;Jia et al., 2017).

Endosymbionts can interact with each other either directly or indirectly through their extended phenotype (Douglas, 2016;Almand et al., 2017). Between bacteria and viruses, direct interactions are usually seen as exploitation of bacteria by viruses. For instance when orally challenged with poliovirus, mortality in mice with normal gut microbiota was twice as higher compared to mice without gut microbiota due to an increase in viral titers. Such effects were not present when the poliovirus was injected, as the virus did not have to interact with the microbiota before entering the internal organs. It was also hypothesized that bacterial components (called MAMPs for Microbial Associated Molecular Patterns) like LPS and glycans can increase viral binding and shedding (Kuss et al., 2011;Moore and Jaykus, 2018). Other types of direct interactions seem to affect horizontal and vertical transmission of the plant viruses vectored by insects. For instance, a common obligate bacterial symbiont of leafhoppers, Sulcia, has been demonstrated to allow rice dwarf virus to vertically transmit to their vector's offspring. Rice dwarf virus achieves this by binding to Sulcia's envelopes due to a specific interaction between the viral capsid protein and the Sulcia outer membrane protein (Jia et al., 2017). Horizontal transmission of other plant viruses vectored by aphids and whiteflies could be affected by their hosts microbiota. Notably Potato Leaf Roll Virus (PLRV, luteovirus) transmission by aphids decreased by 70% after antibiotic treatment of aphids' endosymbiont Buchnera (van den Heuvel et al., 1994). The interactions between virus capsid protein and Buchnera's GroEL protein is thought to be responsible for this, as Buchnera GroEL binds to PLRV in vitro but whether it is the mechanism that facilitates virus transmission in vivo is still debated (Bouvaine et al., 2011). However a more compelling example of GroEL and virus capsid interaction impact on virus transmission was observed in whitefly Bemisia tabaci (Gottlieb et al., 2010;Su et al., 2013). B. tabaci has different biotypes harbouring different symbiotic bacteria: both B and Q biotypes harbour Portiera aleyrodidarum and Rickettsia, additionally B biotype harbour Hamiltonella and Q biotype harbour Wolbachia and Arsenophonus. In nature, B Biotype was more competent for the transmission of Tomato Yellow Leaf Curl Virus (TYLCV) suggesting the involvement of Hamiltonella in vector competence (Gottlieb et al., 2010). This involvement has been confirmed by the comparison of Q biotypes with or without Hamiltonella infection, where whiteflies infected with Hamiltonella transmitted TYLCV more efficiently (Su et al., 2013). Hence the possibility of an interaction between GroEL protein of Hamiltonella and the virus transmission has been highlighted.

Indirect interactions can occur either by exploitative competition where different organisms compete for host resources without a direct interaction between them or by apparent competition where one of the organisms modulates the host immune response. In human infections indirect interactions between bacteria and viruses have been shown to generally benefit bacteria when viruses target a particular cell type such as lymphocytes, macrophages and monocytes (Almand et al., 2017).

For example influenza virus has been shown to decrease alveolar macrophages which lead to facilitate bacterial superinfections (Almand et al., 2017).

In insect hosts, the endosymbiotic bacterium Wolbachia (detailed further in Chapter 1.5.1 Wolbachia) is proposed to do both. Indeed, Wolbachia have been reported to indirectly compete with an RNA virus (Drosophila C Virus, DCV) by using dietary cholesterol thus increasing D. melanogaster life span (Teixeira et al., 2008;Caragata et al., 2013). These effects were reversible by the enrichment of the hosts' diet (Teixeira et al., 2008;Caragata et al., 2013). In mosquitoes, Wolbachia are able to confer protection to their hosts against important human arboviruses, such as Dengue and Chikungunya, putatively via immune priming (Moreira et al., 2009).

1.4! Single-stranded DNA viruses

Single stranded (ss) DNA viruses constitute a supergroup of economically, medically and ecologically important viruses that have been found in a wide range of environmental conditions: air, soil [START_REF] Kim | Amplification of uncultured single-stranded DNA viruses from rice paddy soil[END_REF]Reavy et al., 2015), fresh and marine waters (Rosario et al., 2009;Labonté and Suttle, 2013;Zawar-Reza et al., 2014) including extreme conditions like hot-spring (Mochizuki et al., 2012) and hypersaline habitats (Pietilä et al., 2009). They can infect eukaryotes -such as plants (Howarth and Goodman, 1982), animals and fungi (Yu et al., 2010), bacteria (Székely and Breitbart, 2016) and archaea (Mochizuki et al., 2012). Furthermore paleovirological studies discovered their existence in the genomes of even wider host range than previously expected as endogenous viral elements (Belyi et al., 2010;Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015). Integrated both in vertebrate and arthropod genomes some ssDNA families are thought to be older than 40-50 million years which highlighted their intimate and long term relationship and evolution with their hosts (Belyi et al., 2010;Katzourakis and Gifford, 2010;Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015).

Much like their hosts and environmental conditions they exist in, ssDNA families are highly diverse. They are currently split into eight recognized families: the Anelloviridae, Circoviridae, Bidnaviri-dae and Parvoviridae infecting animals, the Geminiviridae and Nanoviridae infecting plants and the Microviridae and Inoviridae infecting bacteria (https://talk.ictvonline.org/ taxonomy/, ICTV 9th report online). Their genome structures are equally diverse and can be linear or circular, with single or multiple components (Martin et al., 2011). They are usually small viruses (<10 kb, except of an archaeal ssDNA virus with 24.9 kb genome size) (Krupovic, 2013). Their small sized genome is thought to be a result of their high nucleotide substitution rate which is closer to that of RNA viruses than double-stranded DNA (dsDNA) viruses. Indeed this substitution rate can be as high as 10 -4 to 10 -3 substitutions/site/year (Shackelton et al., 2005;Firth et al., 2009;Krupovic, 2013), a feature that can lead to the accumulation of deleterious mutations when the genome size gets bigger. It is also possible that their genome size is limited by the encapsidation capacity of their small capsid (a diameter of 18-27 nm) (Krupovic, 2013). Extensive evidence of recombination have also been found for ssDNA viruses and likely to have a big role in the generation of new genera (Shackelton et al., 2007;Lefeuvre et al., 2009;Martin et al., 2011;Koonin et al., 2015;Martynova et al., 2016;Tijssen et al., 2016;Kazlauskas et al., 2018).

Most of the ssDNA viruses code for two types of genes: non-structural (NS/Rep) and structural capsid proteins (VP/CP). NS genes generally play a role in the replication of the viral genome, an endonuclease encoded by viral NS gene starts the replication by nicking the viral DNA. This replication mechanism, named rolling-circle or rolling hairpin mechanism, used by most of the ssDNA viruses is also used by many plasmids and some transposons (Chandler et al., 2013;Krupovic, 2013;Krupovic and Forterre, 2015;Rosario et al., 2012). Surprisingly, ssDNA NS proteins phylogenetically cluster with proteins from different groups of plasmids and are not monophyletic (Koonin et al., 2015). While VP genes of some of the ssDNA families thought to originate from ssRNA viruses on different occasions (Krupovic, 2013). This suggests that current ssDNA virus diversity is of polyphyletic origin, probably resulted from separate recombination events between RNA and DNA donors and highlights that different families might have originated in different times and evolve further with their hosts (Krupovic, 2013;Koonin et al., 2015). For instance, Parvoviridae family, with their wide host range infecting both vertebrates and arthropods, could have appeared before the rise of major eukaryotic kingdoms (Koonin et al., 2015;Krupovic and Koonin, 2015). On the other hand Bidnaviridae family, lacking the rolling circle/hairpin mechanism, is thought to have evolved much later and possibly by acquiring genes from four different groups of viruses (Koonin et al., 2015;Krupovic and Koonin, 2015).

Both for their ancient origin and wide host range Parvoviridae family is interesting to study in terms of their interactions with their hosts and host microbiota. While the mechanistic aspects of the virus-host interactions and viral pathogenesis of Parvoviridae family are well studied, the ecological interactions between the virus and host as well as the host microbiota and their outcomes are largely unknown. Like it has been observed with bacterial symbionts, the nature of these interactions could be context dependent and they can shape microbial communities and host populations by influencing infection outcomes and can drive the evolution of the different partners involved.

1.4.1! Parvoviridae family

Parvoviridae family consists of small, isometric non-enveloped viruses with linear ssDNA genomes which can range between 4 and 6 kb in length (Kerr et al., 2005). Their genome terminates in hairpin structures at each end, which can either be different from each other in terms of sequence and predicted structure or be the exact same nucleotide sequence inverted, i.e. inverted terminal repeat (ITR) (Kerr et al., 2005). These hairpins serve as primer for host's DNA repair mechanism that convert ssDNA to dsDNA (Berns, 1990). Following this, dsDNA is transcribed to viral mRNAs to be translated into viral proteins during host cells' S phase (Berns, 1990). Replication is started by viral endonuclease NS1 (Rep) which cuts between the hairpin structure and coding sequences (Berns, 1990).

Parvoviruses can cause a wide range of acute or chronic diseases; many, however, are not known to be associated with any disease but to cause persistent infections (Kerr et al., 2005;Liu et al., 2011). The family is divided into two subfamilies based on host range: the Parvovirinae infect vertebrates and the Densovirinae infect insects and other arthropods (Tijssen et al., 2016). Further taxonomic classification within the family is made by the construction of phylogenetic relationships using the conserved SF3 helicase domain within NS1 (Cotmore et al., 2014). SF3 helicase domain consists of a contiguous set of functional subdomains, called Walker boxes that stretch between 100 and 103 amino acids of all known parvoviruses, while they are not found in cellular genomes (Kerr et al., 2005). Capsid proteins (VPs) have also been used to understand phylogenetic relationships within parvovirus family (Cotmore et al., 2014;François et al., 2016). While it is not as contiguous as SF3 domains, VPs also have very conserved domain called PLA2 (Phospoliphase A2), which has a role in viral infectivity (Zádori et al., 2001), and the eight-membered ß-barrel that forms the core of the icosahedral capsid shell (Chapman and Rossmann, 1993).

Our knowledge about this family of viruses mainly come from viruses that are pathogens of humans or economically important animals. However recent studies on public transcriptomic and genomic databases highlighted their ubiquity and diversity, especially of densoviruses, in wider range of hosts (Liu et al., 2011;François et al., 2016). This also suggests that the nature of densovirusarthropod host relationships could also be different than strict antagonism usually observed, making them good candidates to study viruses as endosymbionts of arthropods.

1.4.2! Densoviruses

Densoviruses get their name from the cellular pathology they can cause, "densonucleosis" referring to the electron dense observation of the infected cell's nuclei where the viruses replicate (Meynadier et al., 1964;Gosselin Grenet et al., 2015). They are non-enveloped viruses with icosahedral capsids and Maximum likelihood tree is prepared by Sarah François using SF3 helicase domain within NS1, statistical support is given as bootstrap values (François et al. in prep). Variola D5 protein is used as outgroup. CpDV (red) is phylogenetically far from other mosquito densoviruses (red, brevidensovirus) and closer to lepidopteran and blattodean densoviruses. Viltain virus that has been recently detected in genomics studies in Drosophila seem to be closely related to CpDV but our knowledge about it limited. &::#: : : :

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are among the smallest viruses (18-25nm) (Tijssen et al., 2016). Densoviruses have been isolated mainly from insects (orders of Lepidoptera, Diptera, Hymenoptera, Blattodea, Hemiptera, Orthoptera) but also found in malacostraca (shrimps and crayfish) and echinoderms (starfish and sea urchins)(Figure 1) (Tijssen et al., 2016). They are also integrated in many invertebrate genomes and recent studies suggested that densoviruses may be as diverse and ancient as arthropods (Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015;François et al., 2016)(Figure 1).

While this subfamily keeps expending, the categorization of its genera gets more and more challenging. So far there are 5 defined genera (i.e. Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus) within the Densovirinae subfamily, based on the sequence identity of NS (Rep) gene (Cotmore et al., 2014;Tijssen et al., 2016) (Figure 1).

Densoviruses have originally been discovered

and studied following the observation of mass mortality in economically important arthropods, such as silk worms or shrimps (Watanabe and Maeda, 1981;Lightner and Redman, 1985). Hence most of the DVs so far isolated are lethal for their natural hosts, the first symptoms being anorexia and lethargy followed by flaccidity, paralysis, slow melanisation and death (Tijssen et al., 2016). Most of them exhibit a tissue polytropism replicating in many tissues like the fat body, hypodermis, epidermis, central nervous system, muscular and tracheal cells, gut, hemocytes, ovaries and are considered to be lethal for their natural hosts (Tijssen et al., 2016). Once in the population, these infections can spread quickly as observed in farms of arthropods of economic importance such as crickets (Szelei et al., 2011;Weissman et al., 2012) and shrimps (Lightner, 1996). Moreover, the recent outbreak of sea star wasting disease was also caused by a densovirus (Hewson et al., 2014). Due to their observed pathogenicity, narrow host range and sustainability out of their host-cells in nature, they attracted scientific attention as biological control tools especially against crop pests like lepidopteran pests, and insects of medical and veterinary importance like mosquitoes (Carlson et al., 2006;Mutuel et al., 2010;Gosselin Grenet et al., 2015;Suzuki et al., 2015;Johnson and Rasgon, 2018). For instance, DVs have been successfully used to control Limacodidae larvae in oil palm plantations (Genty and Mariau, 1975;Fédière, 1996). As mentioned until recently their discovery was driven by the observation of epizootics in arthropods of importance and only with the advances in sequencing technologies and growing data from natural populations they have been shown in a wider range of hosts natural populations. Although thus far the research on them still mostly focus on their pathogenicity in experimental infections, virus-host interactions could be much more diverse and complicated than expected in natural populations. Knowledge of the prevalence and epidemiology of these viruses in nature could help us understand how they evolve and interact with their hosts better, making their control or manipulation easier I will further focus on two of the genera of Densovirinae subfamily -Ambidensoviruses and Brevidensoviruses -as they are most relevant for the present dissertation.

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General information

Brevidensoviruses have the smallest genomes among the other genera of the Densovirinae subfamily with their genomes about ~4kb in length, (Kerr et al., 2005). Their genome is monosense, Open Reading Frames (ORFs) for the structural (VP) and non-structural (NS) proteins are on the same strand (Kerr et al., 2005)(Figure 2A). Negative strands constitute the majority of the DNA strains encapsidated in the virion populations: 85% negative strand 15% positive strand (Kerr et al., 2005). Unlike the other densoviruses their genome does not contain any recognizable PLA2 domain in their VP gene (Kerr et al., 2005) (Figure 2A).

There are two recognized species within the genus, namely Dipteran Brevidensovirus 1 and 2, but several variants of these distinct species have also been discovered (Cotmore et al., 2014). All the First MDV has been found in Russia, in laboratory colonies of Aedes aegypti mosquitoes originated from Sout-East Asia in 1972 (Lebedeva et al., 1972). This MDV, later taxonomically classified as belonging to Dipteran Brevidensovirus 1 species (Cotmore et al., 2014), and other very similar (sequence homology) variants have later been discovered in mosquito cell lines, laboratory colonies or natural populations of Ae. albopictus [Ae. albopictus densovirus 1, AalDV1, (Chen et al., 2004)],Ae. aegypti [Ae. aegypti densovirus 1, AaeDV1 (Afanasiev et al., 1991); Ae. aegypti densovirus 2, AaeDV2, (Sivaram et al., 2009); Ae. aegypti Thai strain (Kittayapong et al., 1999)], Anopheles minimus (Rwegoshora et al., 2000), Anopheles gambiae [An. gambiae densovirus, AgDV, (Ren et al., 2008)], Culex pipiens pallens [Culex pipiens pallens densovirus, CppDV, (Zhai et al., 2008)]. Dipteran brevidensovirus 2 species consists of AalDV2, then named as AaPV, that have been isolated for the first time from C6/36 cell lines (Jousset et al., 1993). O'Neill et al. (1995) demonstrated similar variants of this species persistently infecting different mosquito cell lines including Haemagogus equinus and Toxorhynchites amboinensis cell lines [H. equinus densovirus, HeDV, (O'Neill et al., 1995)].

Experimental infections

To study the interactions of the MDVs with their hosts, mosquito larvae have been experimentally infected orally by breeding them in the water either where infected crushed larvae or infected cultured cells were added. Both Dipteran Brevidensovirus 1 and 2 species have been studied for their infectivity in experimentally infected mosquito lines. AaeDV1 virus have been shown to successfully infect and cause mortality in Aedes, Culex and Culiseta species (Buchatsky, 1989). Ae. aegypti larvae were infected using either infectious clones cultured in C6/36 cells or infected dead larvae. In both cases mortality increased with increased virus dose up to 75% with majority of mortality observed during the larval stage (51%) (Ledermann et al., 2004). Longevity and survival were reduced in the adults that survived the AaeDV1 infection (Suchman et al., 2006). The effects of AaeDV1 depended on the viral dose and when the larvae were infected. Larvae infected during the 1 st instar were affected more than the larvae infected during 3 rd instar (Suchman et al., 2006). Infected adults were able to transmit the virus to approximately 70% of the offspring (Suchman et al., 2006). However, vertical transmission was affected by when the larvae was infected at the first place. Indeed, larvae that were experimentally infected during the 3 rd instar transmitted the virus less (Suchman et al., 2006). AaeDV virus Thai strain has also been shown to cause 51% and 82% mortality in orally infected Ae. aegypti and Ae. albopictus mosquitoes (Kittayapong et al., 1999). Surviving adults were also able to transmit the virus to their offspring (Kittayapong et al., 1999). AalDV2 (dipteran brevidensovirus 2), has been shown to cause mortality when Ae. aegypti larvae were infected using naturally infected larvae or viruses cultured in cells (Barreau et al., 1996). The infection method had a large effect on the infection dynamics, larvae fed on the persistently infected cells showed 90% mortality between 6 th and 10 th day while larvae infected by dead larvae reached this mortality around only on the 20 th day. Larval mortality caused by AalDV2 also increased i) when the larvae were infected during first instar larval stage compared to 3 rd larval stage, ii) when host population density increased. The fact that mortality usually occurs during life stage change, that requires molting, can possibly be caused by the energetic cost of these changes. Among the mosquitoes that were able to survive the AalDV2 infection and become adults 17% died before laying eggs. However, there was no effect (i.e. virulence) of the infection on the fecundity of the females (Barreau et al., 1997). These females were able to transmit the virus vertically to their offspring. The vertical transmission rate depended on the virus titer of the parental female and ranged between 28% -55%. This vertical transmission did not persist beyond the second generation. There was also a low percentage of venereal transmission from infected males to uninfected females. Overall the results highlighted that AaDV2 were transmitted horizontally, both from larvae to larvae and venereally between adult mosquitoes, and vertically to the offspring.

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HeDV infection of Ae. aegypti larvae on the other hand resulted in low mortality (10%) but higher infection rate in adults where HeDV were still able to replicate (Ledermann et al., 2004). This finding was interesting because HeDV has been shown to be highly cytopathic in C6/36 cell line but was not as pathogenic in Ae. aegypti larvae, in contrast with Dipteran Brevidensovirus 1 that can cause persistent infection in the cell lines but is highly pathogenic for larvae (Ledermann et al., 2004;Paterson et al., 2005).

Ecological studies, or lack thereof

Similar to the differences between in vivo and in vitro studies, experimental infections using these viruses -while useful to understand the virus-host interactions -might not be reflecting how these viruses interact with their hosts in nature. Although there are not many studies focusing on the epidemiology of densoviruses in natural populations, MDVs are thought to be widespread in mosquito populations (Ma et al., 2011;Ng et al., 2011;Zhang et al., 2013). AeDV Thai strain is the most studied MDV in natural populations to date. In Ae. aegypti adults collected from Thailand (n=97), AeDV Thai strain prevalence was 44.3% while in Ae. albopictus adults tested (n=79) were all negative (Kittayapong et al., 1999). Interestingly AeDV Thai strain have been shown to successfully infect both mosquito species in laboratory conditions, suggesting either the difference between the laboratory and natural conditions or the possibility of a higher virulence in Ae. albopictus larvae preventing their development into adults. If true, this would suggest that AeDV Thai strain infection can shape the structuring of Ae. albopictus and Ae. aegypti populations in nature (Kittayapong et al., 1999).

For further investigation on the seasonality of the infection by AeDV in Ae. aegypti adults, a total of adults has been collected monthly for a total of 844 individuals. The results showed a monthly variance with a prevalence peak in July (35.1%, n=308) and lowest prevalence in August (6.5%, n=31) and September (8.0%,n=138). Naturally infected Ae. aegypti were also able to transmit the virus vertically to a 57.5% of surviving progeny. Furthermore, vertical transmission in naturally infected lines seems to persist for more generations (up to 6 generations) than it has been seen in experimental infections (2 generations) (Barreau et al., 1997;Kittayapong et al., 1999), highlighting that vertical transmission can play a big role in the persistence of MDV infections in the natural populations. Another study also detected the AeDV Thai strain infection in An. minimus adults and larvae, with an overall infection prevalence around 19% in larvae and 15% in adults (Rwegoshora et al., 2000). Larval infection also showed a positive correlation with rainfall two months prior to larval collection (Rwegoshora et al., 2000). Interestingly the infection prevalence in larvae seem to affect the next month's prevalence in adults negatively, suggesting that higher mortality in larvae may cause lower prevalence in adult mosquitoes (Rwegoshora et al., 2000. MDVs are really stable in nature and have been shown to stay infective after a year of introduction to a water body (Buchatsky, 1989). MDVs can be already present in the water where female mosquitoes lay their eggs and can infect the hatching larvae. Infected larvae by releasing virions into the water can horizontally transmit the virus to other larvae (Barreau et al., 1996;Ledermann et al., 2004). Depending on the dose they have received and the larval stage when they got infected, larvae can show varying rates of mortality (Barreau et al., 1996;Suchman et al., 2006). Surviving larvae can emerge as adults and vertically transmit the infection to their offspring, or horizontally to new oviposition sites and venereally to other adults (Barreau et al., 1997;Suchman et al., 2006;Wise de Valdez et al., 2010). The infection rates and intensity can possibly be affected by rainfall and host density (Barreau et al., 1996;Kittayapong et al., 1999;Rwegoshora et al., 2000) (Figure 3). Experimentally, AalDV and Dengue viruses have been shown to persistently co-infect C6/36 cells. However, AeDV replication has been increased after the infection with Dengue virus, while AeDV presence decreased the Dengue infection (Wei et al., 2006).

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Ambidensoviruses get their name from the ambisense organization of their genomes: ORFs of their NS and VP proteins are on the 5' halves of the complementary strands and virion populations encapsidate equal numbers of both single positive and negative strands (Kerr et al., 2005). Their genomes are about 6kb in length and contain about 500 nucleotide long inverted terminal repeats (ITR) (Kerr et al., 2005). Most ambidensoviruses have three non-structural proteins (NS1-NS2-NS3) while other genera of densoviruses have only two (Kerr et al., 2005). NS3 polypeptides are thought to be acquired by horizontal transmission early in the evolution of ambidensoviruses, as similar proteins have been found in other viruses such as

Cryptophlebia leucotreta granulovirus from different virus families (Baculoviridae) (Kerr et al., 2005). They all have Ca ++ binding loop and PLA2 motifs in their VP region that are conserved in all ambidensoviruses (Zádori et al., 2001). Ambidensoviruses have been found in five insect orders Lepidoptera, Diptera, Hemiptera, Blattodea, Orthoptera (Figure 1) (Cotmore et al., 2014). Some of these like GmDV are restricted to their original host, Galleria mellonella, some such as AdDV (Acheta domestica densovirus) and PfDV (Periplaneta fuliginosa densovirus) are restricted to closely related hosts, while others, e.g. JcDV (Junonia coenia densovirus), MlDV (Mythimna loreyi densovirus) can infect many different hosts (Tijssen et al., 2016).

Lepidopteran ambidensovirus strains are generally really closely related and very pathogenic for their hosts. Diatraea saccharalis densovirus (DsDV) induced symptoms that starts at the 4th day following the infection of the larvae and mortality reached 100%, 21 days after the infection (Kouassi et al., 2007). In the case of GmDV, the virus is so virulent and contagious that it has been discovered after a population decline in the mass rearing of Galleria mellonella larvae for fishing bait (Meynadier et al., 1964). MlDV which shows 95% sequence identity to GmDV, can infect many lepidopteran pests. First isolated from Mythimna loreyi. MlDV was also found in field populations of Agrotis ipsilon, Agrotis spinifera, Autographa gamma, S. exigua and S. littoralis (El-Mergawy et al., 2003). Another ambidensovirus that can infect several lepidopteran species is JcDV and has been shown to replicate in Aglais urticae, Bombyx mori, Chrysodeixis chalcites, Lymantria dispar, Mamestra brassicae Mamestra oleracea, Scotia ipsilon, Spodoptera exigua, Spodoptera littoralis (but not in G. mellonella ) and kill these hosts 18-27 days after infection (Rivers and Longworth, 1968;Mutuel et al., 2010). Remarkably Helicoverpa armigera densovirus-1 is the only not pathogenic lepidopteran ambidensovirus. In fact it increases the developmental rates of their hosts both during larval and pupal stages, lengthens their lifespan and increases female fecundity (Xu et al., 2014).

Moreover in field studies they have been shown to increase the resistance of their pest host against biological population control tools, such as Bacillus thuringiensis and a pathogenic baculovirus (Xu et al., 2014). Interestingly, mutualistic HeDV was found to be vertically transmitted to 100% of the offspring.

Similar mutualistic interactions between ambidensoviruses and their hosts have also been reported for hemipteran ambidensoviruses. Dysaphis plantaginea densovirus (Hemipteran ambidensovirus 2) infects rosy apple aphid (Ryabov et al., 2009). Asexually reproducing populations of these aphids, having the same genotype can display two different phenotypes: wingless or winged.! While winged aphids are smaller and have lower fecundity than their wingless counterparts, wingless aphids are necessary for the survival of the clonal population in some ecological conditions (Ryabov et al., 2009). For a long time scientists thought crowding of the host plant and poor quality diet were the reason behind the apparition of winged aphids (Wadley, 1923). Instead, Ryabov et al. (2009) demonstrated that DplDV caused the apparition of these winged aphids in poor diet and crowded conditions allowing aphids to spread to a new host ensuring the survival of their clonal population (Ryabov et al., 2009). Indeed DplDV-free aphid populations were not able to produce wiged morphs (Ryabov et al., 2009). Moreover, aphids infected with DplDV could still produce some virus-free offspring -hence wingless and with higher fecunditywhich allows a fast population growth on the newly colonized plant (Ryabov et al., 2009). Myzus persicae nicotianae densovirus (MpnDV, Hemipteran ambidensovirus 3) on the other hand slightly slowed down the development of the aphids and decreased their body weight which is often correlated with fecundity although they were not highly pathogenic for their hosts. They were also vertically transmitted to the offspring of their hosts. In natural populations of aphids in China, the prevalence of MpnDV was 34%. Phylogenetic analysis revealed that the MpnDV were structured geographically with low level gene flow between locations, suggesting their high vertical transmission and putative co-evolution with their hosts (Song et al., 2016) Both Blattodean ambidensovirus 1 (Periplaneta fuliginosa densovirus) and 2 (Blattella germanica densovirus 1) were found highly pathogenic to their hosts (Mukha et al., 2006;Jiang et al., 2008). Similarly Orthopteran ambidensovirus 1,

Acheta domesticus

AdDV, caused outbreaks of very high mortality in rearings in North America (1988,2009) and Europe (1977Europe ( , 2004Europe ( , 2006Europe ( , 2009) (Szelei et al., 2011). Infected A. domesticus were also smaller and less active suggesting that AdDV could impact host ecology in natural populations. Interestingly sequences of AdDV isolates from different outbreaks in North America in 2009 were identical, suggesting the efficient dispersal of the virus between these locations. On the other hand, isolates from European "1977European " , 2004European " , 2006European " , 2007European " , 2009) ) and North American (2009) outbreaks differed slightly and are estimated to have diverged in 2006 (Szelei et al., 2011).

1.4.3! Culex pipiens densovirus (CpDV)

CpDV, isolated from Cx. pipiens mosquitoes (Jousset et al., 2000), is an ambidensovirus, unlike all the other mosquito densoviruses -which are brevidensoviruses. CpDV indeed shares the characteristics of other ambidensoviruses having a larger genome size than brevidensovirus (~6kb compared to ~4kb), an ambisense genome organization and significant sequence homology at both capsid and NS gene levels (Figure2) (Jousset et al., 2000;Baquerizo-Audiot et al., 2009).

. CpDV putatively encodes for three non-structural proteins: NS3, NS1 and NS2 (Figure 4) (Baquerizo-Audiot et al., 2009). Unlike other ambidensoviruses CpDV NS1 and NS2 putative proteins are encoded by four ORFs with introns in between (NS1-NS1, NS2-NS2'; Figure 4) (Baquerizo-Audiot et al., 2009). Another ORF also has been found between NS3 and NS1, however due to the lack of homology with known NS proteins it is not clear whether it is functional (Baquerizo-Audiot et al., 2009) (Figure 4, grey arrows in NS)Their capsid is ~20 nm, similar to other densoviruses, occasionally arranged into paracristalline arrays (Jousset et al., 2000). Like lepidopteran ambidensoviruses, CpDV has four capsid polypeptides (90, 64, 57 and 12 kDa) designated as VP1 to VP4 (Jousset et al., 2000), although there is only two ORFs found (one large 2253 nt shown with purple box and one smaller 443 nt shown with grey arrows in Figure 4) on the 5' side of the complementary strain (Baquerizo-Audiot et al., 2009). Due to lack of homology with known densovirus proteins, it is unknown if the smaller ORF also encodes for VP proteins (grey arrows, Figure 4). CpDV has been discovered following the observation of mass mortality in laboratory colonies of Cx. pipiens kept in the insectaries of ISEM in Montpellier University. Symptoms, like distorted and comma shaped bodies, appeared in 3 rd instar larvae and usually ended up in death in 4 th instar larvae (Jousset et al., 2000). Thick sections of infected larvae showed hyperthrophied nuclei, a clear sign of densonucleosis (Jousset et al., 2000). Insect cell lines were infected with CpDV and replication of it was checked by immunofluorescence (Jousset et al., 2000). No CpDV-positive cells were observed in D. melonagaster and S. littoralis cells (Jousset et al., 2000). The number of fluorescent cells were low in Ae. albopictus but 1% of An. gambiae and Culex cells showed immunofluorescence, suggesting Ae. gambiae larvae could also be susceptible to CpDV (Jousset et al., 2000). After serial passages, CpDV-positive cell amount decreased and no positive cells were observed at the third passage (Jousset et al., 2000).

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Recently a genetically similar densovirus have been found in genomic studies of natural populations of D. melonagaster (Figure 1) (Kapun et al., 2018). Our knowledge about it is so far limited although this suggested there might be similar viruses to CpDV in nature that have not been detected so far. Unfortunately, this discovery has been made too late for this thesis and thus could not be included in the phylogenetic analysis of CpDV in the following chapters.

1.5! Culex pipiens -Wolbachia

Model System

1.5.1! Wolbachia

Among the endosymbiotic bacteria that infect arthropods and influence their phenotype to a large extend, Wolbachia are the most widespread as they may infect up to 50% of the arthropod species (Hilgenboecker et al., 2008;Zug and Hammerstein, 2012;Weinert et al., 2015). They are vertically transmitted from mother to offspring. Hence the success of their transmission relies on the increase of infected females' proportion in the host populations. Such increase can theoretically be achieved either by conferring some fitness benefits to their hosts or by manipulating the reproduction of their hosts in different ways. Wolbachia can use both strategies.

Wolbachia manipulate the host-sex ratio directly or indirectly increasing its spread by inducing cytoplasmic incompatibility, parthenogenesis, male killing or feminizing males (Figure 5) (Werren et al., 2008). Most common of these strategies, Cytoplasmic Incompatibility (CI), has been described in several arachnids, isopods and insect orders (Werren et al., 2008). The mechanism of Wolbachia induced CI is conceptualized as resulting from a modification factor secreted by the bacteria, possibly a toxin, deposited in the sperm that can be rescued in embryos resulting from eggs infected with a compatible strain (Serbus et al., 2008). Infected females can reproduce successfully with both infected and uninfected males, while uninfected females can only have offspring with uninfected males (Figure 5). Another manipulation called feminization consists for Wolbachia of forcing the development of infected genetic male embryos into functional females, thus becoming able to transmit Wolbachia, resulting in a female bias in the population (Figure 5) (Bouchon et al., 2008).

In addition to the ability of the Wolbachia to manipulate their hosts reproduction, some interactions have been reported to be detrimental on various host life history traits, including body size (Hoffmann and Turelli, 1988), fecundity (Hoffmann et al., 1990;Fleury et al., 2000), survival (Fleury et al., 2000;Tagami et al., 2001), larval competitiveness (Huigens et al., 2004), mating choice (Rigaud and Moreau, 2004) as well as the hosts' immune parameters (Fytrou et al., 2006;Braquart-Varnier et al., 2008;Sicard et al., 2010).

Although reproduction manipulation strategies appear efficient for Wolbachia to become widespread in host populations, conferring beneficial traits to their hosts and increasing their host's fitness could give complementary advantage to invade populations. It has been theoretically shown that mutualistic strains should outcompete nonmutualistic ones (Turelli, 1994). In accordance with these predictions, recent studies showed that Wolbachia is not always conflictual and manipulative but may also act as a mutualist with its hosts by being "protective" (i.e. increasing their host's survival to pathogenic challenge) (Teixeira et al., 2008;Gross et al., 2009;Bian et al., 2010;Glaser and Meola, 2010;Zélé et al., 2012;Eleftherianos et al., 2013) or improving nutrition (Hosokawa et al., 2010). The first "protective" mutualistic effect of the presence of a native Wolbachia (wMel, wMel-Pop, wMelCS) against several viruses has been demonstrated in D. melanogaster (Hedges et al., 2008;Teixeira et al., 2008). However, this protection seems limited to RNA viruses (Teixeira et al., 2008;Rottschaefer and Lazzaro, 2012;Ye et al., 2013). On the other hand, in mosquitoes, in addition to the reports of a protective effect against viruses (i.e. Dengue, Chikungunya in Ae. aegypti and Ae. albopictus) (Moreira et al., 2009), protection against protozoans (Moreira et al., 2009), filarial nematodes (Kambris et al., 2009), as well as two bacteria species (Kambris et al., 2009;Ye et al., Cx. pipiens complex (Rasgon and Scott, 2003;Dumas et al., 2013), Ae. albopictus and some An.

gambiae (Baldini et al., 2014;Gomes et al., 2017). Furthermore it is possible to create mosquito lines stably transinfected with Wolbachia either from other mosquito species or Drosophila (Hughes and Rasgon, 2014). In fact, Ae. aegypti (Walker et al., 2011), Ae. albopictus (Blagrove et al., 2012), Cx.

pipiens, and An. stephensi (Bian et al., 2013) have been successfully transinfected this way. Mosquitoes can also be transiently transinfected with

Wolbachia by injection of adult mosquitoes (Kambris et al., 2010;Dodson et al., 2014).

In the natural Wolbachia-host associations, Wolbachia is usually found in lower density, and rarely interferes with pathogens (Johnson, 2015b;Zug and Hammerstein, 2015). For instance, native Wolbachia of Ae. albopictus, wAlbA or wAlbB, did not affect viral replication of Dengue or Chikungunya. However dengue dissemination to salivary glands was slightly reduced (Mousson et al., 2010(Mousson et al., , 2012)). On the other hand, when Ae. albopictus were stably infected with wMel from D. melanogaster both Dengue and Chikungunya transmission were reduced (Blagrove et al., 2012(Blagrove et al., , 2013)). Interestingly when wAlbB was used to stably transinfect naive Ae. aegypti colonies, it reduced the Dengue infection rate, virus load and transmission (Bian et al., 2010), suggesting that pathogen interference was not a characteristic of the host or Wolbachia but rather their interaction. Similarly, both wMel-Pop and wMel, Wolbachia strains from Drosophila, reduced the infection rate and virus load of Dengue, Chikungunya, West Nile and Yellow Fever viruses in stably transinfected Ae. aegypti lines (Moreira et al., 2009;Walker et al., 2011;Hussain et al., 2012;van den Hurk et al., 2012). This important distinction between the protective phenotypes that native and non-native Wolbachia cause, is thought to be related to the higher density that the bacteria reaches when transinfected. In contrast, in natural Wolbachia-host associations bacteria stay at lower densities. Indeed Wolbachia density and protective phenotype correlation have also been shown in mosquito cells against Dengue and West Nile virus (Hussain et al., 2012;Lu et al., 2012;Frentiu et al., 2014) and in Drosophila against RNA viruses (Martinez et al., 2017) Wolbachia have also been studied for their interactions with Plasmodium parasites. Stably transinfected An. stephensi with wAlbB conferred resistance against Plasmodium falciparum (Bian et al., 2013) and reduced Plasmodium yoelii oocyst intensity at 28°C while there was no effect of wAlbB at 20°C (Murdock et al., 2014). In natural Wolbachia-Cx. pipiens system, Wolbachia increased the mosquitoes' susceptibility to Plasmodium relictum infection (Zélé et al., 2014). Recently Wolbachia infected Anopheles mosquitoes have also been reported in Burkina Faso and Mali (Baldini et al., 2014;Shaw et al., 2016;Gomes et al., 2017). In field studies a lower prevalence of Plasmodium infection in An. coluzzii infected with Wolbachia have been reported (Shaw et al., 2016;Gomes et al., 2017). Interestingly in this case this negative correlation between native Wolbachia and Plasmodium seem to occur despite the very low density of Wolbachia (Gomes et al., 2017).

Though interesting, many of these situations do not represent natural symbiotic systems where Wolbachia would have evolved towards being mutualistic by conferring protection to their host against their natural enemies. Wolbachia conferred protection in mosquitoes have mostly been investigated against human pathogens (that mosquitoes are vectors for, typically arboviruses), owing to the prospective applications in disease control. However, the interactions between Wolbachia and pathogens for which the mosquitoes are the main hosts could be really different, although these are not very well studied. The insect-specific flaviviruses only recently gained attention following the appreciation of their ubiquity in mosquitoes and the discovery of the possible impact on arbovirus transmission (Bolling et al., 2012;Hobson-Peters et al., 2013;Kenney et al., 2014;Vasilakis and Tesh, 2015). However, their basic ecology in nature, their potential effects on mosquitoes and arbovirus transmission is largely unknown. On their interactions with Wolbachia, there are only two recent studies. Newly discovered insect specific virus, Anphevirus (AeAV) infection have been enhanced by Wolbachia in Ae. aegypti cells (Parry and Asgari, 2018). Similarly, field studies in release sites of stably transinfected Ae. aegypti mosquitoes were associated with higher insect specific flavivirus infection rates and density compared to the Wolbachia-free mosquitoes (Amuzu et al., 2018). Much like insect-specific flaviviruses (RNA viruses), effects of DNA viruses on the vector competence of mosquitoes and their interactions with host associated Wolbachia is poorly investigated. However, Wolbachia have been shown to enhance DNA virus infection in Drosophila (Teixeira et al., 2008) and in Spodoptera exempta (Graham et al., 2012).

Although mechanistic bases of Wolbachia's pathogen interference are not well understood, it is thought to be either caused i) indirectly by modulation of the host immune system or modification of host cellular structures resulting in a less hospitable environment for viral replication or ii) by direct competition between Wolbachia and pathogens for host resources (Terradas and McGraw, 2017). Enhancement of the pathogen replication can similarly be caused by the modulation of the immune system (i.e. reduction in resistance) of the host by Wolbachia or to a better tolerance (i.e. survival to higher pathogens load) when they are already infected with Wolbachia. Possibly it could also mean that once infected with a deadly pathogen, host loses the control of their endosymbiont hence causing an increase in Wolbachia density. In the case of insect-specific viruses Wolbachia could also be interacting directly with the viruses during their covertical transmission. Further studies on these interactions are needed both to understand ecology and evolution of different partners of these multi-partite systems and for the use of Wolbachia and insect specific viruses in natural host populations as vector or disease control strategies.

1.5.2! Wolbachia in Culex pipiens mosquitoes

Consisting of several species, including important disease vectors with worldwide distribution (e.g.

Cx. pipiens and Cx. quinquefasciatus) (Farajollahi et al., 2011), mosquitoes of Cx. pipiens species complex have a unique relationship with their endosymbiont Wolbachia (wPip). Indeed, wPip is fixed in natural Cx. pipiens s.l. populations where they induce the most complex CI relationships yet described among arthropods, including uni-and bidirectional incompatibility (Laven et al., 1967;Duron et al., 2006;Wolfgang et al., 2009;Atyame, Delsuc, et al., 2011;Atyame et al., 2014). In contrast to this observed diversity of CI patterns, Cx. Pipiens' wPip strains are closely related and all belong to a clade within Wolbachia B super group (Guillemaud et al., 1997;Baldo et al., 2006;Atyame, Delsuc, et al., 2011). However, recent studies of fast evolving markers showed the presence of many genetically distinct wPip strains in Cx. pipiens s.l. mosquitoes (Duron et al., 2006(Duron et al., , 2007;;Atyame, Delsuc, et al., 2011) distributed in five distinct phylogenetic groups (wPip-I to V) (Atyame, Delsuc, et al., 2011). Using a PCR/RFLP assay based on pk1 gene, encoding proteins with ankyrin motifs, a wPip strain can be assigned to one of these five groups (Atyame, Delsuc, et al., 2011;Dumas et al., 2013). A study of the wPip worldwide distribution showed an important spatial structure of wPip groups (Dumas et al., 2013).

For instance, only wPip-I was found in Sub-Saharan Africa, South America and Southeast Asia, while wPip-III was mainly observed in North America. Strains belonging to wPip-II group were mostly found in Western Europe and wPip-V in Asia.

wPip-IV group strains exhibit a patchy distribution in Europe, North Africa and Asia (Dumas et al., 2013). In addition, Wolbachia genetic diversityespecially diversity of a loci called cida/cidB (Atyame et al., 2014;Bonneau et al., 2018) strongly correlate with their CI patterns. Most wPip strains from the same group render their host compatible with each other (with the exception of few unidirectional incompatibilities) whereas those from different groups often lead to unidirectional or bidirectional incompatibilities (Atyame et al., 2014). No effect of host genetic background on the CI patterns (Duron et al., 2012) and no multiple infections by several strains have ever been shown (Atyame, Delsuc, et al., 2011;Atyame, Duron, et al., 2011;Dumas et al., 2013).

1.6! Objectives of this thesis

As mentioned before, bacteria have been studied

for some time now as intimate partners of their hosts. This perspective, taking microorganisms into account, revolutionized many aspects of science including applied and medical sciences. Recent advances of sequencing technologies also showed the ubiquity and diversity of viruses. It is thus now possible to imagine that viruses can have long-term intimate relationships with their hosts and these relationships can evolve to become antagonistic or mutualistic, or anywhere on the symbiotic spectrum. A lot of factors can shape this evolution including abiotic factors and biotic factors, such as the host or the interactions with the rest of the microbiota. Hosts can be seen as habitats where ecological interactions happen in different scales (Douglas, 2016).

In this context arthropods represent really interesting study models not only because they are ancient and really diverse but also they are able to establish symbiotic relationships with endosymbionts and sometimes to support multiple endosymbionts in their cells. Densoviruses, as ancient and diverse as their arthropod hosts, are also ideal candidates to investigate host-virus-bacteria relationships.

During my PhD, I studied these interactions in a natural host-bacteria-virus system, that is Cx.

pipiens-Wolbachia-CpDV, both in natural populations and laboratory colonies. Interactions within Cx. pipiens, wPip and CpDV could be intricate (Figure 6). Mosquito hosts could control the density of their endosymbiont Wolbachia and Wolbachia in return can modulate host immune system, affect host fitness positively or negatively, and manipulate host reproduction (Figure 6). Host immune system stimulated/supressed by Wolbachia could suppress/enhance CpDV. CpDV could also have fitness effects on their hosts or modulate host immune system hence indirectly influencing Wolbachia density. Wolbachia and CpDV could also compete for resources within the host resulting in the decrease in density of one of them (Figure 6). Alternatively costs of co-infection could be too high and cause disease leading to higher densities of both (Figure 6). Vertically transmitted Wolbachia could possibly have an effect on the transmission of CpDV (Figure 6).

In natural populations, the interactions between Wolbachia and CpDV can be diverse. One major parameter would be the interaction outcome between CpDV and their hosts which could range from mutualism to antagonism. If CpDV are harmful for their hosts, wPip groups which can confer a better protection to their hosts could invade populations easier. On the other hand, CpDV could be mutualistic or commensal with its host. In this case wPip strain which supports the CpDV infection could be favoured. wPip could also be allowing or facilitating vertical transmission of CpDV in which case it might be possible observe their co-divergence together.

First of all, as the knowledge on CpDV was limited to its first discovery in ISEM's insectary in Montpellier University (Jousset et al., 2000), we investigated their prevalence in natural populations of Cx. pipiens worldwide to understand their relevance for Cx. pipiens-wPip system in natural populations (Chapter 2). For this I first established a specific PCR diagnostic test. This approach made the use of many DNA samples, that have been collected by our team (EVAS) over the years for different studies, possible (Chapter 2). I then further studied their diversity and evolution in natural populations(Chapter 2).

Secondly, given their fundamental differences with the other mosquito densoviruses described so far, I have tried to understand their life cycle and dynamics in our laboratory colonies naturally infected with both Wolbachia and CpDV (Chap-

CHAPTER 2:

Prevalence and evolution of CpDV

Introduction

Single-stranded DNA (ssDNA) viruses infect hosts in all domains of life and environmental conditions (Pietilä et al., 2009;Rosario and Breitbart, 2011;Mochizuki et al., 2012;Krupovic, 2013). They can also be found integrated in their host chromosomes, as endogenous viral elements, in many vertebrates and invertebrates suggesting their long-term evolution with animals (Belyi et al., 2010;Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015).

The ssDNA virus genome size, typically below 10kb, is thought to be constraint by their small capsid size (a diameter of 18-27 nm) (Krupovic, 2013). The evolution of their genomes is shaped by high punctual mutation rates (Shackelton et al., 2005)-that might also restrain their genome size due to fast accumulation of deleterious mutationsand frequent recombination both by DNA and RNA donours (Shackelton et al., 2007;Martin et al., 2011;Martynova et al., 2016;Tijssen et al., 2016). Indeed, ssDNA multiple origins are thought result from independent recombination events between bacterial plasmids and RNA viruses (Krupovic, 2013;Koonin et al., 2015). Parvoviridae family represents one of the most widely distributed ssDNA virus families in animals (Cotmore et al., 2014). They infect a wide range of metazoans including vertebrates (Parvovirinae subfamily) and invertebrates, mostly arthropods (Densovirinae subfamily) (Cotmore et al., 2014).

The study of their diversity has been largely biased towards medically and economically important viruses, especially the ones affecting mammals (Cotmore et al., 2014). With the advances in viral metagenomics and next generation sequencing techniques, their high diversity and prevalence in wider host range, particularly in arthropods, have been unravelled (François et al., 2016). Densovirinae indeed are expected to be more diverse than their vertebrate counterparts (Parvovirinae subfamily), as their arthropod hosts, are also more diversified and ancient. Densovirinae subfamily so far consists of 5 genera (i.e. Iteradensovirus, Brevidensovirus, Ambidensovirus, Penstyldensovirus, Hepandensovirus) that have been isolated mostly from insects (e.g. lepidopteran, dipteran, orthopteran, and dictyopteran) and malascostracean (e.g. prawns and crayfish) (Kerr, J. and Cotmore, S. and Bloom, 2005;Tijssen et al., 2016). They are usually pathogenic for their hosts causing epizootics in the infected host populations. Therefore they have long been considered as possible population control tools against agricultural pests such as lepidopterans or as human disease vectors such as dipterans (Carlson et al., 2006;Mutuel et al., 2010;Gosselin Grenet et al., 2015;Suzuki et al., 2015).

Mosquitoes being the vectors of many human diseases, their associated viruses have been studied, originally with a priori diagnostic tests designed for human RNA arboviruses (Kuno, 1998). With the recent advances in viral metagenomics, mosquito virome could now be explored without a priori.

Nevertheless mosquito virome studies are yet still mostly focused on RNA viruses transmissible to humans and lately on insect specific RNA viruses while DNA virus diversity in mosquitoes stayed even less explored (Bolling et al., 2012;Junglen and Drosten, 2013;Blitvich and Firth, 2015). However, the only study which focused on the discovery of mosquitoes' DNA viruses revealed their contribution to mosquito's viromes, especially highlighting the importance of Densovirinae (Ng et al., 2011).

Mosquito densoviruses (MDVs) have been isolated from several natural populations and laboratory colonies of different species of mosquitoes and found to persistently infect many mosquito cell lines (Afanasiev et al., 1991;Jousset et al., 1993Jousset et al., , 2000;;Boublik et al., 1994;O'Neill et al., 1995;Kittayapong et al., 1999;Rwegoshora et al., 2000;Ng et al., 2011), although MDVs have never been investigated in terms of their general prevalence, diversity and putative impact on vector ecology in natural populations.

Most of the MDVs discovered so far are closely related (82-99% sequence homology) and considered to be different strains from two distinct species (so called Dipteran Brevidensovirus 1 and 2) belonging to the genus Brevidensovirus, except Culex pipiens densovirus (CpDV) (Cotmore et al., 2014). CpDV has been isolated from Culex pipiens laboratory colonies following high larval mortality periods (Jousset et al., 2000). With its ambisense genomic organization and larger genome size (~6kb) compared to brevidensoviruses (~4kb), CpDV belongs to Ambidensovirus genus (Baquerizo-Audiot et al., 2009;Cotmore et al., 2014). Like other members of the Parvoviridae family, CpDV genome only encodes two types of proteins: nonstructural proteins (NS or Rep for replication proteins) that have a role in the viral gene expression and DNA replication, and structural proteins (VP for viral proteins or Cap for capsid proteins) that constitute the components of the viral capsids (Figure 1) (Kerr et al., 2005).

First isolated two decades ago following unusually high larval mortality in Culex pipiens colonies (Jousset et al., 2000), CpDV has later been detected to persist in seemingly healthy laboratory colonies in all life stages of mosquitoes ( Chapter3). Their covert infection, in low amounts, combined with vertical transmission explained their long-term persistence in laboratory colonies and suggested that these viruses could also be prevalent in natural populations of mosquitoes (Chapter 3).

To test this hypothesis, we designed a CpDV specific PCR-based diagnostic test to investigate CpDV prevalence in Culex pipiens mosquitoes collected worldwide for more than forty years in natural populations (Dumas et al., 2013;Atyame et al., 2015;Altinli et al., 2018). Sequencing amplicons from Open Reading Frames (ORF) encoding for partial VP and NS allowed us to analyse the worldwide evolution of CpDVs in natural populations and laboratory colonies.

! Methods

! !"#$%&'()%%&(*+),'",-'./0'&1*2"(*+),3' Samples from natural Cx. pipiens (s.l.) populations have been collected between 1975-2018 (Appendix Table 1). Samples that have been sequenced were chosen among the different populations to cover the diversity in different continents. Slab, laboratory mosquito line, that has been initially collected from USA in 1954 (Georghiou et al., 1966) has been reared in our insectary from 1985 to present . We used individuals kept in liquid nitrogen between 1990 and 2018 to follow genetic variations between CpDVs through this time period (Appendix Table 1). ! 4$.5'6&,)#&3'$"2*+"%'3&78&,(+,6'' CpDV have three putative NS genes (Figure 1). For the detection of CpDV, CpDV specific set of primers (Appendix Table 2) was designed from position 1606 to 1843 (238pb) in NS2 and NS2' overlapping ORFs that are expected to be CpDV specific and very conserved (Figure 1). A PCR diagnostic test using these primers was established (Appendix Table 2).

In NS1 region we designed the primers to amplify the whole NS1' (1681-2648; 967nt, Figure 1) which encompasses conserved SF3 helicase domain used for taxonomic classification of densovirus genuses (Cotmore et al., 2014). We also sequenced partial VP ORF including conserved PLA2 domain and stretches for 1389 bp (3570-4959; 1389 bp, Figure 1). With both of these NS and VP fragments amplified, we covered about 40% of the CpDV genome (2.356 over 6kb). A total of 54 samples were amplified and sequenced for NS1 gene, and 39 samples were amplified and sequenced for both genes. To improve the amplification of both NS1 and VP regions semi-nested PCRs were conducted with two PCR steps (Appendix Table 2). PCR products were purified using AMPure (Agencourt) with Biomek 4000. Following the purification, sequencing reaction has been performed using 100ng of DNA. After the completion of sequencing reaction (96°C for 5min, 30 cycles of 96°C 15sec, 50°C 10sec and60°C 4mn All sequences were aligned using MAFFT v7.273 (Katoh and Standley, 2013). In order to avoid misleading phylogenetic signal due to recombinant parts of sequences, signatures of recombination were searched with help of RDP, GENECONV, Bootscan, MaxChi, and LARD methods with default parameters, as implemented in the RDP4 software (Martin et al., 2015). Six sequences were identified as potential recombinants by more than two methods. These sequences were removed from the dataset.

To build maximum likelihood phylogenetic tree of CpDV, we started by using modelfinder (Kalyaanamoorthy et al., 2017) in order to find the model which best fits our dataset while avoiding over-parameterization (Lemmon and Moriarty, 2004). The HKY85 + \Gamma model of sequence evolution using RAxML v8.2 was chosen (Stamatakis, 2014). This model estimates different substitution rates for transitions and transversions (Hasegawa et al., 1985), and allows for a Gamma distribution of evolutionary rates among sites (Yang, 1994). Statistical support of the phylogenetic nodes was computed using a bootstrap procedure, 100 iterations (Felsenstein, 1985). Both concatenated (i.e. VP and NS) and individual gene trees were computed using the same protocol.

Coding regions of our sequences including, ORF of NS1' (1764-2512; 750 bp) and VP (3545-4867; 1326 bp), have been analysed for selection using DATAMONKEY web server (Weaver et al., 2018). Individual sites have been tested for i) episodic positive or negative selection using MEME (Mixed Effects Model of Evolution) (Murrell et al., 2012) and ii) pervasive positive or negative selection using FEL (Fixed Effects Likelihood)(Kosakovsky Pond and Frost, 2005). Distance between CpDV variants and different clades were calculated using Jukes-Cantor distance model implemented in Mega7.

! ><=%)-=,"#+(3'",-'-+3$&23"%')?'4$.5' Bayesian Markov Chain Monte Carlo approach implemented in BEAST v1.8.4 package (Suchard et al., 2018) was used to estimate the evolution rate, the time to the most recent common ancestor (tMRCA) and internal nodes, and the geographic movement of CpDV. For these analyses we only considered samples where both VP and NS1' amplicons were obtained (n=39, length=2076bp).

Both concatenated, and separate NS1'-VP data sets were analysed as these two parts of the genome could be evolving differently.

Exponential growth model has been used as the tree prior with a relaxed uncorrelated lognormal molecular clock model (Drummond et al., 2006) and HKY85 substitution model. The symmetric substitution model, a standard continuoustime Markov chain (CTMC) in which the transition rates between locations are symmetric, has been used as the discrete trait substitution model to infer migratory events. As we had many sampling sites from some geographical areas(e.g. four in Sub-Saharan Africa) and less samples from others (e.g. New York one location), we have grouped the samples from the same geographic area to decrease the sampling bias and inferred the major dispersion routes between these areas (i.e. Turkey and Armenia as Western Asia; Porto Rico, Haiti and Venezuela as Central America; China, Vietnam, Thailand as Eastern Asia; Sub-Saharan African countries as Sub-Saharan Africa; Montpellier natural populations and laboratory, as well as one sample from Moscow as Europe).

The Bayesian stochastic search variable selection (BSSVS) approach was used to identify wellsupported rates using Bayes Factors test using Spread3 (Bielejec et al., 2016). Markov Chain Monte Carlo was ran for 100 million generations and convergence (Effective Sample Size >200) of all parameters was verified with TRACER v1.6.0 (Rambaut et al., 2018). TreeAnnotator v1.8 was used to obtain the maximum clade credibility tree after discarding 10,000,000 states (10%) as burn-in. The estimated uncertainty of the results is reported as 95% highest probability density (HPD) values. For each node posterior probability have also been reported to . We verified that our results were not shaped only by the priors by running the MCMC chains on the data sets with the same prior parameters but without the sequence data.

! Results

! 4$.5'$2&@"%&,(&'+,'!"#$%&'('($)*'$)$8; %"*+),3'A)2%-A+-&'

To investigate the general prevalence of CpDV in Cx. pipiens populations, a total of 2843 individual larvae samples from 136 different locations that have been collected between 1975-2017 were subjected to a diagnostic CpDV specific PCR test (Figure 1, Appendix Table 1). 48% of the samples were CpDV positive and displayed an amplicon at expected size (Figure 2, Appendix Table 1). In Primers within NS1 and VP genes have been designed to infer the diversity of CpDV genomes worldwide as well as their diversity and evolution within one laboratory line through a 28 years period. A total of 54 samples were selected due to their location, amplified and sequenced for NS1 gene, then 39 of them were successfully amplified and sequenced for VP gene. As for some samples it was not possible to amplify VP gene fragment, putatively because of i) the higher diversity of this region that prevents primers to be suitable for all the samples, or ii) VP, which is a longer DNA fragment compared to NS, was more difficult to amplify due to poor DNA conservation over the years. we had too many sampling sites for some specific areas such as Turkey, Tunisia and Mayotte we were not able to show all 136 populations on the map separately and rather grouped these sampling sites together. (see for the full Appendix, Table 1 for the list of sampling sites and CpDV prevalence). 48% of these samples showed a PCR amplification. Only in 29 out of 136 locations there was no CpDV amplification in any of the individuals.

Recombination events

Several studies highlighted the negative impact of recombination on phylogenetic reconstruction of viruses evolutionary histories (Scheffler et al., 2006;Arenas and Posada, 2010;Pérez-Losada et al., 2015). We thus checked recombination in both NS1

and VP gene datasets, as well as in the concatenated one. There was no recombination detected within the sequenced NS1 gene fragment. Within VP region, we have detected 2 different recombinant sequences (between Haiti 2 and Bangui 3, Appendix Figure 1). However, within the concatenated dataset, we observed 4 other recombinant sequences. Breakpoints of recombination were similar in many of the recombinants (Appendix Figure 1 shown as New York 2, but also found in samples from Venezuela 2, Karaoke, Porto Rico 2) generally located around 900 nt position. As these positions correspond to where NS1 sequence ends and VP sequence starts it highlighted the phylogenetic incongruence between these two parts of the CpDV genome (Appendix Figure 1).

Phylogeny

We have constructed a midpoint rooted maximum likelihood phylogenetic tree with the concatenated dataset of NS1' and VP sequences after the removal of the recombinant sequences (n=48). Three distinct and well supported groups were discriminated within CpDV: CpDV1, CpDV2 and CpDV3 (Figure 3). One of the groups was represented only by one sample from Beijing collected in 2003. The other two groups were mixed in terms of their collection date and geographical location. We have also built phylogenetic trees using NS1' and VP sequences separately. While most of the phylogenetic relationships were well resolved in both of the gene trees, and generally bootstraps values were better than it is for the concatenated tree.

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General topology of the trees was similar to the concatenated tree, in terms of the two well supported clades, CpDV1-CpDV2, although trees were not totally congruent. Overall mean distance between variants calculated for the concatenated data was 0.02 (± 0.005) nucleotides. Distance between CpDV1 and CpDV2 clades was 0.05 (± 0.01)

nucleotides, confirming all the variants were closely related and belong to CpDV species.

Selection

The

Phylodynamics of CpDV

To understand the evolution, population dynamics and dispersal of CpDV, we used concatenated coding sequence matrix of the samples. As recombination analysis showed a breakpoint between NS1 and VP sequences we have also analysed these genes separately.

The resulting tip-dated phylogenetic trees using both gene fragments and concatenated datasets (with and without recombinant regions) were similar in topology to the maximum likelihood tree (Figure 4, Appendix Figure 4). Only major difference is that as we were missing Beijing 2 sample which represented CpDV3 in probabilistic phylogenetic tree. The two well-supported clades in maximum likelihood tree were present in the tip dated Bayesian tree as well, however CpDV-2 showed lower Bayesian posterior probability (Figure 4). We have detected major differences between the estimated most recent common ancestor (tMRCA) of different gene trees and concatenated datasets. This was mainly caused by the incongruence between NS1 and VP coding regions. This incongruence could be a result of recombination that were mainly identified in NS1 region (Appendix Figure 6). Hence the exclusion of these regions meant exclusion of NS1 fragment from the dataset. Models were run with and without data to make sure that the results were not only shaped by the prior parameters and that the data had enough temporal information. Both NS1 and concatenated datasets showed sufficient temporal structure while it was less true for VP dataset. With the concatenated dataset time to most recent common ancestor (tMRCA) was estimated to be 71 years before present (1947; HPD 95% 40-108 years before present, Table 1) and mean substitution rate was 6.18x10 -4 (Table1). The mean substitution rate was lower when the analysis was based on only NS1 region (Table 1, 9.3.10 -5 substitution/site/year). tMRCA for this gene was approximately 245 years before present although the confidence of this estimate was lower compared to concatenated dataset (HPD 95%, 50-540 years before present). Due to the lack of temporal information VP region a lower substitution rate and higher uncertainty for the estimation of tMRCA (Table 1).

The NS1', VP and concatenated datasets were also analyzed using exponential growth model to determine the fluctuations in growth of the effective population size between the 1990-2018. We have observed a constant population size through time for both NS and VP fragments while for the concatenated dataset, there was a slight decrease in population size (see Appendix Figure 5). CpDV-1 clade was found in Cx. pipiens from all the sampled areas while CpDV-2 was only found in the samples from Europe (Montpellier), Western Asia (Turkey and Armenia) and South-Central America (Venezuela and Haiti). To infer the dispersal of CpDV, we have only used NS1 gene fragment due to the uncertainty due to the recombinants in concatenated dataset. CpDV1 and CpDV2

clades are likely to be diverged in either South-Central America or Sub-Saharan Africa (SSA) and dispersed to worldwide mosquito populations from here. Sub-Saharan Africa appears to be a hub for CpDV 1 clade distribution as a meeting point of highly significant dispersal routes (Table 2; SSA-Eastern Asia, SSA-New York, SSA-South America, SSA-Western Asia). Variants of the CpDV 2 clade seem to have colonized Europe and Eastern Asia in two different occasions although we did not observe any significant dispersal route between these locations (Table 2, Figure 4). Prevalence and diversification of insect ssDNA viruses have never been studied at a worldwide scale.

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Our study revealed that one of them, the CpDV, is detected in 78% of the 136 Cx. pipiens populations and, on average 48% of larvae belonging to these populations. While it is possible this high prevalence is due to the detection of endogenized CpDV sequences in the mosquito genome rather than detection of a CpDV infection, CpDV endogenization has never been reported (Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015;François et al., 2016). Furthermore, the absence of CpDV in some of the samples within the same location would require this endogenization to be really recent and an on-going process. In the laboratory lines, we demonstrated variations in vertical transmission and were able to isolate CpDV particles demonstrating that CpDV is not endogenized ( Chapter3). MDVs had been previously detected in mosquito's natural populations but reports on their prevalence are scarce (Kittayapong et al., 1999;Rwegoshora et al., 2000). Indeed, to our knowledge, two studies on Aedes aegypti Thai strains densovirus showed that 6% to 35% of Ae. aegypti adults were infected in natural populations (Kittayapong et al., 1999) while the same densovirus was later found at 19% prevalence in natural populations of Anopheles minimus larvae (Rwegoshora et al., 2000). The prevalence of the latter Brevidensovirus was influenced by seasonal environmental changes and rainfall, probably due to variations in host population density and consequently transmission rate of the virus (Kittayapong et al., 1999;Rwegoshora et al., 2000). Here, we did not statistically test the influence of environmental factors. However, the 2843 individuals analysed were collected in 33 different countries in quite contrasted environments, the presence of CpDV in half of them, independent of latitudinal or longitudinal distributions, suggests the persistence of CpDV in natural Cx. pipiens populations living in quite different environmental conditions.

The phylogeographic diffusion analysis further demonstrated a significant dispersal of CpDV in Cx. pipiens populations worldwide, with highly significant dispersal routes especially meeting in Sub Saharan Africa. This dispersal of CpDV could be achieved by the migration of mosquitoes of the Cx. pipiens complex by planes and by boats, as either larvae or adults (Farajollahi et al., 2011). Mosquito eggs and larvae can also be dispersed in used tyres (Schaffner, 2003). Emergent infected adult mosquitoes can transmit densoviruses to new larval/oviposition sites horizontally (Wise de Valdez et al., 2010) or to their offspring vertically (Barreau et al., 1997;Ren et al., 2014)(Chapter3). Densoviruses have also been shown to stay active in natural conditions for long amount of time (Buchatsky, 1989) so they could have also been carried environmentally by human activity alone. Another possibility for densovirus dispersal could be via migratory birds. Migratory birds have been associated with emergence of many viruses including West Nile Virus (Reed and Medical, 2003), they also could possibly carry CpDV in low titers. Although densoviruses have previously been found in insectivorous bats faeces (Ge et al., 2012) and in a great tit (Yang et al., 2016), their transmission to mosquitoes through birds have yet never been tested.

In addition to their prevalence and dispersion, we inferred the worldwide CpDV diversity in natural populations using NS and VP markers that cover 40% of the CpDV genome. We demonstrated that CpDV strains are polymorphic on these markers and that all CpDV strains sequenced form three distinct well-supported clades (CpDV 1, CpDV 2, CpDV 3). These clades are closely related suggesting that all CpDV strains reported here belong to one single species within Ambidensovirus genus. Indeed in Densovirinae sub-family, viruses in a species required to show >85% amino acid sequence identity in NS1 protein (Cotmore et al., 2014). Other MDVs, all belonging to Brevidensovirus, have been so far grouped in two species: Dipteran Brevidensovirus 1 (Lebedeva et al., 1972;Chen et al., 2004;Ren et al., 2008;Zhai et al., 2008;Sivaram et al., 2009) and Dipteran Brevidensovirus 2 (Jousset et al., 1993;Boublik et al., 1994;O'Neill et al., 1995;Paterson et al., 2005) with several really closely related (98% sequence similarity) variants within these species. However, phylogenetic analyses conducted on NS and VP separately, even if they both demonstrated the same clades, exhibited some incongruencies. These incongruencies explained by the recombination breakpoint that has been highlighted by our analyses right at the end of NS coding region. Further investigation of the recombinants indicated that NS and VP fragments were more closely related to different parental recombinants. Recombination is known to be fundamental both in the origin and the evolution of ssDNA viruses such as densoviruses (Krupovic, 2013;Koonin et al., 2015). An extreme example of is the origin of ssDNA viruses that is thought to result from ancient recombination events resulted in acquisition of NS from DNA donors (such as bacterial plasmids) and VP from RNA viruses (Krupovic, 2013;Koonin et al., 2015). Moreover, recombination have also been shown to play an important role in microevolution of densoviruses, especially in Ambidensovirus and Iteradensovirus genera (Martynova et al., 2016). Indeed, phylogenetic trees using different gene fragments (NS3, NS1 and VP) had also lead to incongruences in phylogenetic relationships between lepidopteran Ambidensovirus species, such as Junonia coenia densovirus JcDV, Pseudoplusia includens densovirus (PiDV), Diatraea saccharalis densovirus (DsDV), Helicoverpa armigera densovirus 1 (HaDV1) which are closely related to CpDV (Martynova et al., 2016). This suggested that recombination events between ancestral variants could be the major cause of divergence between new "species" (Martynova et al., 2016). As an important evolutionary mechanism that shape virus genomes putatively resulting in convergent evolution, we also tracked positive (diversifying) and negative (purifying) selection signals. In both NS1 and VP ORFs, we mainly detected purifying selection, while there was one site subject to diversifying selection in VP region. In agreement with our results, Martynova et al., (2016) also reported strong signal of purifying selection in other Ambidensovirus species (i.e. JcDV, PiDV, MlDV, GmDV, DsDV, HaDV1) (Martynova et al., 2016).

Phylodynamics methods allowed us to estimate the evolutionary rate of CpDV. NS1 region and concatenated dataset had sufficient temporal structure that allowed an estimation of tMRCA based on these datasets, while it was not the case for VP region. As we have also detected by the analysis of recombination such discordance could result from acquisition of NS or VP parts from different variants of CpDV. However, recombination while obviously one of the main drivers of virus evolution cannot be taken into account by phylodynamics method and constitutes one of the biggest challenge for these methods (Frost et al., 2014). For these reasons, it is difficult to totally rely on our results based on the concatenated dataset. However, while NS1 region dataset (which does not include recombination events) showed slower substitution rate than concatenated one, it still shows high substitution rate compared to most DNA viruses. Variations in evolutionary rates between viruses are usually explained by whether they use the high fidelity DNA polymerases or lowfidelity RNA polymerases (Elena and Sanjuán, 2007;Duffy et al., 2008). Indeed, DNA viruses that replicate using host DNA polymerases have lower mutation rates closer to what is reported for their hosts' genome (10 -9 -10 -8 ) (Duffy et al., 2008). However, the difference between polymerases used during viral replication is not the only explanation of substitution rates variations. Selection also acts on substitution rates (Duffy et al., 2008). For instance, a very high substitution rate (10 -3 ) is reported during host-switching of Canine parvovirus (CPV) from dogs, when it emerged as feline panleukopenia parvovirus (FPLV) in cats and has been explained by high positive selection pressure (Shackelton et al., 2005). Studies on other temporally sampled ssDNA belonging to parvovirus have also shown a high substitution rate (10 -3 to 10 -5 ) nearing the one observed for RNA viruses (10 -2 -10 -5 ) (Shackelton et al., 2005(Shackelton et al., , 2006;;Firth et al., 2009;Streck et al., 2011). First isolated from laboratory lines kept in ISEM's insectary following high larval mortality, CpDV has since been shown to persist in these lines (Chapter 3). Here, we monitor CpDV persistence during almost 30 years from 1990 to 2018 in one laboratory line (Slab). This approach also allowed to monitor CpDV diversification during this period in laboratory conditions. The reference strain (isolated from our laboratory lines in 1994), while exhibiting some differences, is closely related to the strain present in Slab 1990 both belonging to CpDV-2 clade. However, in the following years we The fact that CpDV-1 clade strains i) have since then not been replaced in the laboratory line and ii) are more frequent in nature might also reflect that CpDV-1 clade could be more adapted to their hosts and more competitive.

Viruses are generally seen as pathogens and most of the research focuses mainly on medically or economically important pathogenic viruses. Therefore our understanding of Parvovirus evolution is biased towards pathogenic viruses -such as canine parvovirus, porcine parvovirus (Shackelton et al., 2005;Hoelzer et al., 2008;Firth et al., 2009;Streck et al., 2011). As virus evolution and population dynamics can also be influenced by the nature of their interactions with their hosts (Duffy et al., 2008), considering viruses as symbionts of their hosts (including other possible host-virus interactions than strict antagonism) can deeply improve our understanding of virus and disease evolution as a whole.

Overall our results on CpDV evolution showed no significant change in exponential growth and a r low genetic diversity compared to their relatively high substitution rate, high prevalence and vertical transmission (Chapter 3). Such characteristics might be pointing their persistence in their hosts instead of causing acute infections (Roossinck, 2011). Our current knowledge of the persistence on virus population dynamics is very limited (Roossinck, 2011). In this context CpDV-Culex pipiens system is a good model to study host-virus interactions and their evolution together both in nature and laboratory conditions. More generally, due to the ability of arthropods to host endosymbionts including vertically transmitted ones, the study of arthropod-virus systems can give valuable insights on the effect of this continuum of interactions on the virus population dynamics, and on the emergence of pathogenic or mutualistic viruses.

! Acknowledgements

We would like to thank Vincent Robert for the samples collected in Armenia, Benoit Assogba for the samples from Benin and Beniamino Caputo for samples from Italy. We also thank Sandra Unal and Patrick Makoundou for technical support. Sequencing data were produced at the LabEX "Centre Méditerranéen de l'Environnement et de la Biodiversité" in the GENSEQ platform. !"#$#%&'(')''*+,-'.//0 !"#$#%&'(')''1"2'3456'7 *+,-'.//0')'1"2'3456'7 85,9:'4;'<"=>"%9"'2#:?','5"94@-#%,%:'45#&#% !44:<:5,A'9>:'4;;'BC0 . DDC ...E .FC. 777G H4<#:#4%'#%',+#&%@"%:

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3.1! CpDV persistence in the laboratory lines

Observation of high prevalence of CpDV worldwide (Chapter 2) brought up several questions: How they can persist in natural populations? How do they transmit from host to host and what is the nature of their relationship with their hosts?

CpDV was isolated for the first time from mosquito lines kept in the insectaries of ISEM, following an episode of high mortality during 4 th instar larvae. Even though at the time it was discovered,

CpDV has been shown in really high amount in the larvae and clear signs of densonucleosis were observed, it was not possible to replicate this infection experimentally to date. Hence the nature of the interactions between CpDV and its host is still not clear.

To investigate CpDV-host interactions and whether there is a CpDV-Wolbachia interaction within host, experimental infections were necessary. However, CpDV specific diagnostic PCR test, based on the amplification of partial NS2 gene revealed the presence of CpDV in all the lines that were kept in the insectary. CpDV infection seemed to be mostly in low amounts, displaying weak PCR bands, nevertheless seemed to persist. Further investigation of adults and larvae from some of the insectary lines, showed i) CpDV presence both in larvae and adults, ii) larvae were 40% more likely to be CpDV positive (20-54%, Appendix A Figure 1), iii) adult males and females displayed similar CpDV prevalence (Appendix A, Figure 2).

Attempts to establish CpDV-free mosquito lines by separating females or bleaching egg rafts failed.

3.2! Experimental Infections

There were two major problems on the way to carry on with the experimental infections i) the lack of CpDV free lines and the unpredictable nature of the persistent infection, ii) low amount of CpDV infection in the transinfected cells.

Despite these problems, we have conducted pilot studies for experimental infections were using two common infection techniques i) using crushed infected larvae (Appendix A, Figure 3) ii) using CpDV positive cell cultures (Appendix A, Figure 4). While the results looked promising with the latter, it was not possible to tackle all of these problems during the course of my PhD.

However, we have managed to improve CpDV cell transinfection and isolation protocols, which made it possible to test synthetic CpDV and natural CpDV (isolated from Ich13 lines) both on Ae. aegypti and Cx. pipiens larvae. First trials showed that CpDV (synthetic with reference genome or isolated current variant) did not cause any mortality in Ae. aegypti larvae. Interestingly, in Cx. pipiens larvae synthetic CpDV with ancestral genome caused higher mortality than negative control while larvae survived better than negative control when the isolated current variant added. If true these results could suggest either that synthetic CpDV is more infectious than the natural one or that the pathogenic CpDV has been replaced by a less infectious variant in the insectary populations. Although the effects of co-infection might be at play due to the already established persistent CpDV infection in the tested larvae (we would be testing persisting CpDV infection + synthetic CpDV vs. persisting CpDV infection + natural isolate), these results looked promising.

Impossibility of cleaning the lines from CpDV after bleaching egg rafts, highlighted the possibility of transovarial vertical transmission of CpDV. Therefore, we have tested vertical transmission of CpDV in naturally infected Cx. pipiens mosquito lines and investigated the effect of their endosymbiotic bacteria, Wolbachia.

3.3! Vertical transmission of

Culex pipiens Densovirus

This study has been submitted for review as: Altinli, M., Soms J., Ravallec M., Justy F., Bonneau M., Weill M., Gosselin-Grenet A.S., Sicard M.

Sharing cells with

Sharing cells with Wolbachia: the transovarian vertical transmission of Culex pipiens densovirus

Abstract. Culex pipiens densovirus (CpDV), a single stranded DNA virus, has been isolated from Culex pipiens mosquitoes but differs from other mosquito densoviruses in terms of genome structure and sequence identity. Its transmission from host to host, the nature of its interactions with both its host and host's endosymbiotic bacteria Wolbachia are not known. Here we report the presence of CpDV in the ovaries and eggs of Cx. pipiens mosquitoes in close encounters with Wolbachia. In the ovaries, CpDV quantity significantly differed between mosquito lines harboring different strains of Wolbachia and these differences were not linked to variations in Wolbachia densities. CpDV was vertically transmitted in all laboratory lines to 17%-20% of the offspring. For some females, however, the vertical transmission reached 90%. Antibiotic treatment that cured the host from Wolbachia also significantly affected both CpDV quantity and vertical transmission suggesting an impact of host microbiota, including Wolbachia, on CpDV transmission. Overall our results show that CpDV is transmitted vertically via transovarian path along with Wolbachia with which it shares the same cells. Our results are primordial to understand the dynamics of densovirus infection, their persistence and spread in populations considering their potential use in the regulation of mosquito vector populations.

3.3.1! Introduction

Densoviruses are non-enveloped, single stranded DNA viruses. They constitute a diverse subfamily within the Parvoviridae family that includes both vertebrate (Parvovirinae) and invertebrate (Densovirinae) viruses (Cotmore et al., 2014;Tijssen et al., 2016). Isolated from many insects (e.g. butterflies, moths, mosquitoes, crickets, grasshoppers and cockroaches) and crustaceans (e.g. prawns and crayfish), they are thought to be widely distributed among arthropods (Kerr et al., 2005;Tijssen et al., 2016). Recent studies, investigating publicly available transcriptomic and genomic databases, have shown their presence in even broader range of nonvertebrate hosts (e.g., molluscs, annelids, nematodes, cnidarians and sea stars) (François et al., 2016;[START_REF] Kang | Densoviruses in oyster Crassostrea ariakensis[END_REF]. In arthropods, most of the densoviruses known so far exhibit a tissue polytropism replicating in many tissues -such as the fat body, hypodermis, epidermis, central nervous system, muscular and tracheal cells, gut, hemocytes, ovaries-and are considered to be lethal for their natural hosts (Tijssen et al., 2016). Due to their observed pathogenicity, narrow host range and sustainability out of their host-cells in nature, they attracted scientific attention as biological control tools especially against mosquitoes (Carlson et al., 2006;Mutuel et al., 2010;Gosselin Grenet et al., 2015;Suzuki et al., 2015;Johnson and Rasgon, 2018).

Mosquito densoviruses (MDVs) have been described in natural populations (Afanasiev et al., 1991;Boublik et al., 1994;Kittayapong et al., 1999;Rwegoshora et al., 2000;Ng et al., 2011), laboratory colonies (Jousset et al., 2000) and established mosquito cell lines (Jousset et al., 1993;O'Neill et al., 1995). The first described mosquito densoviruses were Aedes aegypti densovirus (AeDV) (Lebedeva et al., 1972) and Aedes albopictus densovirus (AalDV) (Jousset et al., 1993). Thereafter many other MDV strains, namely Toxorhynchites amboinensis densovirus (TaDV), Haemagogus equinus densovirus (HeDV), Ae. aegypti Thai strain densovirus (AThDV), Ae. Peruvian densovirus (APeDV) have been identified (O'Neill et al., 1995;Kittayapong et al., 1999;Rwegoshora et al., 2000;Ledermann et al., 2004). Studies on MDV pathogenicity and transmission modes have generally shown a dose and age dependent infectivity and virulence (Barreau et al., 1996;Kittayapong et al., 1999;Ledermann et al., 2004;Rwegoshora and Kittayapong, 2004;Becnel and White, 2007). Vertical transmission of these viruses has only been observed when using sub-lethal doses that reduced the adult mosquitoes' longevity and fertility but did not kill them (O'Neill et al., 1995;Barreau et al., 1996Barreau et al., , 1997;;Kittayapong et al., 1999;Rwegoshora and Kittayapong, 2004;Becnel and White, 2007). In contrast, Anopheles gambiae densovirus (AgDV), a recently discovered MDV, which exhibits a negligible pathogenic effect on its host A. gambiae mosquitoes (Ren et al., 2008(Ren et al., , 2014) ) has been shown to replicate in its host and to be vertically transmitted to the offspring highlighting its potential as transducing agents for mosquito control (Ren et al., 2008;Suzuki et al., 2014).

All of the abovementioned MDVs described so far are closely related (82-99% sequence homology), and are considered as strains from two distinct species (dipteran brevidensovirus 1 and 2) belonging to the genus Brevidensovirus (Cotmore et al., 2014). To date, the only known densovirus to infect mosquitoes, but does not belong to Brevidensovirus genus, is the Culex pipiens densovirus (CpDV). CpDV is a dipteran ambidensovirus (Jousset et al., 2000;Baquerizo-Audiot et al., 2009) which differs significantly from dipteran brevidensoviruses by the lack of sequence homology (<30% in Non-structural (NS1/Rep) gene) and antigenic cross-reactivity (Jousset et al., 2000;Baquerizo-Audiot et al., 2009). It belongs to Ambidensovirus genus along with lepidopteran densoviruses, such as Junonia coenia densovirus (JcDV) and Galleria melonella densovirus (GmDV). Ambidensoviruses have not yet been described in other dipteran species suggesting that they could have been acquired in Cx.

pipiens by horizontal transfer. CpDV indeed shares the characteristics of other ambidensoviruses having a larger genome size than brevidensovirus (~6kb compared to ~4kb), an ambisense genome organization and significant sequence homology at both capsid and NS gene levels (Jousset et al., 2000;Baquerizo-Audiot et al., 2009).

Two decades after its first isolation from Cx. pipiens laboratory colonies, due to an unusually high larval mortality, we detected the presence and persistence of CpDV in all life stages of seemingly healthy Cx. pipiens lines by a PCR diagnostic test (data not shown). This long-term persistence of CpDV in laboratory colonies through generations certainly relies on an efficient transmission. Transmission modes are generally categorized as i) horizontal, transmission from host to host via the environment, ii) vertical transmission from the host parent to offspring, iii) mixed-mode, both horizontal and vertical transmission (Ebert, 2013). Transmission modes have long been investigated in many symbiotic systems especially to understand their impact on virulence evolution [START_REF] Ewald | Host-parasite relations, vectors, and the evolution of disease severity[END_REF][START_REF] Ewald | Transmission modes and evolution of the parasitism-mutualism continuum[END_REF][START_REF] Alizon | Virulence evolution and the trade-off hypothesis: History, current state of affairs and the future[END_REF]Cressler et al., 2016;[START_REF] Clec'h | Phenotypic shift in Wolbachia virulence towards its native host across serial horizontal passages[END_REF]. Empirical studies mostly showed that increased horizontal transmission led to increased virulence, while dominance of vertical transmission mode mostly reduced the virulence [reviewed in (Cressler et al., 2016)]. Therefore transmission modes can have a major impact on the dynamics of virus infection, their persistence and spread in populations.

To understand CpDV infection dynamics, especially its maintenance through generations, we investigated the ability of Cx. pipiens mosquitoes to transmit CpDV vertically. Cx. pipiens are all naturally infected with Wolbachia strains belonging to one of the five genetically distinct phylogenetic group wPipI to wPipIV (Atyame et al. 2011). These wPip strains induce a wide diversity of cytoplasmic incompatibility phenotypes in their mosquito host (Atyame et al. 2014). In addition to their ability to manipulate host reproduction to benefit their own vertical transmission (Stouthamer et al., 1999;Werren et al., 2008;Sicard et al., 2014), Wolbachia are also known to interact with several of their hosts' viruses (Hedges et al., 2008;Teixeira et al., 2008;Zug and Hammerstein, 2015). While putative difference in protective ability between the wPip groups identified in Cx. pipiens has not yet been investigated, a general protection by wPip has been shown against West Nile Virus infection (Micieli and Glaser, 2014). To date, no Wolbachia-mediated "protection" against DNA viruses have been shown in any insects. Contrarily, one study demonstrate that Wolbachia makes their lepidopteran host more susceptible against a Baculovirus (Graham et al., 2012).

We thus examined the influence of Wolbachia on CpDV density and vertical transmission. To do that, we monitored CpDV density and location in the ovaries and eggs, and then quantified its vertical transmission in Cx. pipiens mosquitoes naturally infected with both Wolbachia and CpDV. Given their importance in Cx. pipiens' phenotype both in natural populations and laboratory colonies, we took Wolbachia into account in each step of the study and further investigated the possible effects of their absence on CpDV presence and vertical transmission.

3.3.2! Results

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CpDV was always present, along with Wolbachia, in the ovaries of females from ten laboratory lines tested by qPCR and its amount varied between different lines (glm, df=13, dev=82.448, p<0.001, Figure 1). Only two pools of ovaries -one from Ich13 and another from Ich09 line-over 50 pools tested were CpDV negative. We did not detect any statistically significant effect of Wolbachia amount (glm, df=1, dev=0.007, p= 0.967, Figure 1A) on the amount of CpDV in ovaries. To create backcrossed lines for further experimentation, we used "Harash" mosquito line with the highest amount of CpDV in ovaries (120!114 CpDV/host genome, Appendix B Table 1, Figure 1B) and "Tunis" line with less CpDV in their ovaries (0.005! 0.002 CpDV/host genome, Tunis, Appendix B Table 1, Figure 1B).

To confirm the presence of virus particles -and not only of the viral genome (i.e. as detected by qPCR)-and to investigate their localization in the ovaries, we dissected ovaries from 6 days old Harash females and performed immunolabelling of CpDV using an anti-capsid antibody for confocal observation (Figure 2 and Appendix B Figure 1). The labelling of CpDV was mainly observed in follicular cells surrounding the follicles (Figure 2A-B-C) and occasionally in some muscle cells of the sheath enclosing the ovariole (Figure 2D). In both cell types, the labelling is mainly nuclear with some dots present in the cytoplasm of follicular cells The mosquito ovaries are paired and composed of functional units called ovarioles, containing primary (F1) and secondary follicles (F2) [START_REF] Clements | The biology of mosquitoes[END_REF].

Each primary follicle contains seven nurse cells and one oocyte, both surrounded by follicular epithelium. The secondary follicle is composed of undifferentiated cells surrounded by follicular epithelium. The secondary follicle will turn into a primary follicle after oviposition of the oocyte contained in the previous primary follicle [START_REF] Raikhel | Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: An ultrastructural and immunocytochemical study[END_REF]. The sheath that enclosed each ovariole (Figure 2B to E). Within the follicles, the labelling of CpDV in the nurse cells was not as apparent and rather similar to the background immunofluorescence (Figure 2D-E). Although we cannot entirely exclude a lack of permeability and accessibility of the nurse cells to the antibodies, a clear labelling was nevertheless observed in a well-defined area of the nucleus of the oocyte (Figure 2E). As all the Cx. pipiens lines were infected, the specificity of the CpDV labeling was tested on ovaries isolated from PCR tested CpDV-free Ae. albopictus mosquitoes and no labelling was detected (Appendix B Figure 2).

To examine the CpDV replication, which takes place in the nucleus, we prepared ultrathin sections of ovaries and analyzed the nuclear ultrastructure with transmission electron microscopy (TEM)

(Figure 3). Although no unequivocal densonucleosis was observed, follicular and nurse cells showed hypertrophied nuclei (Figure 3A and G). Intranuclear structures that could be interpreted as viral factories were observed in several follicular cells (Figure 3B), nurse cells (Figure 3E) and oocytes (Figure 3H). Particles with the expected diameter of CpDV (about 25 nm) were also present in the nucleoplasm (Figures 3C andF).

To confirm the presence of viral particles in ovaries, we prepared filtered homogenate of the full organ for observation using TEM. Micrographs revealed the presence of both icosahedral empty capsids and CpDV virions with the expected diameter (Figure 3I). The same experiments were performed on freshly laid dechorionated eggs from Harash females (Figure 4). Immunolocalization of CpDV and DAPI staining of Wolbachia cells at early stage of the embryo development showed a Wolbachia localization especially in the poles, and a CpDV 4C). However, at the early stage of development nuclei were rare, meaning CpDV could only be observed in the cytoplasm where the ribosomes are also present (Figure 4C). As ribosomes and CpDV have the same diameter, it is not possible to firmly distinguish them (Figure 4C). Table 1). We then quantified the amount of Wolbachia and CpDV in these lines as well as in Harash, Tunis and Slab lines (Figure 5). Wolbachia amount in the ovaries was not significantly different between the Cx. pipiens lines no matter what the Wolbachia strain and host genetic background were (Wolbachia strain: glm, df=2, dev=17.703, p=0.875; host genetic background: df=1 dev=0.034, p=0.981, Figure 5A). Indeed, there was no significant effect of the Wolbachia amount in the ovaries on CpDV density (lme, dev=0.001, df=1, p=0.9). However, Wolbachia strain (i.e. the genotype) infecting the mosquito lines explained a Mixed effect linear model showed no effect of Wolbachia amount or genetic background on the CpDV amount in the ovaries (Wolbachia amount dev=0.001, df=1, p=0.9, genetic background: dev=1.602, df=2, p= 0.449). Wolbachia strain, however significantly affected the CpDV amount (dev=12.236, df=1, p<0.001). CpDV vertical transmission rate (C) Females from each line were collected 5 days after their blood meal and isolated to lay eggs. Resulting L1 offspring was tested for CpDV prevalence and vertical transmission rate was calculated per female. A binomial generalized mixed effect model was fitted with host genetic background, Wolbachia strain as well as

CpDV and Wolbachia amount in females' bodies as fixed effects and replicates as random effects. None of these variables had a significant effect on the vertical transmission rate. As all the Cx. pipiens isofemale lines were stably infected with wPip (Wo+), to test the effect of the Wolbachia absence on CpDV presence and vertical transmission, we treated our Slab, BcHarash, and BcTunis lines, all having the same genetic background, with tetracycline (TC) for three generations to cure Wolbachia. We quantified Wolbachia and CpDV in dissected ovaries and carcasses of Wo+ (before TC treatment) and Wo-females (after TC treatment), as well as females during the 1 st and 3 rd generation of TC treatment (Figure 6).

There was no significant change between 1 st and 3 rd generations in terms of CpDV amount (t = -0.643, df = 25.918, p-value = 0.525, Figure 6B).

We thus grouped them together in a "TC treatment' group for further statistical analyses and fig-

ures.

Prior to tetracycline treatment Wolbachia were found in higher density in the ovaries compared to the carcasses (Wilcoxon rank sum test, W= 225, p<0.001; Figure 6A). By the end of the third generation of tetracycline treatment, as expected, Wolbachia totally disappeared both from the ovaries and the carcasses (Figure 6A).

In non-treated Wo+ females, higher amount of CpDV were measured in ovaries compared to carcasses (Wilcoxon rank sum test, W = 168, p = 0.021, Figure 6B). In the carcasses, TC treatment did not cause any significant effects on CpDV amount (Figure 6B CpDV vertical transmission decreased significantly from an average of 25% to an average of 13% during tetracycline treatment (Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. TC treatment, W= 488, p= 0.004, Figure 7). Three generations after the end of tetracycline treatment, vertical transmission in Wo-lines stayed marginally lower than non-treated Wo+ lines (Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. Wo-, W= 576.5, p= 0.05, Figure 7).

3.3.3! Discussion

CpDV was isolated for the first time from Cx.

pipiens larvae almost two decades ago (Jousset et al., 2000). The features of densonucleosis (hypertropied nuclei and electron-dense virogenic stroma) was then observed in larval tissues (hypodermis, fat body, midgut, imaginal disks and nervous tissue) suggesting CpDV's role on the larval mortality (Jousset et al., 2000). Here, we showed that CpDV was also present in apparently healthy adults especially in their ovaries along with endosymbiotic bacteria Wolbachia. Even if the densonucleosis was not unequivocal, intranuclear structures similar to viral factories were observed and viral particles were isolated from ovaries. Furthermore, immunolabelling revealed virus particles in the follicular cells and the oocytes. Only couple of studies reported the presence of densoviruses in the ovaries.

Helicoverpa armigera densovirus (Ambidensovirus) and A. gambiae densovirus (Brevidensovirus) have been shown to reach high densities in ovaries of their hosts while they were also able to infect other host tissues (Ren et al., 2008;Suzuki et al., 2014;Xu et al., 2014).

The presence of CpDV capsids in the ovaries and eggs highlighted the possibility of vertical transovarial transmission. Transmission mechanisms of densoviruses are generally thought to be horizontal causing epizootics (Tijssen et al., 2016). Nevertheless, vertical transmission has been shown both for some ambidensoviruses and brevidensoviruses. Interestingly, ambidensoviruses with different vertical transmission rates had different types of symbiotic interactions: while the 100% vertically transmitted ambidensovirus of H. armigera is mutualistic (Xu et al., 2014), the Myzus persicae densovirus which is transmitted to 30-40% of the offspring has been shown to affect its hosts adversely [START_REF] Van Munster | Characterization of a new densovirus infecting the green peach aphid Myzus persicae[END_REF]. In mosquitoes experimentally infected with brevidensoviruses, the vertical transmission ranged between 28 to 62%: i)

for Ae. albopictus densovirus, the vertical transmission rate ranged between 28-55% depending on the virus titer in parental females (Barreau et al., 1997); ii) for Ae. aegypti densovirus vertical transmission ranged from 42% to 62% depending on the timing of the horizontal infection of the larvae (the larvae that were infected during L3 stage transmitted the virus less, as they became adults, than the larvae infected during L1 stage) (Suchman et al., 2006), and iii) for A. gambiae densovirus vertical transmission was detected in 28% of larval offspring (Ren et al., 2008). We showed that CpDV was transmitted to an average of 17% of the larval offspring in naturally infected Cx. pipiens lines.

Even if the vertical transmission rate varied between individuals, this rate was quite stable (15%-21%) between the Cx. pipiens-Wolbachia lines tested through the different replicates. The importance of inter-individual variability in vertical transmission rate of CpDV revealed the complexity of the factors affecting vertical transmission. Individual differences in vertical transmission, for instance, could be due to when females have been infected by horizontal transmission (Suchman et al., 2006), or whether they have been infected vertically or horizontally. Due to the lack of CpDV free lines, it was not possible to quantify the horizontal transmission or venereal transmission by experimental infections. Nevertheless, the infection rate changed from 17% at the L1 stage to 100% in tested mothers suggesting a high level of horizontal transmission.

The co-existence of endosymbiotic bacteria

Wolbachia and CpDV within the host ovary cells suggested that they might be interacting. Indeed, symbiotic bacteria and viruses have been shown to interact in several ways. Firstly, symbiotic bacteria can "protect" their hosts against pathogens by either modulating the host immunity or tolerance, or by competing for host resources. A famous example of such interaction is the Wolbachia mediated protection of insect hosts against RNA viruses (Hedges et al., 2008;Teixeira et al., 2008). In Drosophila this "protection" strongly changed according to

Wolbachia strains differing in their titers (Martinez et al., 2017). Similar "protection" has been reported in mosquitoes, especially in Ae. aegypti transinfected with non-native Wolbachia strains from Drosophila, against several arboviruses such as Dengue, Chikungunya, West Nile and Yellow fever viruses (Moreira et al., 2009;Mousson et al., 2010;Hussain et al., 2012). On the other hand, native Wolbachia in Ae. albopictus that exhibit low densities, even in the ovaries, do not influence virus replication (Mousson et al., 2010;Lu et al., 2012).

A global pattern both in mosquitoes and Drosophila is that Wolbachia-mediated antiviral protection seems to depend on Wolbachia density (Lu et al., 2012;Osborne et al., 2012;Bian et al., 2013). Our results showed, for the first time, a significant effect of Wolbachia strain on the quantity of a DNA virus, the CpDV, in the ovaries suggesting an interaction between these two symbionts sharing the same cells. Indeed, by using a backcrossing protocol in mosquitoes naturally infected both with their respective wPip strain and CpDV, we have demonstrated that CpDV density in the ovaries was not controlled by host nuclear genetic background but most likely by maternally transmitted cytoplasmic factors such as Wolbachia. However, Wolbachia density was really stable between naturally infected

Cx. pipiens lines for different Wolbachia strains tested suggesting variations of symbiont titer was not responsible for variations in CpDV titers. Nevertheless, further research including the establishment of CpDV free lines from putative uninfected natural populations followed by experimental infections would be useful to delineate the complex tripartite interactions in this new mosquito-virus-

Wolbachia natural system.
To further investigate whether CpDV and Wolbachia had reciprocal interactions and whether CpDV could still be vertically transmitted in the absence of Wolbachia, we treated our lines with antibiotics. This treatment decreased both the amount of CpDV in the ovaries and the vertical transmission alike. However, following the end of the treatment CpDV amount in the ovaries increased in the Wolbachia-free lines. This suggested that this sudden decrease could be caused by either the direct influence of antibiotics on the host metabolism, or by the rest of the microbiota that putatively recovered in the generations following the end of the antibiotic treatment. Nevertheless, even though CpDV was able to be transmitted vertically in the absence of Wolbachia, the vertical transmission was lower in Wolbachia-free lines, suggesting their effect on CpDV transmission. Several endosymbiotic bacteria have been reported to modulate virus transmission. For instance Tomato yellow leaf curl virus' (a single stranded DNA plant virus) transmission is facilitated by GroEL protein secreted by its vector's endosymbiotic bacteria Hamiltonella (Gottlieb et al., 2010). More recently, Jia et al. (2017) showed that Rice Dwarf virus (a plant double stranded RNA virus) binds to the envelopes of endosymbiotic bacteria Sulcia improving its vertical transmission in leafhoppers. Similarly, vertical transmission of Wolbachia could be hitchhiked by CpDV in Cx. pipiens. CpDV could potentially achieve this through the interactions of its capsids with bacterial surface glycans. Densovirus virions, as of many non-enveloped viruses, are constituted by a single major capsid protein with supersecondary structures which can act as interaction motifs (e.g. Single Jelly Roll) [START_REF] Neu | Viruses and sialic acids: Rules of engagement[END_REF][START_REF] Huang | Parvovirus glycan interactions[END_REF]. These motifs can attach to bacteria's surface glycans which can serve as initial attachment factors for many viruses [START_REF] Neu | Viruses and sialic acids: Rules of engagement[END_REF][START_REF] Huang | Parvovirus glycan interactions[END_REF]. Thus, CpDV capsids could potentially interact with a wide array of glycans, including Wolbachia's surface glycans and such interactions could influence the infection outcome and lead to their co-vertical transmission (Kuss et al., 2011;Berger and Mainou, 2018). Overall, we observed a low amount of CpDV in our samples highlighting a covert infection in laboratory Cx. pipiens mosquitoes. Viruses exhibiting covert infections can differ in their replication strategy and virulence [START_REF] Kane | Common threads in persistent viral infections[END_REF]Williams et al., 2017). They can either cause persistent infections with low levels of virus replication, producing virions but only causing sub-lethal disease or causing latent infections, without any production of virions and limited number of viral gene expression [START_REF] Kane | Common threads in persistent viral infections[END_REF]Williams et al., 2017). In the case of CpDV, we were able to observe CpDV virions but not a particularly high mortality in the mosquito lines even in the heavily infected Harash line. This type of covert infection combined with the mixed mode transmission can allow the persistence of CpDV in laboratory as well as natural populations of Cx.

pipiens mosquitoes. In Cx. pipiens mosquitoes even low rate vertical transmission can have a large effect on the epidemiology of CpDV by allowing the transmission of the virus even after acute reduction of host density, e.g. during overwintering diapause (Ebert, 2013;Williams et al., 2017). While still not well studied this type of covert persistent infections by insect specific viruses can largely affect the ecology of the insect populations and vector competence.

3.3.4! Methods

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All the mosquito lines used in this study (Supplementary Table 1) have been maintained in insectary conditions (at 25 ± 2 °C and 75 ± 2% relative humidity and a 12:12 h photoperiod). During larval stage, they have been fed with a mixture of shrimp powder and rabbit pellets. Adults have been kept in 65 dm 3 cages and have been fed with a honey solution. Females were fed weekly with turkey blood using a Hemotek membrane feeding system (Discovery Workshops, United Kingdom).

Backcrosses

To test the effect of the Wolbachia genetic differences on CpDV amount and their vertical transmission, we introduced the cytoplasm of Tunis and Harash lines, along with their respective wPip strains, into the Slab nuclear background as described by Duron et al (Duron et al., 2012). With this backcrossing protocol we have established BcTunis and BcHarash lines with the same genetic background (over 97% of genome replacement of the original lines by Slab nuclear genome is estimated) and different Wolbachia types (wPipI and wPipIV respectively, Table 1). wPip types were verified with a PCR/RFLP test on pool of larvae using pk1 primers as previously described (Altinli et al., 2018).

Wolbachia free lines

BcTunis, Slab and BcHarash lines, all stably infected with wPip (Wo+), were treated with tetracycline (50 mL/L for larval treatment using a 0.4g/L solution) for 3 generations to create Wolbachia free lines (Wo-: BCTunisTC, SlabTC, BCHarashTC, Table1). Experiments were conducted using the females from: 1) the first generation right after we started the antibiotic treatment, 2) the third generation during the antibiotic treatment to be able to follow the effect of the treatment and 3) from at least three generations after the end of the antibiotic treatment, to exclude the direct effect of TC treatment on host metabolism, and consequently on CpDV quantity and transmission (Wo-).

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Immunolabeling were performed on ovaries from 6 days old females and dechorionated eggs. The egg rafts were collected in 1-4 hours following egg laying. The egg-rafts were bleached (active ingredient, 9.6% of sodium hypochlorite) to separate the individual eggs and rinsed with distilled water.

They were then fixed with 3.2% para-formaldehyde diluted in PBS-T (PBS 1X with Tween 0,02%) for 2 hours, and rinsed with PBS 1X. The chorion of each individual egg was removed manually with a needle under an optical microscope (Leica MZ 8).

All the samples were fixed for 1 h in 4% paraformaldehyde (PFA) and washed 3 times with PBS, then treated with 50 mM of NH 4 Cl for 30 min to minimize autofluorescence. Ovaries and eggs were permeabilized with PBS containing 0.5% Triton and blocking was performed with 1% bovine serum albumin (BSA) in PBS for 1 h. Immunolabelling of CpDV were performed using a mouse primary JcDV anti-capsid antibody that co-reacts with CpDV capsids (Jousset et al., 2000), diluted at 1:300 in PBS with 0.2% Tween and incubated overnight à 4°C. Secondary antibodies (Alexa Fluor® 488 and 568; 1:500; Invitrogen) were used at 1:500 dilutions and incubated for 1 h (ovaries) or 6 h (eggs). Samples were incubated 30 min with Phalloidin-FITC (1:300; Sigma) to label actin cytoskeleton, and 5 min with DAPI (1 µg/ml; Invitrogen) to label nuclei and Wolbachia. Finally, labeled tissues and eggs were mounted in Dako (Sigma) on coverslips for observation with a Leica SPE confocal microscope equipped with suitable lasers and bandpass for the dyes (excitation 405 and emission 420-480 for DAPI; excitation 488 and emission 505-550 for Alexa Fluor® 488; emission 532 and emission 580-650 for TexasRed), using a 20X/0.55 HCX APO L W and a 40X/1.15 ACS APO oil objectives. We used the LAS AF (Leica) software to operate the microscope and all images were processed with ImageJ software.

"#"#=#"! 5<+.7*0-!:/.*0,.0'?!! To observe CpDV particles, a pool of ovaries from 6 days old females were dissected, crushed in PBS and filtered with a 0.22 µm pore size filter. The filtered homogenate was subjected to a negative staining using 2% phosphotungstic acid (PTA, Electron Microscopy Sciences) for observation using transmission electron microscopy (TEM, see above).

Ultrathin PCR amplification conditions were as following: i) for CpDVquantiF and CpDVquantiR primers initial denaturation for 5 min at 94 °C, followed by 33 cycles of denaturation, annealing and elongation respectively at 94 °C for 30 s, 56 °C for 30s, and 72 °C for 90 s, and a final elongation at 72 °C for 5 min ii) for ace2quanti primers initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation, annealing and elongation respectively at 94 °C for 30 s, 52 °C for 30s, and 72 °C for 90 s, and a final elongation at 72 °C for 5 min.

The proportion of CpDV-positive over CpDVnegative L1 larvae per female is calculated as vertical transmission rate.

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Ovaries were dissected from 6 days old female mosquitoes. Three ovaries were pooled together and 5 replicates were done per line. Qiagen extraction kit was used to extract DNA of ovary pools according to the manufacturer's instructions. DNA was eluted in 55 µl PCR gradient water. Negative controls were made to confirm that there was no contamination from the extraction kit and were always negative. Additionally to check the effect of Wolbachia and CpDV amount in the females on vertical transmission rate, DNA from the females that laid eggs have been extracted using CTAB technique (Rogers and Bendich, 1989).

Real-time quantitative PCR (qPCR) was used to estimate the amount of Wolbachia and CpDV in each sample. qPCRs were performed using SYBR Green I Master (Roche) and analysed on a Light-Cycler 480 Instrument (Roche) using the Light-Cycler 480 software (version 1.5.1.62). On each DNA sample three PCR reactions were performed in triplicates using i) NS2 protein coding region specific primers to quantify CpDV (CpDVquantiF: 5'-CATTGGAGGAGAAAGTTGGAA-3', CpDV quantiR:5'-TCCCCATTATCTGCTGTCG-3'), ii) wsp specific primers to quantify Wolbachia (wolpipdir 5'-AGAATTGACGGCATTGAATA-3' and wolpiprev 5'-CGTCGTTTTTGTTTAGTT-GTG-3' (Berticat et al., 2002)) and iii) Culex Ace-2 locus specific primers to quantify host genome copy as a reference (Acequantidir: 5'-GCAGCAC-CAGTCCAAGG-3', Acequantirev:5'-CTTCAGG-CCGTTCAAGTAG-3') (Weill et al., 2000). Standard curves were created using dilutions of a pCR®4-TOPO-Vector (Invitrogen) containing a single copy of each ace-2, wsp and ns2 gene fragments. Plates were designed to have all the compared groups of a biological replicate and prepared using Echo 525 Liquid Handler, Labcyte Inc. The amount of Wolbachia and CpDV were normalized with the amount of Ace-2 copies in order to obtain a number of virus or Wolbachia per cell.

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All data analyses have been performed in R version 3.3.1 (R Core Team, 2016).

To analyse the effects of Wolbachia amount and diversity, on the amount of CpDV in the ovaries, one highly infected outlier from Harash line has been removed from the analysis and normality of the continuous response variable (log10 transformed amount of CpDV genome/ amount of host genome) has been checked with Shapiro-Wilk normality test. A linear mixed effect model is fitted with genetic background (Slab, Harash, Tunis), Wolbachia type (wPip 1, wPip 3 and wPip 4) and Wolbachia amount as fixed effects and replicates as random effects using lme4 package [START_REF] Bates | Mixed models in R using the lme4 package Part 5 : Inference based on profiled deviance[END_REF].

Homoscedasticity and normality of the residuals were checked using Breusch-Pagan test (ncvTest, in car package [START_REF] Fox | An {R} Companion to Applied Regression Second[END_REF]) and Shapiro-Wilk test respectively. Likelihood ratio tests of the full model against the model without a given effect were used to obtain deviance and pvalues.

To analyse the effects of Wolbachia diversity on vertical transmission of CpDV, a binomial generalized mixed effect model is fitted with genetic background (Slab, Harash, Tunis), Wolbachia type (wPip 1, wPip 3 and wPip 4), Wolbachia amount and CpDV amount in the full body of the female as fixed effects and replicates as random effects.

CpDV in females were checked for the first and third generation of tetracycline treatment, they were grouped under during tetracycline treatment group given that these two groups didn't show any significant difference in terms of CpDV amount in the ovaries and represented the same treatment status. A Dunnett's test (glhrt function in multcomp package) [START_REF] Hothorn | Simultaneous inference in general parametric models[END_REF] have been performed for pairwise comparison of treatment stages to the non-treated control group.

In this model genetic background (Slab, Harash, Tunis) and treatment status (no treatment, during TC treatment and after TC treatment) were fixed effects and different replicates are added as random effects. This last dataset also has been analysed using pairwise Wilcoxon test with Bonferroni correction to compare the different treatment stages to the non-treated control group.

3.4! Acknowledgments

We thank Sandra Unal and Patrick Makoundou for technical support. We thank Margareth Capurro for providing us with the "Brasil" Culex line. We About 500 larvae were collected from different lines in PBS and has been filtered with syringe filters 0.45 mm. 1/10 dilution of the homogenate has been tested using PCR. The ones that tested negative on PCR were used as negative control CpDV-while the ones tested positive used as CpDV+. Three different doses (25ul, 50ul, 70ul) have been tested.

For each group (line_Wolbachia status_CpDV status_dose) 6 larvae were tested which added up to a total of 144 larvae. Data was analysed using R and survival curves estimated with Cox proportional hazards model implemented in Coxme package.

There was no significant difference between CpDV-positive and CpDV-negative treatment groups (Cox proportional hazards: χ2=1.765678 df=1 p>0.1, Figure A). On the other hand, survival decreased with the increase of the dose (Cox proportional hazards: χ2= 15.6863 df=2 p< 0.001Figure B), suggesting the toxicity of the filtered ground larvae in higher doses.

Figure 3 Experimental infections using crushed larvae

Infection trials with synthetic CpDV

C636 cells were transinfected with synthesized CpDV plasmid by our collaborators Anne-Sophie Gosselin-Grenet and Doriane Mutuel.

BcTU, BcHA, SlabTC, Ha total of 24 for each group CpDV-positive and CpDV negative tested. The amount of CpDV was quantified and 4e+6 geq/larvae was added to hemolysis tubes. Same amount of CpDV negative cell cultures have been added to hemolysis tubes for the negative control groups. 90ul of each added in 1 ml water-food mixture with 3 rd instar larvae.

3.6.3! References

Alout, H., Labbé, P., Berthomieu, A., Pasteur, N., and Weill, M. ( 2009) Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations. Insect Biochem. Mol. Biol. 39: 884-891. Atyame, C.M., Labbé, P., Dumas, E., Milesi, P., Charlat, S., Fort, P., and Weill, M. (2014) Wolbachia divergence and the evolution of cytoplasmic incompatibility in Culex pipiens . PLoS One 9: e87336. Bonneau, M., Atyame, C., Beji, M., Justy, F., Cohen-Gonsaud, M., Sicard, M., and Weill, M. (2018) Culex pipiens crossing type diversity is governed 1 by an amplified and polymorphic operon in Wolbachia genome. Nat. Commun. 9: 1-10. Duron, O., Lagnel, J., Raymond, M., Bourtzis, K., Fort, P., and Weill, M. (2005)

4.1! Introduction

Situated in Mediterranean basin between three continents -Asia, Europe and Africa-Turkey is a hotspot for biodiversity. Due to this unique geographical location and its suitable climate, Turkey can also provide ideal conditions for arthropod vectors and arthropod borne viruses. Hence it also represents a critical area for the control efforts of such arthropod borne viruses. Indeed a high diversity of mosquitoes have been revealed in the area, as well as the circulation of different strains of West-Nile virus and the diversity of insect-specific RNA viruses (Ergunay et al., 2011(Ergunay et al., , 2013(Ergunay et al., , 2014(Ergunay et al., , 2015;;Erdem et al., 2014;Ergünay et al., 2016;Failloux et al., 2017;Öncü et al., 2018).

Important vectors of West Nile and St. Louis encephalitis viruses and avian malaria (Plasmodium spp.), mosquitoes of Cx. pipiens complex are always found infected with Wolbachia wPip of the five genetically different groups (wPip-I to V), which induce very complex cytoplasmic incompatibility patterns in their hosts including uni-and bidirectional incompatibility (Farajollahi et al., 2011;Atyame et al., 2014). The worldwide distribution of these groups have already been investigated, although samples from Turkey were restricted to one site in Istanbul (Dumas et al., 2013).

Previous studies by our team in the ISEM, have showed that samples from Istanbul harbour wPip from the group-IV which showed an interesting incompatibility pattern with other wPip groups. Indeed wPip group-IV carrying male mosquitoes were able to sterilize the females carrying other wPip groups suggesting that this strain could be used in an 'Incompatible Insect Technique' (IIT) vector control strategy (Atyame, Pasteur, et al., 2011;Atyame, Cattel, et al., 2015). This speciesspecific approach consists of sterilization of field female mosquitoes by inundative releases of incompatible males which can lead to the reduction of mosquito population densities (Atyame, Pasteur, et al., 2011;Atyame, Cattel, et al., 2015).

Belonging to Densovirinae subfamily of Parvoviridae family, CpDV represents a unique mosquito densovirus: they differ from all the other mosquito densoviruses so far isolated in terms of their genome structure and sequence homology (Jousset et al., 2000;Cotmore et al., 2014). First isolated two decades ago following the observation of unusually high larval mortality in ISEM's insectary lines,

CpDV has later been detected to persist in seemingly healthy laboratory colonies in all life stages of Cx. pipiens mosquitoes (Chapter 3). Their covert infection combined with vertical transmission could explain their long-term persistence in laboratory colonies and suggested that these viruses could also be really prevalent in natural mosquito populations (Chapter3). Indeed we have previously reported a high prevalence of CpDV in worldwide distributed natural populations of Cx. pipiens (Chapter 2), using a PCR diagnostic test. Furthermore, CpDV-wPip co-existence in the same host cells and their co-vertical transmission (Chapter 2) suggested that CpDV and Wolbachia could interact in their shared host environment.

Wolbachia pathogen inference leading to a decrease in pathogen density and infection frequency has previously been shown in several arthropod species (Hedges et al., 2008;Teixeira et al., 2008;Moreira et al., 2009;Mousson et al., 2010;Hussain et al., 2012;Braquart-Varnier et al., 2015). In Cx. pipiens s.l. mosquitoes, wPip can reduce the West Nile Virus infection depending on the Wolbachia density (Micieli and Glaser, 2014).

Wolbachia-CpDV interactions could, for instance, influence CpDV prevalence and diversification or Wolbachia wPip group distribution in natural populations and eventually shape host populations. Especially where two different wPip groups with different incompatibility patterns co-exist the impact of these interactions could be more important. In natural Cx. pipiens populations co-existing wPip strains are more likely to be compatible with each other (Rousset et al., 1991;Duron et al., 2005Duron et al., , 2006Duron et al., , 2011;;Engelstädter and Telschow, 2009;Atyame, Duron, et al., 2011). In fact theory predicts that when the bi-directionally incompatible wPip strains co-exist, the most prevalent strain should eventually invade the population (Rousset et al., 1991;Engelstädter and Telschow, 2009). On the other hand when co-existing wPip strains are unidirectionally incompatible, the CI-inducing strain is expected to invade the population once above a frequency threshold (Engelstädter and Telschow, 2009;Atyame, Labbé, et al., 2015). On the other hand, densoviruses could also influence these dynamics. If densoviruses are pathogenic for their host a more protective wPip strain could invade the host population easier as it would benefit its host. In return probably, such a protective wPip strain can make CpDV presence impossible in this wPip-host combinations. Contrarily if CpDV is beneficial for the host the frequency of wPip-host combination that favours the presence of CpDV would increase in the population.

In this context, natural populations of Cx.

pipiens mosquitoes especially where two different wPip groups with different incompatibility patterns co-exist could be interesting to study Wolbachia-CpDV interactions. Due to its unique geographical location, Turkey represents an area where the diversity from different continent meets and have a potential for investigation of wPip and CpDV diversity and interactions. To this end, we explored first the Wolbachia diversity and incompatibility patterns (Chapter 4.2., (Altinli et al., 2018)). Secondly we focused on the prevalence of CpDV and diversity in natural populations of Cx. pipiens (s.l.) in Turkey.

4.2! Wolbachia diversity and incompatibility patterns in

Background

First discovered in Culex pipiens (s.l.) mosquitoes [1], the α-proteobacterium Wolbachia pipientis is one of the most common vertically transmitted cytoplasmic symbionts. Indeed, meta-analysis predicts Wolbachia infection in up to 50% of the arthropod species [START_REF] Weinert | The incidence of bacterial endosymbionts in terrestrial arthropods[END_REF]. The success of their vertical transmission mainly relies on their host reproduction manipulation strategies; parthenogenesis induction, feminization, male killing and cytoplasmic incompatibility (CI) [3]. CI, the most common of these strategies, is modelled by a modification-rescue (mod-resc) system where Wolbachia modifies sperm of infected males (mod function), and only a compatible Wolbachia strain in the eggs can rescue (resc function) this modification [4]. Consequently, Wolbachia causes conditional sterility in crosses either between uninfected females and infected males [START_REF] Hoffmann A | Factors affecting the distribution of cytoplasmic incompatibility in Drosophila simulans[END_REF] or like in the case of Cx. pipiens (s.l.), between females and males infected with incompatible Wolbachia strains [6,7].

Consisting of several species, including important disease vectors with worldwide distribution (e.g. Cx. pipiens and Cx. quinquefasciatus) [START_REF] Farajollahi | Bird biting" mosquitoes and human disease: a review of the role of Culex pipiens complex mosquitoes in epidemiology[END_REF], mosquitoes of Cx. pipiens complex have a unique relationship with their endosymbiont Wolbachia (wPip). wPip is fixed in natural Cx. pipiens (s.l.) populations where they induce the most complex CI relationships yet described among arthropods, including uni and bidirectional incompatibility [7,[START_REF] Laven | Speciation and evolution in Culex pipiens[END_REF][START_REF] Duron | Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens[END_REF][START_REF] Markus | Evidence for low-titre infections in insect symbiosis: Wolbachia in the bark beetle Pityogenes chalcographus (Coleoptera, Scolytinae)[END_REF][START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF]. So far, reciprocal crosses between many isofemale lines and four reference lines showed eight different mod and four different resc functions in Cx. pipiens (s.l.), resulting in the definition of 14 different cytotypes throughout the world [7].

In contrast to this observed diversity of CI patterns, Cx. pipiens wPip strains are closely related, and all belong to a clade within Wolbachia B supergroup [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF][START_REF] Guillemaud | Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens[END_REF][START_REF] Baldo | Multilocus sequence typing system for the endosymbiont Wolbachia pipientis[END_REF]. However, recent studies of fast evolving markers showed the presence of many genetically distinct wPip strains in Cx. pipiens (s.l.) mosquitoes [START_REF] Duron | Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens[END_REF][START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF]15] distributed in five distinct phylogenetic groups (wPip-I to V) [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF]. Using a PCR/RFLP assay based on pk1 gene, encoding proteins with ankyrin motifs, a wPip strain can be assigned to one of these five groups [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF][START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. A study of the wPip worldwide distribution showed an important spatial structure of wPip groups [START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. For instance, only wPip-I was found in sub-Saharan Africa, South America and Southeast Asia, while wPip-III was mainly observed in North America. Strains belonging to the wPip-II group were mostly found in western Europe and wPip-V in Asia. wPip-IV group strains exhibit a patchy distribution in Europe, North Africa and Asia [START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. Also, Wolbachia genetic diversity and their CI patterns strongly correlate; most wPip strains from the same group render their host compatible with each other (except few unidirectional incompatibilities) whereas those from different groups often lead to unidirectional or bidirectional incompatibilities [7]. Recently this huge diversity of CI patterns observed in Cx. pipiens has been explained by the amplification and the diversity of an operon in wPip strains' genomes [START_REF] Bonneau | Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon in Wolbachia genome[END_REF] composed of cidA and cidB genes involved in Wolbachia induced CI [START_REF] Lepage | Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility[END_REF][START_REF] Beckmann | A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility[END_REF]. No effect of host genetic background on the CI patterns [START_REF] Duron | Rapid evolution of Wolbachia incompatibility types[END_REF] and no multiple infections by several strains have ever been shown [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF][START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF][START_REF] Atyame | Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations[END_REF].

Being a natural bridge between Africa, Asia and Europe, Turkey is a critical spot for many emerging and reemerging vector-borne diseases [START_REF] Inci | Tick-borne diseases in Turkey: A review based on one health perspective[END_REF][START_REF] Failloux | Surveillance of arthropod-borne viruses and their vectors in the Mediterranean and Black Sea regions within the MediLabSecure Network[END_REF] and for the diversity of the vectors that transmit these diseases [START_REF] Failloux | Surveillance of arthropod-borne viruses and their vectors in the Mediterranean and Black Sea regions within the MediLabSecure Network[END_REF]. For instance, high diversity and abundance of Cx. pipiens (s.l.) species have been recorded in the area including Cx. quinquefasciatus, Cx. pipiens and its physiological variant Cx. pipiens f. molestus [START_REF] Gunay | Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors[END_REF]. Arboviruses such as West Nile virus, mainly transmitted by these mosquitoes, have also been shown to circulate in Turkey [START_REF] Ergunay | Arboviral surveillance of field-collected mosquitoes reveals circulation of West Nile virus lineage 1 strains in Eastern Thrace, Turkey[END_REF][START_REF] Ergunay | Serological, molecular and entomological surveillance demonstrates widespread circulation of West Nile virus in Turkey[END_REF][START_REF] Ergunay | Close relationship between West Nile virus from Turkey and lineage 1 strain from Central African Republic[END_REF][START_REF] Öncü | West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia[END_REF]. Therefore, understanding wPip diversity and their CI properties of wPip to control Cx. pipiens (s.l.) populations in Turkey is a cornerstone for vector control in the region and prevention of putative epidemics extending through Europe, Asia and northern Africa. This knowledge can contribute to the biological vector control techniques using CI properties such as incompatible insect technique (IIT). IIT, the mass release of males harbouring incompatible Wolbachia into focal populations, has been shown to successfully decrease the female reproduction by sterilisation and reduce the pest/vector populations [START_REF] Knipling | Genetic control of insects of public health importance[END_REF][START_REF] Zabalou | Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control[END_REF][START_REF] Laven | Eradication of Culex pipiens fatigans through cytoplasmic incompatibility[END_REF][START_REF] Chen | Naturally occurring incompatibilities between different Culex pipiens pallens populations as the basis of potential mosquito control measures[END_REF][START_REF] Atyame | Cytoplasmic incompatibility as a means of controlling Culex pipiens quinquefasciatus mosquito in the islands of the South-Western Indian Ocean[END_REF][START_REF] Atyame | Wolbachia-based population control strategy targeting Culex quinquefasciatus mosquitoes proves efficient under semi-field conditions[END_REF]. Nevertheless, our knowledge of the wPip genetic diversity and the CI patterns in Turkey is yet limited to only one line established with samples collected in Istanbul in 2003 [START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF].

Here, we collected and analysed 753 Cx. pipiens (s.l.) individuals (larvae and adults) from natural populations across Turkey. We studied (i) the wPip diversity in this geographically critical region in the crossroads of three continents, (ii) the CI relationships between Cx. pipiens lines from Turkey and (iii) the CI relationships between Turkish lines and reference lines to compare their CI properties to previously characterized mod (male crossing type) and resc (female crossing type) functions. Taken together, these results might be used in integrated vector control programs against Cx. pipiens (s.l.) in Turkey.

Methods

Sample collection and identification

A total of 753 samples from 59 different sampling sites in Turkey were tested for Wolbachia diversity. Most of these samples (n = 677) were collected during the larval stage, between July to September 2016 (Table 1). The rest of the samples has been collected as adults, using adult light traps, from May to September (2012-2015) (Table 1, Fig. 1). All of the sampling sites were situated outdoors with the only exception of sample site 16 (Table 1). Collected larvae and adults were morphologically identified as Culex pipiens (s.l.) / Cx. torrentium [START_REF] Schaffner | The mosquitoes of Europe[END_REF]. As the samples were only morphologically identified, we used Cx. pipiens (s.l.) to refer to Cx pipiens assemblage that includes both Cx. pipiens and Cx. quinquefasciatus, their hybrids and physiological forms [START_REF] Atyame | Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations[END_REF][START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF]. Samples were stored in 70% ethanol until DNA extraction before testing them for the wPip presence and genetic characterisation.

Isofemale lines

For analysing the CI patterns induced by the wPip strains belonging to different groups found in Turkey, egg rafts and larvae were collected from a population in Thrace region of Turkey, in Tekirdag (Table 1 sampling site 52, Fig. 1). Collected larvae were reared to adults in insectary conditions (at 25 ± 2 °C and 75 ± 2% relative

Crossing experiments between Turkish Culex pipiens lines

Tek wPip-I and Tek wPip-II isofemale mosquito lines were reared for at least four generations in insectary conditions to allow their acclimatisation before crossing experiments. Mosquitoes were isolated during pupal stage, and emerging adults were sexed. Then, 2-5 days old virgin males and females (n = were used to carry out reciprocal crosses between them and with Istanbul wPip-IV line. Females were fed with turkey blood using a Hemotek membrane feeding system (Discovery Workshops, United Kingdom) on the sixth day following caging and were allowed to lay eggs five days after the blood meal. Egg rafts were then isolated individually in 24 well plates filled with tap water until hatching. Embryonic development of all the unhatched egg rafts was verified to differentiate between nonfertilized egg rafts and CI induced embryonic death as previously described [START_REF] Duron | Wolbachia infection influences the development of Culex pipiens embryo in incompatible crosses[END_REF].

The crossing relationships were identified as following [7]: compatible (C) when > 90% of the rafts hatched in the two reciprocal crosses; and incompatible (IC), with two CI patterns: (i) unidirectionally incompatible crosses: when between 0-10% of the rafts hatched in one of the reciprocal crosses and > 90% in the other; and (ii) bidirectionally incompatible crosses: when less than 10% of the rafts hatched in both reciprocal crosses.

Crossing experiments to infer mod and resc functions

The mod (male crossing type) and resc (female crossing type) functions caused by many wPip strains, which belong to different wPip groups (I-V), have been identified by reciprocal crosses with 4 reference lines: LaVar (wPip-II), MaClo, Slab (wPip-III) and Istanbul (wPip-IV) [7]. Here, we used same four reference lines to define the mod-resc functions of Tek wPip-I and Tek wPip-II isofemale lines and to compare them to previously defined ones [7].

Identification of Wolbachia diversity

DNA was extracted from the samples using CTAB method [START_REF] Rogers | Extraction of DNA from plant tissues[END_REF]. PCR assays were conducted using pk1 primers (PK1 Forward: 5'-CCA CTA CAT TGC GCT ATA GA-3' and PK1 Reverse: 5'-ACA GTA GAA CTA CAC TCC TCC A-3'-AM397079 [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF]), which amplify a 1.3-kilobase (kb) fragment from ankyrin domain coding gene of Wolbachia. PCR amplifications were made in following conditions: initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation, annealing and elongation respectively at 94 °C for 30 s, 52 °C for 30s, and 72 °C for 90 s, and a final elongation at 72 °C for 5mn. Resulting PCR products then used in RFLP assays first with TaqαI enzyme to discriminate specific wPip alleles "a" or "e" (wPip-I or wPip-V; 991, 251, 107 bp), "b" (wPip-III; 669, 665 bp), "c" (wPip-II; 851, 498 bp) and "d" (wPip-IV; 497, 251, 107 bp) [7,[START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. Secondly, since TaqαI digestion of "a" and "e" alleles show the same digestion pattern, pk1 PCR products of the samples showing this pattern were digested with PstI enzyme to further discriminate "a" 903,303,141 bp) and "e" (wPip-V; 903, 430 bp) alleles [7,[START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. Digested amplified fragments were separated by agarose gel electrophoresis (2%), stained with ethidium bromide (1 μg/ml) and visualized with UV light. Samples from laboratory mosquito lines with different wPip groups and tetracyclinecured Wolbachia negative lines were included in every reaction as positive and negative controls, respectively, and always gave the expected result.

Statistical analyses

The occurrence of different wPip groups was compared by a Chi-square test using R software (version 3.3.1).

Results

Diversity and distribution of wPip groups

Wolbachia wPip was present in all of the 753 Cx. pipiens (s.l.) individuals tested and they were further identifiable to one of the five previously described groups (wPip-I to V). Co-infection of one individual by different wPip groups was never observed. Out of five wPip groups identified so far in the world, three of them (i.e. wPip-I-II and IV) were represented in the studied area (Fig. 1). The abundance of these groups was significantly different from each other (χ 2 = 474.99, df = 2, P < 0.0001). While wPip-II was the most dominant (n = 500, 66% of the samples, Table 1, Fig. 1) and widespread group (39 sampling sites out of 59 total) in Turkey; wPip-IV was found only in two locations, both in Thrace Region (in Edirne, sampling site 21 and Istanbul sampling site 34, Table 1; Fig. 1), and was the least abundant group (n = 12, 1% of the samples, Table 1; Fig. 1). wPip-I was found in 31 locations and a total of 241 individuals.

Co-existence of wPip strains in different individuals from the same sampling sites

In 20 % of the sampling sites wPip-I and wPip-II co-existed (Table 1, Fig. 1). wPip-IV was only found co-existing with wPip-II in one sampling site but never found in the same sampling site with wPip-I, even though they were sampled from nearby sites (~8 km) in north western Turkey (in Edirne, sampling site 21 and 23, Table 1; Fig. 1).

Naturally occurring CI patterns in Turkey

Two isofemale lines (Tek wPip-I and Tek wPip-II), harbouring two different wPip strains from two different groups, were established from north western Turkey (Tekirdag Province, Table 1 sampling site 52, Fig. 1) to identify CI patterns caused by different wPip groups in the region. Reciprocal crosses between these lines showed that Tek wPip-I and Tek wPip-II were fully compatible with each other (Table 2). Both lines were bidirectionally incompatible with the line harbouring Istanbul strain (wPip-IV, Table 2).

Mod and resc properties of Turkish wPip strains

To compare mod and resc functions of Turkish wPip strains with mod and resc functions of worldwide collected wPip strains, we performed reciprocal crosses of Tek wPip-I and Tek wPip-II lines with the four reference lines [LaVar (wPip-II), MaClo (wPip-III), Slab (wPip-III) and Istanbul (wPip-IV)]. Tek wPip-I males were compatible with LaVar (wPip-II) and MaClo (wPip-III) females while incompatible with Slab (wPip-III) and Istanbul (wPip-IV) females (Table 2). This type of mod property, inferred from similar crosses, has already been shown for the wPip-I group from Tunisia; numbered "vi" [7]. Contrarily, Tek wPip-II males demonstrated a new mod property, as they were incompatible with LaVar and Istanbul, and compatible with MaClo and Slab females (Table 2). We numbered this new mod as "ix" to continue the previously published numeration [7]. Both Tek wPip-I and Tek wPip-II lines showed the same resc type, which was characterised by the compatible crosses of females of these lines with all the males from the reference lines except Istanbul (Table 2). This resc type (resc "2") is the most common resc type found worldwide for wPip-I and wPip-II groups [7].

Discussion

In Turkey, all tested Cx. pipiens were infected with Wolbachia wPip. Such fixation of wPip has been demonstrated worldwide, including in the neighbouring country Iran [START_REF] Karami | Wolbachia endobacteria in natural populations of Culex pipiens of Iran and its phylogenetic congruence[END_REF], in Cx. pipiens and Cx. quinquefasciatus populations [START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF][START_REF] Rasgon | Cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: parameter estimates and infection dynamics in natural populations[END_REF][START_REF] Fedorova | Morphological and molecular-genetic distinctions between adult mosquitoes Culex torrentium Martini and C. pipiens Linnaeus (Diptera, Culicidae) from Moscow Province[END_REF][START_REF] Khrabrova | The distribution of strains of endosymbiotic bacteria Wolbachia pipientis in natural populations of Culex pipiens mosquitoes (Diptera: Culicidae)[END_REF][START_REF] Duron | Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito[END_REF][START_REF] Raharimalala | Molecular detection of six (endo-) symbiotic bacteria in Belgian mosquitoes: first step towards the selection of appropriate paratransgenesis candidates[END_REF]. A previous study in Turkey, however, showed a lower prevalence of wPip [START_REF] Yildirim | Detection and molecular characterization of the Wolbachia endobacteria in the Culex pipiens (Diptera: Culicidae) specimens collected from Kayseri province of Turkey[END_REF]. This might be caused by the misidentification of a recently described cryptic species within Cx. pipiens complex that has been shown to lack wPip infection and to be reproductively isolated from the other members of the complex [START_REF] Rasgon | Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking[END_REF][START_REF] Dumas | Molecular data reveal a cryptic species within the Culex pipiens mosquito complex[END_REF]. Similarly, Cx. torrentium, which is difficult to differentiate morphologically from Cx. pipiens (s.l.) mosquitoes is not infected with Wolbachia [START_REF] Fedorova | Morphological and molecular-genetic distinctions between adult mosquitoes Culex torrentium Martini and C. pipiens Linnaeus (Diptera, Culicidae) from Moscow Province[END_REF][START_REF] Raharimalala | Molecular detection of six (endo-) symbiotic bacteria in Belgian mosquitoes: first step towards the selection of appropriate paratransgenesis candidates[END_REF][START_REF] Leggewie | Culex torrentium mosquitoes from Germany are negative for Wolbachia[END_REF]. Therefore 100% wPip infection rate of our samples confirmed that we only analysed Cx. pipiens complex members (excluding both previously mentioned cryptic species and Cx. torrentium) in the present study.

The identification of the Cx. pipiens taxa was left out of the scope of this study for several reasons. Previous studies on the diversification of wPip in Cx. pipiens (s.l.) have proved that their diversity is not directly related to the nuclear genetic background of the mosquitoes, meaning that no wPip group was specific for a Cx. pipiens sibling species [START_REF] Duron | Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens[END_REF][START_REF] Atyame | Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations[END_REF]49]. It rather follows the same distribution as mitochondrial diversity (mtDNA) of mosquitoes, as wPip are maternally transmitted to the next generation through the egg cytoplasm along with mitochondria [START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF][START_REF] Rasgon | Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking[END_REF]. Moreover, CI properties are independent of the genetic background of Cx. pipiens (s.l.) and directly dictated by their Wolbachia [START_REF] Duron | Rapid evolution of Wolbachia incompatibility types[END_REF]. Recent studies on Cx. pipiens (s.l.) in Turkey had shown that both Cx. quinquefasciatus, Cx. pipiens and its form Cx. pipiens f. molestus, were present in Turkey [START_REF] Gunay | Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors[END_REF]. The co-existence of these sibling species in same sampling sites [START_REF] Gunay | Barcoding Turkish Culex mosquitoes to facilitate arbovirus vector incrimination studies reveals hidden diversity and new potential vectors[END_REF][START_REF] Shaikevich | Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: contribution of Cx. quinquefasciatus in Mediterranean populations[END_REF] and the existence of hybrids [START_REF] Shaikevich | Genetic diversity of Culex pipiens mosquitoes in distinct populations from Europe: contribution of Cx. quinquefasciatus in Mediterranean populations[END_REF][START_REF] Shaikevich | The discovery of a hybrid population of mosquitoes of the Culex pipiens L. complex (Diptera, Culicidae) on the Kos Island (Greece) by means of molecular markers[END_REF][START_REF] Smith | Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae)[END_REF] suggest that they can exchange wPip strains easily in natural populations.

We have identified three different wPip groups, i.e. wPip-I-II and IV in Cx. pipiens (s.l.) mosquito populations in Turkey. The only previous sample from Turkey, which has been assigned to wPip groups, was a wPip-IV group strain collected in Istanbul in 2003 [START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF][START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF]. Other than this single case, the wPip diversity in Turkish Cx. 24) IC ( 24) C ( 9) C ( 17) C ( 34)

Tek II 2 C ( 26) IC ( 32) C ( 26) C ( 20) C ( 14) pipiens populations was to date completely unknown.

Istanbul 3 IC (
Although wPip diversity was investigated in regions around Turkey [START_REF] Dumas | Population structure of Wolbachia and cytoplasmic introgression in a complex of mosquito species[END_REF]. Dumas et al. [16] have found wPip-I strains in Middle East (Lebanon, Israel, Jordan) and in northern Africa (Tunisia), wPip-II strains widely distributed in eastern Europe and Cyprus, and wPip-IV strains in a patchy distribution in Europe -in areas dominated by other wPip groups. We demonstrated that wPip strains belonging to wPip-I, wPip-II and wPip-IV, previously identified near Turkey, are all present in the Cx. pipiens (s.l.) populations within this country, suggesting that Turkey is a crossroads for wPip strains from eastern Europe, Africa and Middle East as for their vector hosts.

The most widespread groups in Turkey, wPip-I and wPip-II, induce reciprocal compatibility between their hosts and co-exist in many populations. Indeed, different wPip strains can co-exist in a single natural Cx. pipiens (s.l.) population [START_REF] Atyame | Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito[END_REF][START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF][START_REF] Duron | Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito[END_REF][START_REF] Duron | Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the mosquito Culex pipiens[END_REF] and these coexisting strains are usually compatible with each other [START_REF] Duron | Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito[END_REF]. Mathematical models confirm that only compatible strains can stably coexist in unstructured and panmictic host populations when the fitness costs related to infection by different wPip strains are the same [START_REF] Rousset | Cytoplasmic incompatibility in the mosquito Culex pipiens: how to explain a cytotype polymorphism?[END_REF][START_REF] Engelstädter | Cytoplasmic incompatibility and host population structure[END_REF]. When bidirectional incompatibility inducing wPip strains co-exist in one population the most prevalent strain is expected to eventually invade the population [START_REF] Rousset | Cytoplasmic incompatibility in the mosquito Culex pipiens: how to explain a cytotype polymorphism?[END_REF][START_REF] Engelstädter | Cytoplasmic incompatibility and host population structure[END_REF] and when unidirectional incompatibility inducing wPip strains co-exist, CI-inducing strain is expected to invade the population once above a frequency threshold [49,[START_REF] Engelstädter | Cytoplasmic incompatibility and host population structure[END_REF]. Therefore, a stable co-existence of incompatible strains is predicted to be rare. However, an example of the co-existence of unidirectional incompatibility inducing strains, belonging to a wPip-I group and wPip-IV group, has been shown in Tunisia while mathematical models predicted that wPip-I should have invaded this area in only 4 generations [START_REF] Hoffmann A | Factors affecting the distribution of cytoplasmic incompatibility in Drosophila simulans[END_REF]49]. Atyame et al. [49] hypothesized that low dispersal and extinction-recolonization events could explain this stable co-existence. In one site in Turkey, we observed the co-existence of bidirectional incompatibility inducing strains (wPip-IV and wPip-II). The low prevalence of wPip-IV and its incompatibilities suggest that it should disappear from the population. However, we have evidence that wPip-IV strains were already present at least 13 years ago at Istanbul since it has been sampled in 2003 [START_REF] Duron | Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination[END_REF]. This persistence of wPip-IV, at low frequencies, could be explained by higher fitness costs associated with wPip-I and wPip-II infections or by extinction-recolonization events of wPip-IV-infected individuals as it has been suspected in Tunisia [START_REF] Duron | Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito[END_REF]49,[START_REF] Engelstädter | Cytoplasmic incompatibility and host population structure[END_REF]. Fitness difference could be, for instance, linked to differences in fecundity [START_REF] Poinsot | Wolbachia infection in Drosophila simulans: does the female host bear a physiological cost?[END_REF][START_REF] Bordenstein | Do Wolbachia influence fecundity in Nasonia vitripennis?[END_REF] or to a possible ability of the different wPip strains to protect their hosts against other microbial infections [START_REF] Hedges | Wolbachia and virus protection in insects[END_REF][START_REF] Brownlie | Symbiont-mediated protection in insect hosts[END_REF][START_REF] Hughes | Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae[END_REF][START_REF] Braquart-Varnier | The mutualistic side of Wolbachia-isopod interactions: Wolbachia mediated protection against pathogenic intracellular bacteria[END_REF][START_REF] Teixeira | The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster[END_REF]. Further studies on the differences between wPip strains in terms of infection costs and pathogen protection might help to understand stable co-existence of bidirectionally incompatible wPip-IV strains observed in Turkey.

To study the phenotypical diversity of crossing types in Turkey, we crossed Turkish isofemale lines harbouring wPip-I and wPip-II strains with four reference lines defined by Atyame et al. [7]. We inferred both their mod and resc functions and compared them to the eight mod and four resc functions already described worldwide. The Tek wPip-I line showed the most common resc functions for a wPip-I infected line (i.e. resc 2) but a rare mod function previously defined in few lines harbouring wPip-I or wPip-II strains (i.e. mod "vi"). The Tek wPip-II line showed the same resc function as wPip-I (i.e. resc 2) but a totally new mod function (i.e. mod "ix"). Our findings are consistent with theoretical predictions and empirical data suggesting new mod functions can more easily evolve and spread in the population than new resc functions [7,[START_REF] Charlat | On the mod resc model and the evolution of Wolbachia compatibility types[END_REF].

Natural CI properties induced by Wolbachia can be used to control the vector populations: the mass release of males harbouring incompatible Wolbachia into the natural populations can decrease the female reproduction and eradicate the pest/ vector populations (IIT) [START_REF] Knipling | Genetic control of insects of public health importance[END_REF][START_REF] Zabalou | Wolbachia-induced cytoplasmic incompatibility as a means for insect pest population control[END_REF]. Indeed, wPip induced CI has been used against Cx. quinquefasciatus (formerly named Cx. pipiens fatigans) for the first time in 1967 to control filariasis in Southeast Asia [START_REF] Laven | Eradication of Culex pipiens fatigans through cytoplasmic incompatibility[END_REF]. More recently, natural CI properties caused by wPip infection have been found promising to control Cx. pallens (no longer considered as a valid species) in China [START_REF] Chen | Naturally occurring incompatibilities between different Culex pipiens pallens populations as the basis of potential mosquito control measures[END_REF] and Cx. pipiens populations in La Réunion Island [START_REF] Atyame | Cytoplasmic incompatibility as a means of controlling Culex pipiens quinquefasciatus mosquito in the islands of the South-Western Indian Ocean[END_REF][START_REF] Atyame | Wolbachia-based population control strategy targeting Culex quinquefasciatus mosquitoes proves efficient under semi-field conditions[END_REF]. In the latter study, a wPip-IV strain from Istanbul has been successfully used to sterilise wPip-I females in semifield conditions. We demonstrated that this Istanbul strain also induces bidirectional incompatibility with mosquitoes harbouring wPip-I or wPip-II in Turkey. This means that most Cx. pipiens females in Turkey, except in few sites in the Thrace region, can be sterilised by the release of males infected with Istanbul strain. Although further studies on intrapopulation CI variability, mating choice, hatching rate and population dynamics in semi field populations are needed for wPip-IV Istanbul to be used in future vector control programs in Turkey, a critical region for vector-borne diseases, our results suggest that it could constitute a good candidate.

Conclusions

We identified wPip diversity in natural Cx. pipiens (s.l.) populations in Turkey. The previously described wPip-IV group was in fact restricted to only two populations while wPip-I and wPip-II group are widely distributed and coexist in many populations all over the country. The wPip-IV strain Istanbul was found bidirectionally incompatible with individuals harbouring wPip-I or wPip-II from Turkey. This highlights the potential of wPip-IV harbouring males as a vector control to sterilise local Cx. pipiens populations, particularly where only wPip-I or wPip-II harbouring females were found. were also investigated for the prevalence and diversity of CpDV. The samples collected as adults from May to September in 2012-2015 using adult light traps, have been tested before the second collection and no CpDV was detected in these samples (n=76). To investigate whether the life stage of the mosquito can have an effect on the prevalence of CpDV, I collected new larvae samples from July to September 2016 some of which I let to become adults in insectary conditions in the water from which have been collected and stored as emerged adults in 70% ETOH.

4.3! CpDV Prevalence and Diversity in Turkey

!"#"$"2! 3,4+50&61-7/0.+/*7/+ CpDV genome contains three non-structural (NS) genes; NS3 ORF which is on the 5' side of NS1, and NS2 ORF overlapping the 5' half of the NS1 ORFs (Baquerizo-Audiot et al., 2009)((Figure 1). In CpDV genome, NS1 and NS2 coding sequences split into four ORFs, instead of two ORFs like in other ambidensoviruses (Kerr et al., 2005;Baquerizo-Audiot et al., 2009) (Figure 1). NS1 gene includes highly conserved endonuclease called SF3 helicase which is generally used for densovirus taxonomy (Kerr et al., 2005)(Figure 1).

We designed a diagnostic PCR using CpDV specific primers which amplify a 238 bp fragment (Chapter 2) within NS1-NS2 region (Figure 1). A total of 236 adults and 430 larvae were tested.

To investigate the diversity of CpDV in natural populations of Cx. pipiens mosquitoes in Turkey, specific primers to amplify fragments from both NS1 and VP coding region of CpDV genome have been used (Figure 1). These primers amplified most of the NS1' ORF of NS1 gene (Figure 1) that encompasses the SF3 helicase domain in the 5'end of NS1 and as well as other less conserved sites (1681-2648; 967 bp) and a part of VP region of CpDV that includes both conserved PLA2 and stretches for 1389 bp (3570-4959; 1389 bp, Figure 1). To increase the chance of amplifying these fragments, semi-nested PCRs were conducted with previously described protocols (Chapter 2, see Supplementary Table 2 for protocols and primers).

We have further designed specific primers within NS3 area to be able to have access to much more polymorphic region to discriminate CpDV between geographically close samples (Figure 1). For this a 1566 nt long fragment, including NS3 coding region, has been amplified using CpDV_355_F (5'-GGTCTGAATTGGCTGATGC-3') and CpDV_1902_R

(5'-GTGTCCCAG-CAACTTCTC GA-3') primers. Resulting PCR products have been used as a template to amplify a 1072 nt long fragment using CpDV_355_F and CpDV_1387_R(5'-CGAAATTAGCATTT-GACTCTCC-3'). For both PCR reactions protocol was as following: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 56 °C for 30s, and 72 °C for 90 s, and a final elongation at 72 °C for 5 min.

With three of these fragments amplified for most of the samples, we covered about 54% of the CpDV genome (3256 over 6000 nt, Figure 1).

All the PCR products were purified using AM-Pure (Agencourt) with Biomek 4000. Following the purification, DNA was quantified by nanodrop and sequencing reaction has been performed using 100ng of DNA (50ng/kb). After the completion of sequencing reaction (96°C for 5min, 30 cycles of 96°C 15 sec, 50°C 10sec and 60°C 4mn) products have been purified using CleanSEQ (Agencourt) with Pipeteur Biomek 4000, before the capillary electrophoresis of the sequences.

!"#"$"#! 8&/&+91&):707+

For probabilistic phylogenetic analyses we have used concatenated matrix of all the NS3, NS1 and VP amplicons, from the Turkish populations. We have also added samples from three phylogenetic groups found in the worldwide phylogeny of CpDV (Chapter 2)(Slab, Armenia, Montpellier) and Reference genome, to be able to compare the diversity in Turkey to the worldwide diversity. MAFFT v7.273 (Katoh and Standley, 2013) was used to align the concatenated sequences.

In order to avoid misleading phylogenetic signal due to recombinant parts of sequences, signature of recombination we used the RDP, GENECONV, Bootscan, MaxChi, and LARD methods with default parameters, as implemented in the RDP4 software (Martin et al., 2015). Recombinant regions of the sequences have been excluded while the rest of the sequence was kept. Modelfinder (Kalyaanamoorthy et al., 2017) was then used to find the best fitting model (Lemmon and Moriarty, 2004). Phylogeny of CpDV in Turkey was then inferred under the HKY85 + \Gamma model of sequence evolution using PhyML. Statistical support of the phylogenetic nodes was computed using a bootstrap procedure (Felsenstein, 1985).

4.3.2! Results

!"#"2"$! 3;*<&)*1.*+-=+,(8>"++ Excluding all the adults that were collected between 2012-2015 with adult traps and were negative, 60% of the samples tested showed a presence of CpDV (Figure 2). The proportion of infected larvae was lower compared to infected adults, 58% and 64% respectively. The effect of the life stage was significant however it only explained a low amount of deviance (binomial glm, df=1, dev=18.896,p<0.001, Figure 3A). There was also a significant effect of collection location on the CpDV prevalence (binomial glm, df=36, dev=210.74, p<0.001). There was no effect of wPip group on the prevalence of CpDV (binomial glm, df=2, dev=0.745, p>0.5, Figure 3B). Maximum likelihood tree based on the partial NS3-NS1 and VP sequences showed that there were four distinct CpDV variants in Turkey (depicted with different colours on the map and sample names in Figure 4). Reference CpDV genome, along with other samples from around the world (Slab 2018, Bangui, Tunisia, Armenia, Montpellier) have been used to understand the diversity in Turkey compared to worldwide phylogeny of CpDV ( Chapter2). General CpDV diversity in Turkey grouped in two clades (Figure 4). Most of the sequenced samples belong to CpDV-1 (as defined in Chapter 2, Figure 4). These samples were collected mostly from Northern Turkey and they were really closely related to each other (purple points on the map, Figure 4) and the current laboratory variant Slab 2018. Interestingly all the four variants were present in North-Western Turkey. One variant (shown in red, Figure 4) was really closely related to the samples from Armenia and Montpellier.

Both wPip-I and wPip-II harbouring mosquitoes were found infected with variants from CpDV-1. On the contrary, CpDV-2 was only found in the mosquitoes harbouring wPip-II Wolbachia.

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4.4! Conclusions

A high prevalence of CpDV has been shown worldwide (Chapter 2). Focusing on Turkey we investigated the possible effects of life stage and wPip genetic diversity. Samples that have been collected as adults did not show any CpDV presence, although when larvae have been collected, emergent adults were more likely to be infected. The longer storage time of the samples collected as adults (2012)(2013)(2014)(2015) could have made impossible to detect CpDV but one should keep in mind that we were able to amplify and identify wPip strains using the same DNA extractions. Alternatively, CpDV could be emerging in Turkey only recently, reaching higher frequency in 2016. Another possibility could be that CpDV infected adults are less likely to be collected with adult traps and there could be several reasons for that: i) high mortality in larvae resulting in very low prevalence in adults, ii) reduced adult lifespan, iii) reduced dispersal. A similar pattern has been observed in AeDV Thai strain prevalence in natural populations of Ae. aegypti and Ae. albopictus (Kittayapong et al., 1999). In Ae. aegypti adults collected from Thailand (n=97), AeDV Thai strain prevalence was 44.3% but there were no AeDV in Ae. albopictus adults tested (n=79) although AeDV Thai strain was able to infect both species in experimental infections (Kittayapong et al., 1999). Interestingly, in our laboratory lines we have observed the opposite: randomly sampled adults were less likely to be infected by CpDV compared to larvae (Chapter 3). Given that samples from natural populations were collected as soon as they emerged, a reduced lifespan of the adults could explain the difference observed between natural and insectary populations. Other mosquito densoviruses have been shown to reduce the lifespan of their Ae. aegypti adults (Suchman et al., 2006) although the effect of CpDV on their host is still not clear.

After identifying wPip diversity in Turkey we have also investigated whether there is an effect of wPip diversity on CpDV prevalence. Although the percentage of mosquitoes harboring wPip-I that were also CpDV positive was higher than the other wPip groups, this effect was not significant. In fact most of the variance in the data was explained by the location where the mosquitoes have been collected. The fact that populations where different wPip groups co-existed was rare made further analysis of wPip diversity-CpDV prevalence correlation impossible, especially since we observe such an high impact of the collection location. On the other hand if mosquitoes harboring different wPip groups were collected from the same environmental conditions, both of the groups would have the same probability to be infected, in terms of the effect of the environmental conditions, hence we could compare their CpDV infection status directly.

We have identified two clades of CpDV in Turkey, from three that has been found worldwide (Chapter 2). Similar to what we have observed in worldwide populations, most of the CpDV variants belonged to the CpDV-1 clade. There was no clear association between CpDV and wPip diversity. All of the samples collected from the populations around the Blacksea coast showed the same variant of CpDV suggesting the efficient dispersal of this variant between the populations of this area. It is known that adult mosquitoes can contaminate new oviposition sites with mosquito densoviruses (Wise de Valdez et al., 2010), they can also be transmitted by human activity. It is also important to note that most of these samples collected around Blacksea coast were collected from old tyres filled with rain water. On the other hand the lack of the two other variants (shown in red and brown Figure 5) in this area might suggest a barrier between the populations harbouring the red, brown and purple variants or a more recent introduction of red and brown variants to Turkey.

Our results demonstrated the diversity of both wPip groups and CpDV in Turkey, especially in North-Western part (Thrace region). Both the diversity of insect-specific flaviviruses and the circulation of West-Nile virus vectored mainly by Cx. pipiens mosquitoes have previously been demonstrated in the same region (Ergunay et al., 2013(Ergunay et al., , 2014(Ergunay et al., , 2015;;Öncü et al., 2018). Indeed Turkey with its unique geographical location, climate and arthropod diversity, could represent a suitable habitat for circulating viruses to amplify even more and for emergence of new viruses. Overall our results were also in accordance with the previously reported Cx. pipiens diversity for the area (Gunay et al., 2015) and suggested Turkey could be a place where different populations of Cx. pipiens mix.

This highlights the importance of this area, for surveillance efforts against arboviruses to prevent putative epidemics and for investigation of the ecological interactions between wPip groups, arboviruses and insect-specific viruses in natural mosquito populations.

4.5! References

! ! ! ! ! ! !
Influence of Wolbachia on prevalence, viral load and diversity of Culex pipiens densoviruses Abstract: Culex pipiens densovirus (CpDV), belong to Densovirinae subfamily and have been initially isolated from laboratory Cx. pipiens colonies. Recently their wide prevalence and their ability to vertically transmit were reported, highlighting their long-term associations with their hosts. In nature Cx. pipiens are always found infected with Wolbachia wPip from one of the five phylogenetic groups (wPip-I to V).

Wolbachia are endosymbiotic bacteria which deeply modify their host phenotypes including their reproduction, for instance via cytoplasmic incompatibility, and their survival for instance via viral interference.

Given the importance and co-occurrence of these two Cx. pipiens endosymbionts, we have investigated the putative reciprocal influence between CpDV and Wolbachia in natural populations of Cx. pipiens. For this we have chosen a specific zone in Northern Tunisia where two genetically distinct incompatible wPip groups co-exist in same environmental conditions and where the prevalence of CpDV is high. Our results showed an effect of Wolbachia groups and their titers, on the prevalence, viral load and diversity of CpDV. Overall our results represented a good evidence for Wolbachia and CpDV interactions in natural populations and highlighted the potential importance of bacterial ensodymbiont on insect specific virus ecology.

5.1.1! Introduction

Densoviruses (Densovirinae) are small single stranded DNA viruses of arthropods (Cotmore et al., 2014;Tijssen et al., 2016). They belong to Parvoviridae family, a virus family that consists of animal viruses, including vertebrate (Parvovirinae) viruses (Cotmore et al., 2014;Tijssen et al., 2016).

Their existence in a broad range of invertebrate hosts, sometimes even as endogenous viral elements, suggests that they might have an important influence on their hosts' phenotypes and evolution (Liu et al., 2011;Thézé et al., 2014;Metegnier et al., 2015;François et al., 2016). Mosquito densoviruses (MDVs) have long attracted scientific attention as vector control tools due to the pathogenicity and host specificity reported for some of them (Buchatski! et al., 1987;Carlson et al., 2006;Johnson and Rasgon, 2018). Most of MDVs are closely related (82-99% sequence homology) belonging to the genus Brevidensovirus (Cotmore et al., 2014). However one MDV, namely Culex pipiens densovirus (CpDV), differs extensively from other MDVs and belongs to Ambidensovirus genus (Jousset et al., 2000;Baquerizo-Audiot et al., 2009). Indeed CpDV with their larger genome (6kb compared to 4kb for brevidensoviruses), ambisense genome organization and sequence homology, is closely related to lepidopteran and blattodean Ambidensoviruses but not to other MDVs (Jousset et al., 2000;Baquerizo-Audiot et al., 2009;Cotmore et al., 2014). First discovered during high mortality episodes of laboratory Cx. pipiens larvae (Jousset et al., 2000), CpDV have since been shown to persist at low densities in seemingly healthy Cx.

pipiens lines and found widespread in natural populations worldwide (Chapter 2, Chapter 3).

Moreover, their hosts Cx. pipiens can harbour both

Wolbachia -widespread endosymbiotic bacteria of arthropods-and CpDV in their germinal cells resulting in the vertical co-transmission of both endosymbionts (Chapter 3).

Such co-existence of Wolbachia and CpDV within the same cells in Cx. pipiens, especially during transmission, (Chapter 3) suggests that these endosymbionts may interact and influence their respective prevalence, load and diversity. Both antagonistic and beneficial interactions between Wolbachia and viruses have previously been reported. However, antagonistic interactions have been by far more studied showing that Wolbachia can decrease virus multiplication by indirectly modulating their host resistance or tolerance, or directly by competing with the virus for host resources (Hedges et al., 2008;Teixeira et al., 2008;Moreira et al., 2009;Mousson et al., 2010;Hussain et al., 2012). The strength of this antagonism seems to mostly depend on variations in Wolbachia titers between different strains (Lu et al., 2012;Osborne et al., 2012;Bian et al., 2013;Martinez et al., 2017).

For instance, Wolbachia (wPip), when exhibiting a higher titer in some Cx. pipiens s.l. lines, reduced the West Nile Virus load (Micieli and Glaser, 2014). To date, such antagonistic interactions of Wolbachia have only been revealed against RNA viruses but never against DNA viruses (Teixeira et al., 2008;Graham et al., 2012). On the contrary, Wolbachia seem to increase Spodoptera exempta nucleopolyhedrovirus (SpexNPV) load, a double stranded DNA virus, and decrease the host survival in African armyworm. Furthermore in Drosophila Wolbachia did not have an effect on Insect Iridescent virus (double stranded DNA virus) infection in Drosophila (Teixeira et al., 2008;Graham et al., 2012). As many medically important mosquito species are infected with Wolbachia (Rasgon and Scott, 2003;Dumas et al., 2013;Baldini et al., 2014;Gomes et al., 2017), either naturally or stably transinfected, Wolbachia could be a major determinant of MDVs dynamics in natural mosquito populations. Because all the mosquitoes of Cx. pipiens complex are naturally infected with wPip strains belonging to five genetically distinct phylogenetic groups Wolbachia-CpDV interactions might be frequent both in laboratory colonies and natural populations (Chapter 3). The wPip strains strongly manipulate their host's reproduction by causing cytoplasmic incompatibility (CI), a sperm-egg incompatibility resulting in embryonic death (Werren, 1997;Atyame, Delsuc, et al., 2011). The diversity of wPip strains leads to a huge diversity in CI patterns in Cx. pipiens, including bidirectional (where the incompatibility is reciprocal between different strains) and unidirectional incompatibilities (where only one of the strains leads to an incompatible cross when carried by the male mosquitoes) (Atyame 2014). This diversity has recently been explained by the amplification and the diversification of an operon within WO phage region in wPip genomes (Bonneau et al., 2018). In natural Cx.

pipiens populations, different wPip strains can coexist when they are compatible with each other (Duron et al., 2006(Duron et al., , 2011;;Atyame, Duron, et al., 2011;Altinli et al., 2018) while a stable co-existence of incompatible strains is predicted to be rare in unstructured and panmictic host populations (Rousset et al., 1991;Engelstädter and Telschow, 2009). A rare example of stable co-existence between incompatible wPip strains, belonging to wPip-I and wPip-IV groups, over at least a 7-years period has been shown in Northern Africa. By mapping the distribution of wPip strains in Algeria and Tunisia and describing their CI patterns, Atyame et al. (2015) predicted that the wPip-I should outcompete the wPip-IV in only several generations (Atyame et al., 2015). While the authors further hypothesized that low dispersal and extinction-recolonization events may contribute to explain this stable co-existence, Wolbachia's virus interference could also influence this stability: if wPip groups interact differently with viruses either in antagonistic or beneficial ways, it can also influence their co-existence in addition to their CI properties (Hedges et al., 2008;Teixeira et al., 2008;Brownlie and Johnson, 2009;Fenton et al., 2011;Hughes et al., 2011). The presence of CpDV has recently been revealed in this contact zone between these two wPip groups (Chapter 2). Here, we tested the hypothesis that mosquitoes harbouring wPip-I and wPip-IV groups might differ in terms of their interactions with CpDV may be affecting its dynamics and distribution. To this end, we have studied CpDV prevalence and analysed them together with previously published results of wPip groups distribution in the same samples (Atyame et al., 2015). We additionally quantified both CpDV and wPip loads to study their potential reciprocal influence. Lastly, given their co-vertical transmission (Chapter 3), we investigated putative link between CpDV diversification and wPip diversity in natural populations of Cx. pipiens from this area.

5.1.2! Methods

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All the samples have been collected between 2010 and 2011 from natural populations of Cx. pipiens s.l. in Tunisia (Atyame et al., 2015) during larval stage (Supplementary Table 1). DNA has been extracted as described in Atyame et al. (2015) and stored in -80 °C after its utilization for identification of wPip groups (Atyame et al., 2015). To detect the diversity in geographically close populations, a 1566 nt long fragment including NS3 coding region, has been amplified using CpDV_355_F (5'-GGTCTGAATTGGCTGATG C-3') and CpDV_1902_R (5'-GTGTCCCAG-CAACTTCTCGA -3') primers. Resulting PCR products have been used as a template to amplify a 1072 nt long fragment using CpDV_355_F and CpDV_1387_R (5'-CGAAATTAGCATTTGAC TCTCC-3'). These fragments than sequenced using only CpDV_1387_R primer resulting in 800 nt long sequences.

For both PCR reactions protocol was as following: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 56 °C for 30s, and 72 °C for 90 s, and a final elon-gation at 72 °C for 5 min. PCR products were purified using AMPure (Agencourt) with Biomek 4000. Following the purification, DNA was quantified by nanodrop and sequencing reaction has been performed using 100ng of DNA (50ng/kb). After the completion of sequencing reaction (96°C for 5min, 30 cycles of 96°C 15sec, 50°C 10sec and 60°C 4mn) products have been purified using CleanSEQ (Agencourt) with Pipeteur Biomek 4000, before the capillary electrophoresis of the sequences. 5.1.62) were used to collect and analyse the results. Three PCR reactions were performed in triplicates using for each DNA sample i) NS2 protein coding region specific primers to quantify CpDV (CpDVquantiF: 5'-CATTGGAGGA-GAAAGTTGGAA-3' , CpDVquantiR: 5'-TCCCCATTATCTTGCTGTCG-3'), ii) wsp specific primers to quantify Wolbachia (wolpipdir 5'-AGAATTGACGGCATTGAATA-3' and wolpiprev 5'-CGTCGTTTTTGTTTAGTTGTG-3' (Berticat et al., 2002)) and iii) Culex Ace-2 locus specific primers to quantify host genome copy as a reference (acequantidir: 5'-GCAGCACCAG-TCCAAGG-3', acequantirev: 5'-CTTCAC-GGCCGTTCAAGTAG-3') (Weill et al., 2000). Dilutions of a pCR®4-TOPO-Vector (Invitrogen) containing a single copy of each ace-2, wsp and ns2 gene fragments were used as standard curves. As such the amount of Wolbachia and CpDV were normalized with the amount of ace-2 copies in order to obtain a number of virus or Wolbachia per cell. We have tested 114 samples in total and samples were chosen to balance the years and Wolbachia types (n=114; wPip-I n= 52, wPip-IV n=62; Col-lected in 2010 n=69, Collected in 2011 n=45). Samples that showed CP>33 for any of the PCR reactions have been excluded because of the high uncertainty of the quantification at such low titter levels.

!"#"$"!! 1D:)-;*0*./,+ C0&):<*<+ -4+ 5(67+ 8/3*2</4/,&./-0+ Sequences were aligned using MAFFT v7.273 (Katoh and Standley, 2013). In order to avoid misleading phylogenetic signal due to recombinant parts of sequences, recombinant region was identified and removed using RDP4 software (Martin et al., 2015). Modelfinder (Kalyaanamoorthy et al., 2017) has been used to find the best fitting model (Lemmon and Moriarty, 2004). Phylogenetic tree has been constructed under the HKY85 + \Gamma model of sequence evolution using RAxML v8.2 (Stamatakis, 2014). Statistical support of the phylogenetic nodes was computed using a bootstrap procedure (Felsenstein, 1985). Reference genome of CpDV (Baquerizo-Audiot et al., 2009) has been used as an outgroup to root the phylogenetic tree. Spatial correlation between the distribution of CpDV clades and geographical location of collection sites as well as wPip group distribution have been tested using Mantel test included in ecodist package (Goslee and Urban, 2007). wPip I north-western populations with a narrow contact zone between them. In the samples tested, the distribution of Wolbachia strains was significantly affected by year and population (glm, year: df=3, dev=17.128, p<0.001; population: df=34, dev=1587.3, p<0.001). B. CpDV prevalence and distribution in the area. A total of 933 was infected. In this global analysis of all sampled individual there was no apparent significant link between CpDV prevalence and wPip groups distribution (glm, df=1, dev=0.109, p>0.05).

5.1.3! Results

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We used 1230 samples, that have been collected between 2010-2011 by Atyame et al. (2015) and wPip groups identified (Figure 1A) and further tested them to infer CpDV prevalence in and around the wPip-I/wPip-IV contact zone. In such global analysis of all these samples from different locations (i.e. populations), Wolbachia groups (i.e.

wPip-I versus wPip-IV) had no significant effect on CpDV prevalence (glm, df=1, dev=0.108, p=0.741, Figure 1). However, CpDV prevalence significantly differed between locations (Figure1B, Population: glm, df=34, dev=638.04, p<0.001). CpDV prevalence was quite stable between years 72 % in 2010 (n=676) and 76% in 2011 (n=554). The absence of CpDV in some locations could reflect that these populations never had encountered CpDV because the environmental conditions where not favourable for infection. As this could bias the results, we performed the analysis on a subset of the data only considering the populations where at least one individual was CpDV positive l (per year/per population) and both Wolbachia groups co-existed. This way, we were able to assess wPip I and wPip IV groups effects on CpDV prevalence in same ecological conditions (i.e. the same probability of getting infected with CpDV for Cx. pipiens infected with wPipI and wPipIV).

CpDV prevalence in relation to Wolbachia groups

Main factor that affected the CpDV prevalence, specifically in the contact zone, were the Wolbachia groups (i.e. wPip-I versus wPip-IV) (glm, df=1, dev= 16.492, p< 0.001, Figure 2). Indeed, mosquitoes harbouring the wPip-IV were 34 % (RR=1.338; OR=2.791, 95% CI: 1.709-4.542; p<0.001) more likely to be CpDV positive. This difference was constant between the two years of sampling that were not significantly differing from one another (glm,df=1,dev=2.739,p= 0.097,Figure 2).

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CpDV load got as high as 82900 CpDV/host cell in only one sample from El Manar population (Figure 3A). This sample probably representing a peak of an acute infection was an outlier that we excluded from further analyses. Overall, the average load of CpDV per host cell of Cx. pipiens larvae was 3 (±0.911). However, CpDV load significantly differed between populations where larvae were sampled from (df=8, dev=127.27, p< 0.001, Figure 3A). This was mainly based on the samples from the El Manar population that showed the highest mean of CpDV/host cells (El Manar n=21, mean=14.881±3.925) while samples from Fontaine (n=61, mean=0.055±0.0180) populations showed really low amount.

Average Wolbachia amount per host cell was 0.356 (±0.052). The Wolbachia load was not different between Wolbachia groups or collection years (df=1, dev=0.162, p=0.359 and df=1, dev=0.571, p=0.085, respectively). However, Wolbachia load differed between populations (df=8, dev=3.147, p=0.038).

CpDV load differed with respect to Wolbachia load (df=1, dev=9.839, p< 0.001, Figure 3B), although there was no significant effect of wPip groups on the CpDV load (df=1, dev=0.256, p>0.05). More specifically, CpDV load was positively correlated with Wolbachia load (r 2 =0.11 p< 0.001, Figure 3C) CpDV-Ib (Figure 5). CpDV variants collected from the same populations, while showing some variabilities, generally clustered together (Figure 5). Especially the samples from Oued Melah, El Battan and Tabarka formed clades which were well-supported by bootstrap test (Figure 5). Samples from some other populations also grouped together while their bootstrap values were not as strong (e.g. Utique,

Pompe, Figure 5).

Both CpDV-Ia and CpDV-Ib were found in mosquitoes infected with either one of the wPip groups. However, their distribution correlated with the distribution of wPip groups in the region (Mantel, r= 0.34, p=0.02, Figure 6A-Figure 6B) while was independent from geographical distances between the different populations they have been collected from (Mantel test,r=0.17,p=0.13). These statistical results mean that wPip group variation might be the major ecological variable influencing CpDV-Ia and CpDV-Ib distributions in Cx. pipiens populations.

5.1.4! Discussion

We studied CpDV load, prevalence and diversification in a specific area, Northern Tunisia, where a rare stable contact zone between two incompatible Wolbachia strains have previously been revealed (Atyame et al., 2015). All the studied parameters suggested reciprocal interactions between the two endosymbionts in natural populations.

By studying the viral load in relation to

Wolbachia load, we observed a positive correlation between them independently from Wolbachia group infecting the host. Such positive correlation between symbionts' loads suggests a beneficial interaction between CpDV and Wolbachia. In most of Wolbachia-virus interactions studied so far, especially in Drosophila and mosquitoes, the load of the bacterial symbiont had generally been reported to negatively correlate with the virus load (Hussain et al., 2012;Lu et al., 2012;Frentiu et al., 2014;Martinez et al., 2017). However, a positive interaction between CpDV and Wolbachia have already been revealed in laboratory mosquito lines where mosquitoes infected with Wolbachia tended to exhibit higher CpDV loads than Tetracycline treated ones (Chapter 3). In addition to their effect on CpDV load, another type of Wolbachia and CpDV was also highlighted by differential CpDV prevalence in wPip-I carrying mosquitoes versus wPip-IV carrying ones. While in the whole study area prevalence of CpDV did not appear to be influenced by wPip groups, this influence was observed in a subset of this area, where both wPip groups (wPip-I and wPip-IV) and CpDV co-existed in same sampling sites. Indeed, by analyzing only the individuals living in the similar ecological conditions and certainly having the same probability to get infected by CpDV, we showed that wPip-IV was associated with higher prevalence of CpDV compared to wPip-I. This "beneficial" effect of one Wolbachia genotype on a virus prevalence can be seen, at first, as counterintuitive because of the growing evidence of Wolbachia antagonistic interactions with many RNA viruses such as DENV (Walker et al., 2011;Lu et al., 2012;Bian et al., 2013;Frentiu et al., 2014), Zika (Dutra et al., 2016) and WNV (Glaser and Meola, 2010;Micieli and Glaser, 2014). However, antagonistic Wolbachia-DNA virus (such as Densoviruses) interactions have never been shown. Contrarily, Wolbachia has been reported to enhance the DNA (Baculovirus) infection in lepidopteran hosts (Graham et al., 2012). While these differences of Wolbachia interference could be due to the differences between their interactions with either DNA or RNA viruses, they could also result from differences in the interactions between insect hosts and viruses. For instance, insects can be the principal host for viral multiplication for some viruses specialized on insects, such as the abovementioned DNA viruses baculoviruses and densoviruses, while they can act only as vectors for some others, such as abovementioned arboviruses DENV, WNV, Zika which are RNA viruses. Interestingly, contrary to the antagonistic interactions reported between Wolbachia and arboviruses, insect specific RNA viruses infec- tion rate and loads increased in Aedes aegypti stably transinfected with wMel compared to their Wolbachia free counterparts in natural populations (Amuzu et al., 2018).

Viruses generally studied for their pathogenicity although they can also be seen as endosymbionts of their hosts with interactions that might range from mutualism to antagonism (Roossinck, 2015;Roossinck and Bazán, 2017). To date CpDV's true nature with their hosts is unknown.

Initially they have been isolated during high mortality periods in laboratory Cx. pipiens larvae (Jousset et al., 2000). However, it was so far impossible to replicate this 'deadly' infection in laboratory conditions (personal communication) and densonucleosis have not been firmly observed in infected adults in laboratory lines (Chapter 3). CpDV, namely Helicoverpa armigera densovirus-1 is beneficial for their hosts as it increases their developmental rate, female fecundity, lifespan and the resistance of their host against microbiological agents of control, such as Bacillus thuringiensis and a pathogenic baculovirus (Xu et al., 2014). MDVs can also have negligible effects on survival of their hosts (Ren et al., 2014) even if many MDVs have been shown to affect their mosquito host adversely (Barreau et al., 1996;Ledermann et al., 2004;Rwegoshora and Kittayapong, 2004;Carlson et al., 2006;Becnel et al., 2007). Moreover, the virulence of the host-virus interactions can evolve through time. Indeed, repetitive challenges by a densovirus from generation to generation can result in a decrease of the pathogenicity of the virus due to a higher resistance of the host or to the emergence of a persistent infection. The latter has been described in Aedes aegypti mosquitoes in which survival increased progressively when they were challenged with AThDV infections for successive generations while the offspring were always as infected (Roekring et al., 2006). While it is difficult to interpret the beneficial interactions between CpDV and Wolbachia endosymbionts with limited knowledge on CpDV-host interactions, the higher prevalence of CpDV in mosquitoes harbouring wPip-IV could be a result of its improved efficient vertical transmission due to the bacterial symbiont (Chapter 3). Indeed, Altinli et al. (submitted, Chapter 3) demonstrated that wPip-IV infected mosquitoes had higher loads of CpDV in their ovaries compared to wPip-I. If CpDV is beneficial for the hosts, the fact that CpDV hitchhikes this wPip-IV strain could be favourable for both symbionts.

Studies on CpDV transmission in laboratory lines

By sequencing a polymorphic NS part of CpDV genome, we investigated their microevolution in geographically close locations. Three distinct CpDV clades (CpDV-I, CpDV-II, CpDV-III) had previously been described in a worldwide study of CpDV diversity (Chapter 2). Samples from Tunisia belonged to CpDV-I clade (Chapter 2). The different CpDV variants found in the area all seem to derive from a common ancestor that further diversified in two closely related but distinct clades as named CpDV-Ia and CpDV-Ib. Furthermore, most of the samples collected from same locations clustered together indicating population structuration and that CpDV diversification occurs at local scales. Within species diversity of MDVs had never been studied to such an extent, although several variants have been discovered from the two recognized MDV species Brevidensovirus, Dipteran brevidensovirus-I and Dipteran brevidensovirus-II (Cotmore et al., 2014). Similarly, the diversity of Ambidensovirus species was poorly studied. In fact it is limited to one study on Myzus persicae nicotinae densovirus (MpnDV) which revealed that the MpnDV were structured geographically with low level gene flow between locations, suggesting their high vertical transmission and putative co-evolution with their hosts (Song et al., 2016). The distribution of CpDV-Ia and CpDV-Ib clades, which diversified in Tunisian populations, was independent from geographical distances between the different populations but correlated with the distribution of wPip groups in the region (Figure 5). Assuming CpDV transmission is mainly vertical from mother to offspring, wPip could drive the distribution and evolution of CpDV in natural populations especially due to their CI properties. Indeed, CI can theoretically prevent gene flow between incompatible hosts which can result in the divergence of the CpDV. Given their co-vertical transmission it is possible that wPip and CpDV co-diverge in the natural Cx. pipiens populations. On the other hand, we have reported CpDV horizontal transmission at high rate in laboratory conditions (Chapter 3), if this is similar in natural conditions co-divergence of these endosymbionts together should be rare in natural populations. The fact that we observe this co-divergence could be related to the ability of CpDV to disperse to new populations. Mosquito densoviruses have been shown to be carried to new oviposition sites by adult mosquitoes but it is usually in really low levels and might not always be enough to infect other larvae horizontally (Wise de Valdez et al., 2010). Hence a CpDV is more likely to infect a new oviposition site via vertical transmission to the offspring of the infected female. Horizontal transmission then might play the major role for the infection of the larvae from uninfected females or females carrying a different CpDV. Therefore, it is expected that whichever variant infects the most females in a given area would also be found more in the larvae in the same location despite the occasional horizontal transmission of CpDV between larvae from different mothers. This could also explain why we do not observe a strict association between wPip-IV or wPip-I with two CpDV variants, but a strong association between their distribution despite the expected high horizontal transmission of CpDV.

Strong variations between different sampling locations in CpDV load and prevalence also suggested that there might be different variants circulating with different infectivity and that co-infections could occur. Although these questions were not in the scope of our study, we did not observe any general patterns between CpDV clades and CpDV load. For instance, CpDV load was lowest in Fontaine and highest in El Manar while both populations exhibited similar diversity of CpDV hosting variants from both clades. Similarly, co-in-fections by different CpDV variants could also result in high CpDV load. Although we did not quantify these co-infections in our study, we observed many double and triple peaks in CpDV sequences of the samples from El Manar population, where we also observed high viral loads.

Both MDVs (Buchatski! et al., 1987;Buchatsky, 1989;Afanasiev et al., 1994;Carlson et al., 2006;Ren and Rasgon, 2010;Johnson and Rasgon, 2018) and Wolbachia (Laven, 1967;Atyame, Pasteur, et al., 2011;Hoffmann et al., 2011;McGraw and O'Neill, 2013;Frentiu et al., 2014) are considered as promising vector control tools. Although more studies have been carried on the effects of Wolbachia on viral infections in the natural populations, these are generally focused on RNA viruses, especially arboviruses, and our knowledge on insect specific viruses is very limited (Amuzu et al., 2018). The prevalence of DNA viruses, especially densoviruses, highlights their importance on their hosts and possibly on their host' microbiota. Our study pointed out the interactions between Wolbachia and CpDV in natural Cx.

pipiens populations, future studies are warranted to investigate these complex tripartite interactions between insect-specific viruses and Wolbachia in other mosquitoes. Indeed, especially in field releases of Wolbachia trans-infected mosquitoes, these tripartite interactions could be crucial for the dynamics of important vectors, their endosymbionts and the viruses they transmit, in natural populations.

Overall our results suggested that CpDV-host interactions could be diverse in nature and CpDV-Wolbachia interactions can influence the outcomes of CpDV infection for the host. The study of the arthropod viruses in the context of symbiosisrather than focusing only on their pathogenicityboth in natural populations and experimental settings could lead to a better understanding of the evolution of mutualism and pathogenicity of viruses, and their interactions with the rest of the microbiota.

6.1! Summary of Results

During my PhD project we have shown:

•! A high prevalence and high diversity of a mosquito densovirus, CpDV in natural host populations (Chapter 2)

•! That the CpDV evolution was shaped by recombination and purifying selection (Chapter

2)

•! Highly significant migration routes and the lack of a geographical structure in CpDV diversity which suggested their efficient dispersal worldwide (Chapter 2)

•! Persistent CpDV infection in apparently healthy laboratory lines (Chapter 3)

•! CpDV vertical transmission and their presence in oocytes along with Wolbachia (Chapter 3)

•! Effect of host microbiota including Wolbachia on the amount of CpDV in the ovaries and on CpDV vertical transmission (Chapter 3)

•! High prevalence and diversity of both wPip and CpDV in Turkey, a critical region for vector control efforts (Chapter 4)

•

6.2! Discussion

The aim of this PhD thesis was first to study the prevalence and diversity of a unique mosquito densovirus, CpDV, in natural populations of Cx.

pipiens mosquitoes. Once their relevance for Cx. pipiens ecology was established, we have further investigated interactions with their hosts and host's endosymbiont Wolbachia both in laboratory lines and natural populations. Mosquito densoviruses (MDV), relatively more studied than many other arthropod viruses, are still rarely studied in an ecological and evolutionary context to date. Indeed, the research around MDVs mainly focused on their pathogenicity and/or their application as a vector control tool (Buchatski! et al., 1987;Carlson et al., 2006;Johnson and Rasgon, 2018). They were nevertheless thought to be widespread in natural populations as their presence in mosquito larvae and adults as well as in insectivorous bat faeces has been reported (Kittayapong et al., 1999a;Rwegoshora et al., 2000;Ma et al., 2011;Ng et al., 2011;Ge et al., 2012). Only for one MDV general high prevalence in natural populations and the effect of abiotic factors on this prevalence has been shown (AaeDV Thai strain) (Kittayapong et al., 1999a;Rwegoshora et al., 2000). CpDV, given their fundamental differences from the rest of MDVs, could be expected to have different life cycles, host interactions and transmission modes. Their genomic differences from other MDVs could also suggest that they have been acquired from another species. Using samples that have been collected at different times worldwide, we have revealed for the first time that a MDV can be prevalent in such a large scale and persistent through the years in Cx. pipiens populations (Chapter 2).

While this prevalence was not as high as it could be observed for some endosymbionts, such as Wolbachia wPip, it highlighted their importance for Cx. pipiens ecology.

By sequencing some of the CpDV variants found in natural populations, we sought to understand their diversity, evolution, dispersal and population dynamics (Chapter 2). Knowledge of within species diversity of densoviruses was limited at best to couple of variants that are sequenced (Cotmore et al., 2014), except one study of MpnDV in China (Song et al., 2016). Authors indeed reported a high diversity of MpnDV which often clustered accordingly to geographically defined aphid host populations (Song et al., 2016). Using methods that are now widely used for fast evolving human viruses such as Zika and influenza [START_REF] Baillie | Evolutionary Dynamics of Local Pandemic H1N1 / 2009 Influenza Virus Lineages Revealed by Whole-Genome Analysis[END_REF][START_REF] Thézé | Genomic Epidemiology Reconstructs the Introduction and Spread of Zika Virus in Central America and Mexico[END_REF], we have also observed a high diversity of CpDV clustered in three clades and their efficient dispersal worldwide. Parvoviruses usually have been reported to have a high substitution rate making their evolution fast and allowing the use of similar methods to estimate the Most Recent Common Ancestor (tMRCA) and population dynamics (Shackelton et al., 2005;Firth et al., 2009;Streck et al., 2011;[START_REF] Lin | Phylodynamic and genetic diversity of canine parvovirus type 2c in Taiwan[END_REF][START_REF] Grecco | Inter-and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America[END_REF]. We demonstrated a prevailing First approach was failed as these filtered homogenates were toxic in higher volumes where we reached an adequate amount of CpDV to experimentally infect other larvae (Chapter 3). Trials using second approach were also impeded as we faced difficulties to culture cells highly infected with synthetic CpDV and most importantly as our mosquito lines were already persistently infected with CpDV (Chapter 3). This persistence suggested they might be vertically transmitted along with their hosts' endosymbiont Wolbachia. MDVs have been known to be transmitted vertically but their interactions with the rest of the microbiota are not yet investigated (O'Neill et al., 1995;Barreau et al., 1997;Kittayapong et al., 1999b;Rwegoshora and Kittayapong, 2004;Suchman et al., 2006;[START_REF] Barik | Factors influencing infection and transmission of Anopheles gambiae densovirus (AgDNV) in mosquitoes[END_REF]. Moreover, as CpDV is genetically closer to ambidensoviruses than brevidensoviruses (other MDVs), whether their biology would be similar to ambidensoviruses or MDVs was not clear. Investigating the vertical transmission in laboratory lines naturally infected with CpDV, we have shown that i) CpDV was vertically transmitted in low amounts, ii) they were inside the oocytes (where also Wolbachia is) and not just on the surface of the eggs, iii) microbiota and Wolbachia in particular seem to have a role on the amount and transmission of CpDV (Chapter 3). Although we were not able to quantify properly due to the lack of CpDV free mosquito lines, horizontal transmission seems to be high given the low amount of vertical transmission to the first instar larvae and high amount of prevalence in the emerged adult females (Chapter 3).

Transmission modes of endosymbionts could

give insights about their evolution as to whether they would evolve towards mutualism or antagonism. In our lines, CpDV was observed to cause Chapter 6 -Discussion 125 densonucleosis and was possibly the reason of high larval mortality when it was first isolated in around 1994 (Jousset et al., 2000). Almost two decades later, CpDV seems to persist in low amounts and be vertically transmitted in our seemingly healthy laboratory lines. This shift in virulence suggests a change in CpDV over the years which made the new variant less pathogenic to their hosts. Another result that might be in support of this hypothesis, came with the improvement of the transinfection and CpDV isolation methods which allowed us to use a synthetic CpDV and a natural isolate from our lines, Ich13 isolate, to perform experimental infections (Chapter 3). While the results are preliminary (only one replicate), they suggested that synthetic CpDV (that is designed based on reference genome) was pathogenic while Ich13 isolate (current variant in the laboratory lines) seemed to improve host survival. Moreover, CpDV sequences from the laboratory lines collected between 1990-2018 indicated a shift from CpDV 2 clade to CpDV 1 in a short period of time between 1994 (reference genome) to 2001 (Slab 2001)(Chapter 2). This change in CpDV could be caused by their evolution with the host, as it is possible that a variant with lower pathogenicity, or even beneficial for the host, emerged and became widespread in the insectary populations. On the other hand it is also possible that another CpDV variant arrived to the insectary with the establishment of a new Cx. pipiens line collected from natural populations and outcompeted the pathogenic CpDV (reference genome). The results of recombination analysis suggest that the truth could be lying somewhere in between these two hypotheses (Chapter 2). Indeed, a new quitoes was unknown (Ergunay et al., 2011(Ergunay et al., , 2013;;Erdem et al., 2014;Ergunay et al., 2015;Gunay et al., 2015;Failloux et al., 2017;Öncü et al., 2018).

We have revealed the diversity of wPip groups and their cytoplasmic incompatibility patterns, and highlighted the potential of wPip-IV harbouring males as a vector control to sterilise local Cx. In Northern Tunisia wPip diversity and their CI patterns have already been studied in detail by our team EVAS at ISEM (Atyame et al., 2015).

Authors discovered a contact zone where mosquitoes harbouring different wPip groups, wPip group I and IV, co-existed. Their CI patterns predicted the invasion of the contact zone by the strain inducing uni-directional CI, namely wPip-I, in only couple of generations although the contact zone was stable over seven years. Using a part of these samples, we have investigated the prevalence of CpDV in the same area and the effect of different wPip groups on this prevalence (Chapter 5). Our initial hypothesis was that a pathogen protection conferred by one of the wPip strains, in this case wPip-IV, could possibly explain why wPip-I could not invade the whole host populations in the contact zone. Conversely, we demonstrated that CpDV was more likely to infect mosquitoes harbouring wPip-IV. While it is difficult to interpret these interactions without knowing the nature of the CpDV-host interactions, this higher prevalence of CpDV in mosquitoes harbouring wPip-IV could be a result of efficient vertical transmission, rather than a more efficient protection by wPip-I. In support of this hypothesis we have observed, in the laboratory (Chapter 3), that wPip-IV infected mosquitoes had higher amount of CpDV in their ovaries compared to wPip-I infected mosquitoes.

Indeed, the density of CpDV seemed to be controlled by a cytoplasmic factor, e.g. Wolbachia, rather than host genetic background (Chapter 3).

However due to the high variance in vertical transmission rate at individual females' level, we were not able to show such a difference in vertical transmission between mosquitoes harbouring different wPip groups (Chapter 3).

In the field, while the prevalence was affected by the wPip group, there was no effect of this group on the CpDV density (Chapter 5). However, the density of CpDV was positively correlated with wPip density (Chapter 5). This suggested that wPip could enhance CpDV infections in natural populations. wPip protection against West Nile Virus (RNA virus) by reducing the virus density has previously been reported (Glaser and Meola, 2010;Micieli and Glaser, 2014). On the other hand Wolbachia appear to enhance DNA virus infections so far which was consistent with our results (Teixeira et al., 2008;Graham et al., 2012). The interactions between Wolbachia and DNA viruses could be different as these viruses are likely to affect their hosts differently. It is possible that arboviruses vectored by mosquitoes have less of an impact on mosquito biology compared to mosquitospecific viruses. Indeed recently, Wolbachia has been reported to enhance an insect specific RNA virus (Anphe virus) in Ae. aegypti cells (Parry and Asgari, 2018). Furthermore, natural populations of Ae. aegypti stably transinfected with Wolbachia displayed higher insect-specific RNA virus infection rates and loads compared to their Wolbachia-free counterparts (Amuzu et al., 2018). These CpDV- Nevertheless, they grouped in two statistically well supported distinct groups (Chapter 5). Mapping these two distinct groups revealed their similar distribution with wPip groups (Chapter 5). Given their co-vertical transmission it is possible that these two endosymbionts co-diverge and as a result their distributions would be similar in natural populations. On the other hand, in the insectary conditions, we have observed a high rate of CpDV horizontal transmission (Chapter 3), if this is similar in natural conditions it would be difficult to observe the co-divergence of these endosymbionts together. Similar distribution of wPip and CpDV could be explained in this case by the way CpDV disperses between different water bodies. Indeed, we know that CpDV can be vertically transmitted to the larvae in a new location where infected females lay eggs (Chapter 3). Although adult mosquitoes have also been shown to contaminate a water body with other MDVs, it is usually in really low levels and often not enough to infect the larvae (Wise de Valdez et al., 2010). Hence it is possible that whichever variant infects the most females in that area would also be found more in the larvae there despite the occasional horizontal transmission of CpDV between larvae from different mothers. This could also explain why we do not observe a strict association between wPip group IV and wPip I with the two CpDV variants while the association is clearer between geographical distribution of Although my PhD focused mainly on CpDV and CpDV-Wolbachia interactions, endosymbiont interactions could be much more intricate in nature than only tripartite interactions. It is possible that the rest of the microbiota also affects CpDV-Wolbachia interactions. For instance, diversity and presence of insect-specific RNA viruses in natural populations of mosquitoes have been shown in the recent years. Nevertheless, their effects on their hosts and host microbiota are still poorly studied. Such a Cx. pipiens associated RNA virus have recently been discovered in Northern Tunisia [START_REF] Bigot | Discovery of Culex pipiens associated tunisia virus: a new ssRNA(+) virus representing a new insect associated virus family[END_REF] offering the possibility to study a DNA virus-RNA virus-Wolbachia-Culex pipiens interactions. We could not investigate this question in the Chapter 5 as it was not possible to take RNA viruses into account in our study due to the use of readily extracted DNA samples. Future studies could possibly take these multi-partner species interactions in environments into account using their co-occurrence and their correlation patterns as it is now being used for bacterial species in microbiota studies.

Wolbachia

MDVs have long been investigated due to their pathogenicity and their narrow host range as a vector control strategy. There are two methods i) their direct use to kill mosquito larvae ii) their use as transducing agents (Buchatski! et al., 1987;Afanasiev et al., 1994;[START_REF] Ward | Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae[END_REF]Carlson et al., 2006;Ren and Rasgon, 2010;Suzuki et al., 2014). First method consists of the application of highly pathogenic MDV directly to the larval habitats (Carlson et al., 2006;Johnson and Rasgon, 2018). High mortality during larval stages could indeed reduce mosquito populations but could also lead to rapid resistant evolution due to high selection pressure caused by MDV (Johnson and Rasgon, 2018). It is argued that the second method could be evolution proof, as the real target would be older mosquitoes which are more likely to carry arthropod borne viruses (Ren and Rasgon, 2010). Sublethal MDVs would not cause as high selective pressure as a pathogenic MDV as they would allow larvae to survive and young adults to reproduce (Ren and Rasgon, 2010). While both methods are promising in terms of mosquito-borne disease control efforts, they are still hindered by the lack of knowledge on the basic biology and ecology of MDVs, and the lack of MDVs specific to all medically important mosquitoes (Johnson and Rasgon, 2018). In future, CpDV, or the knowledge on their effects on host ecology and evolution, could also prove useful for these control strategies. Especially since they might be used as transducing agent due to their persistence in mosquito populations and low pathogenicity against Cx. pipiens (s.l.).

In this context, but also to understand CpDV biology better, it would be useful to study their host specificity. Being similar to the other MDVs in terms of hosts they infect while being genetically closer to lepidopteran ambidensoviruses (Baquerizo-Audiot et al., 2009;Cotmore et al., 2014), it might be possible that they infect both of these hosts. Furthermore a genetically similar densovirus to CpDV has been recently discovered in Drosophila (Kapun et al., 2018). Transinfection of cell cultures with CpDV have previously shown that they were able to amplify in Anopheles and Culex cells while in the Aedes, Drosophila and Spodoptera cells they did not amplify (Jousset et al., 2000). Our preliminary results also showed that CpDV did not cause any mortality in Ae. albopictus larvae (Chapter 3). Future studies on Anopheles mosquitoes, lepidopterans as well as Drosophila could shed more light to host specificity of CpDV.

Virus discovery have long depended on the observation of outbreaks and the research to understand their pathogenicity depended on their culture. The advances of sequencing technology and molecular biology techniques allowed researchers to discover the ubiquity and the diversity of viruses. Despite these advances, virus discovery mainly focused on vertebrate viruses. Arthropods with their ancient evolutionary history represent 80% of animal diversity on our planet. Assuming a correlation between host and virus diversity, this might mean that we are still oblivious to most of the existing virus diversity. Furthermore, even the known diversity of these viruses is poorly studied in term of virus ecology. The ubiquity of endosymbiosis in arthropods and the ability of arthropods to support (sometimes multiple) symbionts suggest that their interactions with their viruses might be of symbiotic nature, intimate long-term associations with outcomes ranging from mutualistic to antagonistic.

In this context, this new approach to study viruses, while still presents many challenges could answer broad range of questions about the ecology and evolution of host-microbiota interactions. Densoviruses, as ancient and diverse as their arthropod hosts, are ubiquitous in nature and have small genomes that are easy to manipulate with basic cloning techniques. Hence, they could be good models to study host-virus-bacteria interactions. In the intersection of these many novel areas, our results, while may have raised more questions than they answered, suggested that CpDV-Cx.pipiens-Wolbachia system could be a good model system to study the ecological interactions between these partners and their evolution together both in the nature and in laboratory conditions. (Buchatski! et al., 1987 ;Carlson et al., 2006 ;Johnson et Rasgon, 2018). On pense néanmoins qu'ils sont largement répandus dans les populations naturelles car leur présence dans les larves de moustiques et les adultes ainsi que dans les fèces de chauves-souris insectivores a été signalée (Kittayapong et al., 1999a ;Rwegoshora et al, 2000 ;Ma et al, 2011 ;Ng et al, 2011 ;Ge et al., 2012). Cependant, seule une étude avait jusqu'à lors décrit la prévalence élevée d'un MDV (la souche AaeDV Thai) dans les populations naturelles et l'effet de facteurs abiotiques sur cette prévalence (Kittayapong et al., 1999a ;Rwegoshora et al., 2000). (Shackelton et al., 2005 ;Firth et al., 2009 ;Streck et al., 2011 ;[START_REF] Lin | Phylodynamic and genetic diversity of canine parvovirus type 2c in Taiwan[END_REF][START_REF] Grecco | Inter-and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America[END_REF] 'Neill et al., 1995 ;Barreau et al., 1997 ;Kittayapong et al, 1999b ;Rwegoshora et Kittayapong, 2004 ;Suchman et al, 2006 ;[START_REF] Barik | Factors influencing infection and transmission of Anopheles gambiae densovirus (AgDNV) in mosquitoes[END_REF]. En étudiant pour la première fois la trans- (Ergunay et al., 2011(Ergunay et al., , 2013(Ergunay et al., , 2015 ; ;Erdem et al, 2014 ;Gunay et al, 2015 ;Failloux et al, 2017 ;Öncü et al, 2018). Cependant, la diversité des chatski! et al., 1987 ;Afanasiev et al., 1994 ;[START_REF] Ward | Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae[END_REF]Carlson et al, 2006 ;Ren et Rasgon, 2010 ;Suzuki et al., 2014). La première méthode consiste à appliquer le MDV pathogène directement dans les habitats larvaires (Carlson et al., 2006 ;Johnson et Rasgon, 2018). Une mortalité élevée au stade larvaire pourrait en effet réduire les populations de moustiques, mais pourrait aussi entraîner une évolution rapide de la résistance en raison des fortes pressions de sélection exercées par des MDV épandus à hautes doses (Johnson et Rasgon, 2018). Certains auteurs soutiennent, qu'au contraire, la deuxième méthode pourrait « résister à l'épreuve de l'évolution », car la véritable cible serait les moustiques plus âgés, qui sont plus susceptibles de transporter des arbovirus et de les transmettre (Ren et Rasgon, 2010). Les MDV fai- Mine ALTINLI submitted the doctoral thesis dealing with the evolution and ecology of a tripartite interaction including an insect, a bacterial symbiont and ssDNA viruses. Given that bacterial symbioses are ubiquitous in insects and that those arthropods are often infected with viruses, Mine ALTINLI asked for a very interesting, innovative and original research questions: (1) are there some interactions between viruses and bacterial symbionts within insect individuals/populations and ( 2) how may evolve an ecological trio composed of an arthropod, a bacterial symbiont and viruses. Viruses have long been considered as microbes causing mortality in infected insect populations. However, interactions between insect and viruses might have be of symbiotic nature, intimidate long-term associations with effects on host individuals ranging from mutualistic to antagonistic. In this context, the PhD work focused on prevalence and diversity of densoviruses in the Culex pipiens mosquito species (CpDV) and their interactions with the symbiotic bacteria Wolbachia harbored by mosquitos. The topic of the thesis is highly relevant for all studies on evolutionary ecology of interspecific interactions, a very important field of biological research.

Quality of the manuscript

The PhD manuscript is mainly written in English, and the results are presented in the form of chapters written as papers that have been or will be submitted/accepted. The report, of high quality, consists of seven chapters, covers 158 pages, including the introduction, four papers, the discussion and a French summary of the thesis. The overall style is clear and the extent of the thesis corresponds to its significance and amount of new knowledge it details, especially considering the fact that results of the thesis are published or submitted in renowned journals.

Introduction section

The first part, the Introduction, gives a good literature review covering the essential background of the thesis and details biological models studied during three years. The main goals of the PhD work are also described but too succinctly. Mine ALTINLI should have detailed the research questions included in her thesis work and the link between those issues.

Results section

The result section contains four subsequent parts, each presenting an article written by the candidate as first author. The first manuscript, not submitted, presents investigation of the CpDV prevalence in natural populations using specific PCR-based diagnostic test and considering both past and contemporary samples. Also, by sequencing amplicons from virus genome, Mine ALTINLI analysed dispersal of CpDVs and their evolution within mosquito's populations. A high prevalence and high diversity of CpDVs were found in studied populations. Highly significant migration paths and lack of geographical structure in CpDVs diversity suggest efficient dispersal worldwide.

Given high prevalence and high dispersal of CpDV viruses in natural populations, second part asked for two main factors influencing infection dynamics of densoviruses within C. pipiens populations: (1) the CpDVs transmission routes and ( 2) nature of their interactions with mosquito hosts. If the effect of densoviruses on host fitness is shortly presented in an Appendix (Appendix A, pp 75-77), the vertical transmission of CpDV is detailed in a paper submitted to Environmental Microbiology. Results show that CpDV are vertically transmitted via transovarian path along with the endosymbiont Wolbachia. Rate of transmission is highly variable at the individual level but not at the mosquito line one and interestingly, is marginally lowest when hosts are uninfected with Wolbachia. Also, the density of CpDV in host's ovaries would be controlled by maternally transmitted cytoplasmic factors: Wolbachia strains would affect density of CpDV.

The third Result's part present studies focused on Wolbachia and CpDV prevalence and diversity in Turkey. Firstly, a published paper details the study focusing on the Wolbachia diversity and cytoplasmic incompatibility in C. pipiens populations in Turkey. Within this country, all individuals were infected with Wolbachia from three different groups with varying frequencies (I, II and IV). The most widespread groups, I and II, induce reciprocal computability between hosts and this would explain their coexistence. In addition, contrary to expectations, coexistence of bidirectional incompatability inducing strains (II and IV) has been observed in one site. Interestingly, mosquito males infected with Wolbachia of group IV may be used to sterilise mosquito females infected with Wolbachia from the two other groups. Secondly, Mine ALTINLI details a study that focused on the prevalence of CpDV and diversity in natural populations of C. pipiens in Turkey. About 60% of individuals were infected with the CpDV, an infection rate that varies between mosquito life stages and host population locations. The Wolbachia group harboured by the mosquitoes has not effect on densoviruses prevalence. CpDV diversity has been found in Western-North Turkey only and there is no clear link between CpDV and Wolbachia diversity.

Finally, to test whether Wolbachia could be a major determinant of CpDV dynamics in mosquito populations, the fourth Result's part present a very interesting study focused on the influence of the bacterial endosymbiont Wolbachia on prevalence, viral load and diversity of CpDV. Reciprocal interactions between those endosymbionts are suggested from results: (1) Wolbachia groups influence prevalence of CpDV in Tunisian mosquito populations but not their load; (2) there is a positive correlation between CpDV and Wolbachia loads; (3) group variation in Wolbachia would influence distribution of CpDV variants in C. pipiens populations, a distribution covariation explained by co-vertical transmission of these microbial endosymbionts.

Discussion section

In the final chapter, a general conclusion and perspectives are offered. In the discussion section of each manuscript, plus this general discussion, Mine ALTINLI clearly demonstrates by her writing that she has thought deeply about the results and attempts to interpret them in regards to the work done by others. This section is very much appreciated and summarises with clarity results obtained. Besides, areas of further research suggested by the findings are also clearly proposed and there clearly remains much to be done in this very interesting field of research. Without any doubt, this thesis will incite further studies on other tripartite interaction between arthropods, bacterial symbionts and viruses.

Recommendation

After carefully reading and studying Mine ALTINLI's thesis, I am fully convinced it represents a significant and original contribution to the scientific field it deals with. The thesis contains original results and ideas, and therefore is a quite important publication. Mine ALTINLI does highlight the novel findings of her studies, and at times indicates their limitations, providing new relevant perspectives. Overall, Mine ALTINLI has demonstrated excellent scientific rigour in her writing and the report shows that Mine ALTINLI masters a number of field, experimental and analytical techniques and academic fields for answering addressed issues, proving to be skilled scientist.

En conclusion, ma recommandation pour l'Université de Montpellier est d'accepter sans aucune réserve que la thèse de Mine ALTINLI soit soutenue.

Rennes, 28 th October 2018 ! ! !!!!!!!!!!!!"#$#%&'()!)%!"#$*+&'()!,)-!.$-)/%)-!0)/%)(1-! !!!!!!!!!2#341%)+)$%!,)-!5414-&%)-!)%!.$-)/%)-!0)/%)(1-! !"#$%&%'%(%

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Ecology and Evolution of Culex pipiens -Wolbachia -Densovirus Tripartite Interactions

Virus discovery has long depended on the observation of outbreaks, and the research to understand their pathogenicity depended on their culture. The advances of sequencing technology and molecular biology techniques allowed researchers to discover the ubiquity and the diversity of viruses. Despite these advances, virus discovery remains mainly focused on vertebrate viruses. Furthermore, even the known diversity of these viruses is poorly studied in term of virus ecology. Arthropods with their ancient evolutionary history represent 80% of animal diversity on our planet. The ubiquity of endosymbiosis in arthropods and their ability to support (sometimes multiple) symbionts suggest that their interactions with their viruses might be of symbiotic nature, intimate long-term associations with outcomes ranging from mutualistic to antagonistic. In this context, this PhD dissertation focuses on a mosquito densovirus, Culex pipiens densovirus (CpDV), to investigate their prevalence and diversity in natural populations of Culex pipiens mosquitoes and their interactions with endosymbiotic bacteria Wolbachia in a natural host-bacteria-virus system (Cx. pipiens -Wolbachia -CpDV) both in natural populations and laboratory colonies. We reveal a high prevalence and diversity of CpDV in worldwide Cx. pipiens populations. Moreover, CpDV persistently infects laboratory lines and are vertically transmitted along with their hosts' endosymbiont Wolbachia. We reveal high CpDV prevalence and diversity in Turkey and Tunisia and further focus on these two countries to investigate Wolbachia-CpDV interactions in natural populations. In Tunisia, in a narrow contact zone where two genetically distinct Wolbachia groups coexist, different Wolbachia groups influences CpDV prevalence and diversity. Moreover, we show a positive correlation between Wolbachia density and CpDV density. Overall, our results suggests that CpDV-Cx.pipiens -Wolbachia system is a good model system to study the ecological interactions between these partners and their evolution together both in the nature and in laboratory conditions. Densoviruses with their diversity and long-term evolution with their hosts, and their easily manipulated small genomes, are good models to study complex hostvirus-bacteria interactions as well as virus ecology and evolution in a symbiotic context.

Écologie et évolution des interactions tripartites entre Culex pipiens, Wolbachia et les Densovirus

  .................................................................................................. 7 1.4.2 DENSOVIRUSES ............................................................................................................... 7

18 1

 18 3 CULEX PIPIENS DENSOVIRUS (CPDV)..............................................................................14 1.5 CULEX PIPIENS -WOLBACHIA MODEL SYSTEM ............................................................ 15 1.5.1 WOLBACHIA ..................................................................................................................15 1.5.1.1 Wolbachia Pathogen Inference in Mosquitoes ........................................................ 17 1.5.2 WOLBACHIA IN CULEX PIPIENS MOSQUITOES ...................................................................

Figure 1 .

 1 Figure 1. Phylogenetic tree of Densovirus Sub-Family. Densovirus sub-family consists of 5 genera infecting mainly insects from five orders (Lepidoptera, Hemiptera, Blattodea, Diptera, Hymenoptera) but also Crustaceans and Echinoderms, endogenous viral elements and viral sequences found in public transcriptomics and genomics databases are also added. Host species are given as images.

1 L4>80B0

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Figure 2 .

 2 Figure 2. Differences between Ambidensovirus and Brevidensovirus genome structures. Brevidensovirus (A) have smallest genome among densoviruses, about 4kb length. Their genome is monosense, only transcribed from one strand. They lack PLA2 domain that is conserved in most of the Parvoviridae family including Ambidensoviruses. Their DNA strands terminate with hairpin structures but lack inverted terminal repeats. Ambidensoviruses (B) have an ambisense genome their ORF of their NS genes are located on plus strand while ORF of VP genes are located on the minus strand. Their capsids encapsidate equal amounts of both strands. Their genome size is larger compared to Brevidensovirus genus (about 6kb). Their genomes are terminated with inverted terminal repeats that forms a hairpin structure. Conserved SF3 domain in NS region and PLA2 domain in VP region are indicated with pink band orange band respectively. Arrows indicate the direction of transcription. diversity grouped in the Brevidensovirus genus has been isolated from mosquitoes and they are thought to be species specific. In fact, all mosquito specific densoviruses (MDVs) belong to this genus except Culex pipiens densovirus, which infects mosquitoes but belong to Ambidensovirus genus (Figure 1, Culex pipiens densovirus discussed in detail later in section 1.4.3).

  logical to try infer the general life cycle of MDVs in nature based on the experimental infections there are still a lot of unknown in terms of their interac-

Figure 3 Life

 3 Figure 3 Life Cycle of Mosquito Densoviruses (MDVs): Infected females can vertically transmit MDV to their offspring(A). These infected larvae could either survive and become adults or die and in both cases excrete virions to the water body they live in (B). Uninfected larvae in the same water body can get infected horizontally(C). Surviving larvae can become adults and carry MDVS to other oviposition sites (D). MDVs can shorten the lifespan of the adults (E) and be transmitted venereally (F). MDV virions can stay active in nature for long time (G).

Figure 4

 4 Figure 4 CpDV genome. CpDV genome encodes for two types of proteins: non-structural NS and structural VP. ORFs of NS and VP proteins are situated on different single strands, which makes CpDV genome ambisense. They have 3 putative NS proteins. NS3, NS2, NS1 (ORFs are shown as blue boxes). ORFs of NS1 and NS2 overlap on the same strain (indicated with dashed lines), and split into two ORFs (NS1-NS1', NS2-NS2'). Two ORFs for which homologous proteins were not found, are shown as gray arrows in NS and VP regions. CpDV genome terminates with inverted terminal repeats (ITR, identical sequences repeated on 3' terminal of both strands shown in blue and 5' terminal shown in yellow) that fold into hairpin structures. Conserved SF3 helicase domain consisting of Walker A and B boxes as part of within NS1 is shown in pink (ATPase motif). Conserved PLA2 and Ca ++ binding motifs in VP have been highlighted as orange and green bands respectively. CpDV has four putative VP proteins (ORFs are shown purple boxes) that are encoded by leaky scanning from single VP ORF, although their location is not certain.

Figure 5

 5 Figure 5 Reproduction Manipulation by Wolbachia. In arthropods, Wolbachia induce four different reproductive phenotypes. Feminization leads infected genetic males to develop as females. By inducing Parthenogenesis Wolbachia can exclude males from reproduction. Male killing results in higher survival of infected female siblings. Cytoplasmic incompatibility (CI) prevents uninfected females to reproduce with infected males (A) while infected females can reproduce with both infected (B) and uninfected males (C). More complex CI patterns are observed in Cx. pipiens complex mosquitoes. Mosquitoes with the same or compatible Wolbachia strain can reproduce together (D). Uni-directional incompatibility occurs when mosquitoes harbouring different Wolbachia strains are compatible in one cross while reciprocal cross results in embryonic death (E). Bi-directional incompatibility is when both of the reciprocal crosses between mosquitoes infected with different Wolbachia strains are incompatible (F).

  Wolbachia natural infections are common in mosquitoes including several arbovirus vectors such as

Figure 6

 6 Figure 6 Possible CpDV-Wolbachia-Cx. pipiens interactions. CpDV and Wolbachia can affect each other's density either by competing for resources or modulating host immune system. They can both cause positive or negative fitness effects on their hosts and beneficial variants could get selected. Wolbachia might also influence CpDV transmission and infection outcomes.

ter3) .

 . Due to the persistent CpDV infection in our mosquito lines, it was difficult to assess the horizontal transmission of CpDV. Therefore, I have mainly focused on their vertical transmission. As Wolbachia is also vertically transmitted I have further investigated whether they might interact during their journey from mother to offspring(Chap-ter3). Finally, I explored Wolbachia-CpDV interactions in natural Cx. pipiens populations by assessing their prevalence and microevolution together (Chapter4 and 5). First, I have investigated the prevalence and diversity of wPip and CpDV in Turkey where the Cx. pipiens diversity is high as populations from Europe, Middle East and Africa are likely to meet (Chapter 4). Secondly, I have focused on a previously determined contact zone where two wPip strains co-exist in Northern Tunisia to investigate the interactions between wPip and CpDV in Cx. pipiens populations (Chapter 5).

  Audiot et al., 2009). Four different VP proteins have been identified for CpDV and thought to be encoded by a single ORF via leaky scanning (Figure1)(Baquerizo-Audiot et al., 2009). To detect the presence of CpDV and to further infer the within species diversity, we designed CpDV specific primers in NS2, NS1', VP regions in a maximum number of samples coming from diverse mosquito populations worldwide. To do this we used an alignment of the reference genome(Baquerizo-Audiot et al., 2009) with a partial consensus genome obtained by next-generation sequencing of CpDV from six different mosquito populations (data not shown).

Figure 1 CpDV

 1 Figure 1 CpDV Genome encodes for two types of proteins: non-structural NS and structural VP. ORFs of NS and VP proteins are situated on different single strands, which makes CpDV genome ambisense. CpDV hs 3 putative NS proteins. NS3, NS2, NS1 (ORFs are shown as blue boxes). Conserved SF3 helicase domain consisting of Walker A and B boxes as part of within NS1 is shown in pink (ATPase motif). Conserved PLA2 in VP have been highlighted in orange CpDV has four putative. Location of primers used in semi-nested PCRs have been shown as arrows. Red part has been amplified for the diagnostic test and green parts are sequenced to infer CpDV diversity.

  only 29 localities out of 136, no CpDV was detected. No apparent spatial or temporal structuration of CpDV prevalence were observed.

Figure 2

 2 Figure 2 Worldwide prevalence of CpDV in Culex pipiens s.l. populations A total of 2843 individual larvae samples collected from 136 different locations worldwide have been tested with a PCR detection test using conserved Ns2 gene in CpDV. As

/

Figure 3

 3 Figure 3 Phylogenetic tree of CpDV variants. Midpoint rooted Maximum Likelihood tree (RaxML) was built using NS1-VP concatenated sequence data set after the exclusion of recombinant sequences. 100 Bootstraps have been made to test the statistical significance of the nodes and nodes that were supported with more than 60% are indicated. There were three well-supported clades within CpDV; CpDV-1, CpDV-2, CpDV-3. CpDV-3 was represented by only one sample from Beijing while the other clades were more populated and diverse.

  purifying (negative) and diversifying (positive) selection putatively acting on both NS1' ORF and VP ORF have been tested. Both pervasive selection (selection detected across the whole phylogeny) and episodic selection (selection detected only at a subset of branches) were also investigated. Within NS1' 23 individual sites and within VP 22 individual sites were subjected to pervasive purifying selection (Appendix Figure 2 and 3 respectively, FEL, p<0.05). No positive selection was detected at any individual sites of NS1'. In VP only one site was subjected to episodic positive selection which only occurred on the branch between Slab 1990 and Reference and caused by one non-synonymous nucleotide change in site 342 (MEME, p=0.01, Glycine to Threonine change, Appendix Figure 3). Contrarily there was no pervasive diversifying selection detected. Additionally, both Walker A and Walker B sites were fully conserved as well as putative start and termination codons of other predicted ORFs within NS1'. Similarly, in VP, Ca++ binding or PLA2 motifs were highly conserved: there was no nucleotide change in Ca++ binding motif while there was only one synonymous nucleotide change in Slab 1990 for PLA2 motif.

Figure 4

 4 Figure 4 Phylodynamics of CpDV. Bayesian maximum clade credibility phylogenetic tree of tip dated NS1 sequence data set was built under an exponential coalescent tree model and HKY substitution model. Tips are colored by the location of the sample collection. As shown in the map some locations were grouped together to decrease sampling bias. the internal nodes are also coloured based on the reconstructed ancestral state as estimated by a symmetric discrete phylogeographic model. x axis shows the time of divergence (for nodes) and sample collection (for tips). Posterior probability >0.5 were given on the left side of the nodes.

  Figure1). Indeed, its VP belongs to CpDV-2 while its NS region belonged to CpDV-1 (Appendix Figure6). This suggested that during this period CpDV-1 and a CpDV-2 variant could have co-existed in the laboratory and co-infected Slab line resulting in a recombination event between them.

Figure. 1

 1 Figure.1 Recombination in CpDV. Bootscan plots of the significant recombination events within the concatenated NS1-VP dataset detected by RDP4 software. Y-axis represents the bootstrap support of each pair of sequences, x-axis shows their position in the alignment. Recombinant regions are shown in pink.Triangles indicate where NS1 region ends and VP starts approximately at 900 nucleotide position. Recombination break points coincided with this point in most of the samples, except Haiti 2 which showed a slightly different recombination pattern.

Figure. 2 .

 2 Figure. 2. Selection on NS1'. Subscript under each nucleotide represents how many variant had a given nucleotide. N 0.5 indicates double peaks. Information on only 25 sequences are shown after clonal sequences were removed. Numbers on the right side represents the nucleotide position within the sequenced fragment.

Figure

  Figure generated using classic.datamonkey.org. Codons under purifying selection are indicated in red boxes.

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Figure. 3 .Figure 4 .

 34 Figure. 3. Selection on VP. Subscript under each nucleotide represents how many variant had a given nucleotide. N 0.5 indicates double peaks. Information on only 25 sequences are shown after clonal sequences were removed. Numbers on the right side represents the nucleotide position within the sequenced fragment. Figure generated using classic.datamonkey.org. Codons under purifying selection are indicated in red boxes and one codon under diversifying selection is shown in green.

Figure 5 .Figure. 6 .

 56 Figure 5. Population dynamics of CpDV estimated for partial NS1(A), VP (B) codons and concatenated dataset (C) using exponential growth model as implemented in BEAST. Y-axis represents the exponential growth line for the median growth rate through time and the 95% HPD intervals for this growth are shown as a solid area.

  Wolbachia: the transovarian vertical transmission of Culex pipiens densovirus.

Figure 1 .

 1 Figure 1. The two symbionts Wolbachia and CpDV coexist in Cx. pipiens ovaries of ten laboratory lines Wolbachia amount (A) Ovaries from three females per line were dissected and pooled. wsp copy number/ace-2 copy number was quantified for 5 pools per line by qPCR assays. CpDV amount (B) NS2 copy number and ace-2 copy number have also been quantified in the same pools. Harash line with the highest quantity of CpDV in ovaries and Tunis line with less CpDV in ovaries were selected for further experimentation.

Figure 2 .

 2 Figure 2. Immunolabeling of CpDV in Cx. pipiens ovaries. Structure of a Cx. pipiens ovariole (A).The mosquito ovaries are paired and composed of functional units called ovarioles, containing primary (F1) and secondary follicles (F2)[START_REF] Clements | The biology of mosquitoes[END_REF].

  is a thin membrane with an outer muscle layer composed of isolated cell bodies interconnected by muscle fibers. Confocal images of ovaries isolated from 6 days old Cx. pipiens females (B, C, D, E). Immunolabeling of CpDV (in red) was performed with an anticapsid antibody, actin was labeled with phalloidin-FITC (green) and nuclei with DAPI (blue). Arrows show CpDV-infected cells and arrowheads point cytoplasmic dots of CpDV labelling. Images of the surface of an ovary and an ovariole (composed of a primary and a secondary follicles) are shown in B and C, respectively. Internal views of primary follicles are shown in D and E.

Figure 3

 3 Figure 3 Ultrathin sections of ovaries isolated from 6 days old females (Harash line) (A to H). Large views of an ovariole (primary follicle) in A, D and G (bar scale: 5 µm). Ultrastructure of the hypertrophied nuclei and putative virus factories in a follicular cell (A and B), in an oocyte (D and E) and in a nurse cell (G and H). Zooms in the nucleoplasm of the follicular cell (C) and in the oocyte (F) show particles with the expected size of CpDV (25 nm). Virus particles observed in a filtered homogenate of ovaries (I). Arrows show icosahedral virus particles and arrowheads point empty particles with the expected size of CpDV (bar scale: 50 nm).

Figure 4 .

 4 Figure 4. Localization of CpDV in Cx. pipiens eggs. Confocal images of dechorionated eggs from Cx. pipiens females (Harash line) (A). CpDV (in red) were immunolabeled with an anti-capsid antibody; Wolbachia and nuclei (in blue) were labeled with DAPI. Top panel shows eggs in the first hour after laying (early developmental stage) and bottom panel eggs 2 hours after laying (late developmental stage). The second part of the Top panel is a zoom of one egg pole in the first hour after laying; the second part of the Bottom panel is a zoom in the egg cytoplam. Arrows show Wolbachia cells at one egg pole. Electron micrographs of ultrathin sections of dechorionated eggs (B and C). In B, a large view of an early egg pole (bar scale: 5 µm), showing the accumulated reserves required for the embryo development (VG, vitellin granule; LD: lipid droplet). In C, a zoom (bar scacle: 500 nm) inside the cytoplasm of the early egg showing numerous Wolbachia (arrowheads). The virus particles cannot be distinguished from the ribosomes because of their similar size.

  To assess the effect of Wolbachia (strains and density) on the amount on CpDV in the ovaries, we have established mosquito lines, with a backcrossing protocol, with the same host nuclear genetic background but different Wolbachia strains (BcTunis, Slab genetic background harbouring wPip I from Tunis line; BcHarash, Slab genetic background harbouring wPip IV from Harash line,

Figure 5

 5 Figure 5 Slab line's genetic background was introgressed into Harash and Tunis lines, to establish BcHarash and BcTunis lines with the same genetic background as Slab (shown in circles) and same Wolbachia strain as Harash (shown in orange) or Tunis (shown in green) respectively. Wolbachia amount in ovaries (A). was not significantly different between the lines no matter what the Wolbachiastrain (df=2, dev=17.703, p=0.87) and host genetic background were (df=1 dev=0.034, p=0.981). CpDV amount in ovaries (B) from three females were dissected and pooled. 5 pools per line have been tested by qPCR assays to quantify CpDV and Wolbachia amount.

Figure

  Figure 5B).

  , Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. during TC treatment, W= 132.5, p=0.08; Wo+ vs. Wo-, W= 100 , p=0.138; during TC treatment vs. Wo-, W=308, p=1). On the contrary, in the ovaries CpDV amount significantly decreased during the TC treatment (Dunnett's test with Bonferroni Correction; Wo+ vs. during TC treatment, t=2.302, p=0.025; Figure 6B). However, in Wo-lines (three generations after the end of the tetracycline treatment) CpDV amount increased and reached a level not significantly different from non-treated Wo+ lines (Dunnett's test with Bonferroni Correction; Wo+ vs. Wo-, t= 0.118, p=1).

Figure 6

 6 Figure 6 Effect of curing Wolbachia by tetracycline treatment on CpDV amount in Cx. pipiens. We have treated Slab, BcHarash and BcTunis lines with tetracycline (TC) for three generations and dissected the ovaries of 6 days old females. Dissections were performed on the non-treated mosquitoes (Wo+), first and third generation of mosquitoes during tetracycline treatment (during TC treatment) and three generations after the tetracycline treatment was finished (Wo-). Effect of tetracycline treatment on the amount of Wolbachia in ovaries and carcasses (A). Wolbachia amount decreased immediately starting from first generation during tetracycline treatment. There were no Wolbachia detected after first treatment generation neither in ovaries nor in carcasses. Effect of tetracycline treatment on CpDV amount (B). CpDV amount in the ovaries significantly decreased starting from the first round of tetracycline treatment but increased when tetracycline treatment stopped (Wo-)(Dunnett's test with Bonferroni Correction; Wo+ vs. during TC treatment, t=2.302, p=0.025; Wo+ vs. Wo-, t= 0.118, p=1). Carcasses had less CpDV compared to ovaries (Wilcoxon rank sum test, W = 168, p = 0.021). The effect of the treatment was not significant on the carcasses (Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. during TC treatment, W= 132.5, p=0.08; Wo+ vs. Wo-, W= 100, p=0.138; during TC treatment vs. Wo-, W=308, p=1).

Figure 7

 7 Figure 7 Effect of Wolbachia-curing tetracycline treatment on CpDV vertical transmission. In BcHarash, BcTunis and Slab lines, Females emerged from the larvae that underwent the first tetracycline treatment were isolated five days after bloodmeal to lay eggs. 10 females per line (total n=30), 10 L1 stage larvae per female were collected and tested for CpDV presence to estimate vertical transmission rate. This procedure was repeated for females emerged after the third generation of TC treatment and for the females Wo-(three generation after the end of tetracycline treatment). There was a significant decrease in vertical transmission during tetracycline treatment (Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. during TC treatment, w= 488, p= 0.004). Three generations after end of the treatment the vertical transmission rate increased but still was marginally significant from the non-treated group (Wilcoxon rank sum test with Bonferroni correction; Wo+ vs. Wo-, w= 576.5, p= 0.05).

  also thank Dr Philippe Clair for his help in the Real Time Quantitative PCR experiments which were performed through the technical facility of the "qPCR Haut Debit (qPHD) Montpellier genomiX" platform. We acknowledge the imaging facility MRI, member of the national infrastructure France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). Mine Altinli received support from Ministère des affaires étrangères (Campus France) via the French 3CpDV specific diagnostic PCR test, based on the amplification of partial NS2 gene revealed the presence of CpDV in all the line that were kept in the insectary.

Figure 1 .

 1 Figure 1.CpDV prevalence in insectary Lines (A). CpDV prevalence was higher in Larvae compared to adults (B)

Figure 2 .

 2 Figure 2. Prevalence in Males vs. Females.

Figure 4 .

 4 Figure 4. Experimental infections using CpDV transinfected C6/36 cells

Figure 5 .

 5 Figure 5. Confocal images of ovaries isolated from 6 days old Cx. pipiens females (Harash line). Immunolabeling of CpDV (in red) was performed with an anti-capsid antibody, actin was labelled with phalloidin-FITC (green) and nuclei with DAPI (blue). Arrows show CpDV-infected cells and arrowheads point cytoplasmic dots of CpDV labelling. Images shows split channels and merges of images from the figure 2.

Figure 6 .

 6 Figure 6. Confocal images of ovaries isolated from 6 days old Ae. albopictus females. Immunolabeling of CpDV (red) was performed with an anti-capsid antibody, actin was labeled with phalloidin-FITC (green) and nuclei with DAPI (blue) in the same conditions as in the supplementary figure 1. No red labeling was observed in Ae. albopictus ovaries.

  Gunay, F., Alten, B., Weill, M., and Sicard, M. (2018) Wolbachia diversity and cytoplasmic incompatibility patterns in Culex pipiens populations in Turkey. Parasites and Vectors 11: 198. *Figure 1 colours have been modified to fit the rest of the thesis.

a

  Data taken from Duron et al.[START_REF] Duron | Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens[END_REF] Note: Reciprocal crosses between Tek wPip-I, Tek wPip-II, Istanbul wPip-IV lines have been performed to identify natural CI patterns induced by these strains in the region. Additional reciprocal crosses between Turkish lines and 4 reference laboratory lines [LaVar (wPip-II), MaClo and Slab (wPip-III) and Istanbul (wPip-IV)] have been performed to define the mod-resc functions of Tek wPip-I and Tek wPip-II isofemale lines and to compare them to previously defined ones by Atyame et al.[7]. Crosses were classified as either compatible (C, raft hatching > 90%) or incompatible (IC, raft hatching = 0-10%). Bidirectionally incompatible crosses are shown in bold. The number of egg-rafts collected for each cross is indicated in parentheses

Figure 1 .

 1 Figure 1.CpDV genome. CpDV has an ambisense genome: its non-structural genes (NS) are on one strand while its structural gene (VP) are on the complementary strand. CpDV has 3 different NS genes. NS1 and NS2 are transcribed from the same strand. Their ORFs overlap on the same strand and both are split into two ORF. ORFs shown with arrows. NS1 and VP genes have very conserved motifs such as PLA2 and endonuclease motifs. In red fragments used for diagnostic PCR test shown, and in green sequenced NS3, NS1 and VP fragments.

Figure 2 .

 2 Figure 2. Prevalence of CpDV in natural populations of Cx. pipiens (s.l.) in Turkey. A PCR test based on the amplification of partial NS2 region is used to detect CpDV. Only samples that have been collected in 2016 are shown. All samples collected between 2012-2015 with adult traps were negative.

Figure 3 Figure 4

 34 Figure 3 Life stage & CpDV prevalence (A). CpDV presence was detected using a PCR diagnostic test amplifying NS2 region. Adults were more likely to be CpDV positive (binomial glm, df=1, dev=18.896, p<0.001). wPip group & CpDV prevalence (B). Mosquitoes that have been tested for their wPip group also tested with PCR diagnostic test and prevalence of CpDV was recorded. Generalized linear model did not show any effect of wPip diversity on (df=2, dev=0.745, p>0.05)

  OF WOLBACHIA ON PREVALENCE, VIRUS LOAD AND DIVERSITY OF CULEX PIPIENS DENSOVIRUSES ................................................................................................ 5.1.1 INTRODUCTION ............................................................................................................ 5.1.2 METHODS ....................................................................................................................

  3 RESULTS...................................................................................................................... 5.1.3.1 Analysis of CpDV prevalence in the all sampling area .......................................... 5.1.3.2 Analysis of CpDV prevalence in host populations where different Wolbachia strains coexisted in similar ecological conditions .................................................................................. 5.1.3.3 Relation between CpDV and Wolbachia loads...................................................... 5.1.3.4 Diversification of CpDV in relation with Wolbachia groups .................................. 5.1.4 DISCUSSION ................................................................................................................. 5.1.5 ACKNOWLEDGEMENTS ................................................................................................. 5.1.6 REFERENCES ...............................................................................................................

A

  CpDV specific PCR based diagnostic test was used to infer CpDV prevalence in the area. This test consists of an amplification of a 238 bp fragment using CpDV-For (5'-ACCACCAC-GGACTGGTATGA-3') and CpDV-Rev (5'-TCCCCATTATCTTGCTGTCG-3') primers within conserved NS2 coding region. PCR was performed with the following conditions: initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation, annealing and elongation respectively at 94 °C for 30 s, 56 °C for 30s, and 72 °C for 90 s, and a final elongation at 72 °C for 5

  the Wolbachia load differed between mosquitoes harbouring different Wolbachia groups (wPip-I and wPip-IV) and whether it affected the CpDV load in the area, we quantified CpDV and Wolbachia amount for the same individuals using Real-time quantitative PCR (qPCR). SYBR Green I Master (Roche) and LightCycler 480 Instrument (Roche), LightCycler 480 software (version 1.

  effects of population, collection year (2010-2011) and Wolbachia groups (wPip-I, wPip-IV) on CpDV prevalence, we first fitted a generalized linear model from binomial family. Likelihood ratio tests of the full model against the model without a given effect were used to obtain deviance and p-values. A subset of the data then created to include only the populations where both Wolbachia strains co-existed and where at least one sample was CpDV positive. Hence in each population tested, the probability of a given sample to be infected by CpDV assumed to be independent from the location where samples were collected from. A binomial generalised linear model was used to test the effect of the collection year (2010 or 2011) and Wolbachia groups (wPip-I, wPip-IV) on CpDV prevalence. To investigate the effects of Wolbachia groups, Wolbachia load and location on CpDV load a generalised linear model was fitted.

Figure 1

 1 Figure 1 Wolbachia groups and CpDV prevalence in the contact zone. A total of 1588 samples collected from 35 different populations between 2008-2011, have been tested. A. wPip strain distribution in the area. (Adapted based on the results of Atyame et al. 2015) There are two distinct wPip strains in the area; wPip IV in south-eastern populations and

Figure 2 .

 2 Figure 2. Effect of Wolbachia group on CpDV prevalence in the Cx. pipiens populations where both wPip-I, wPip-IV and CpDV co-exist in the same environmental conditions. The data set was reduced only to locations where both Wolbachia strains and CpDV present. The proportions of both CpDV positive (in darker colours) and CpDV negative (in lighter colours) mosquitoes were indicated relative to the groups of Wolbachia wPip-I versus wPip-IV they carry. Sample size for each wPip group is shown on the top of the bars. A total of 378 samples over 479 samples were found CpDV positive. CpDV distribution was significantly affected by the Wolbachia group (glm, df=1, dev= 16.492, p<0.001). Mosquitoes harbouring the wPip-IV were 34 % (RR=1.338; OR=2.791, 95% CI: 1.709-4.542; p< 0.001) more likely to be CpDV positive. Year of collection did not have a significant effect in the complete model (glm, df=1, dev=2.739, p>0.05).

Figure 3 .

 3 Figure 3. Relation between Wolbachia and CpDV amounts in the Cx. pipiens populations where both wPip-I, wPip-IV and CpDV co-exist in the same environmental conditions. Both Wolbachia and CpDV densities were quantified by qPCR assays using wsp and NS2 gene copy number, respectively, relative to ace2 mosquito gene in a total of 114 samples. Samples with Cq>33 for any of the qPCR reactions have been excluded from the analysis and the graph because of the high uncertainty of the quantification. A. CpDV load. CpDV load reached 82900 CpDV/host cell in one sample from El Manar population and this was excluded from further analysis as an outlier. CpDV load was affected mainly Wolbachia loads (df=1, dev=9.840, p< 0.001) but also by population (df=8, dev=127.27, p<0.001). Samples from the El Manar population showed the highest mean of CpDV / host cells (El Manar n=21, mean=14.881±3.925) while samples from Fontaine (n=61, mean=0.055±0.0180) showed really low amount. B. Wolbachia load. Outliers from Fontaine population were excluded from analyses. Average Wolbachia amount per host cell was 0.356 (±0.052). Wolbachia load also seemed to be influenced by CpDV load (glm, df=1, dev=3.050, p<0.001) and the population (df=8, dev=3.1472, p<0.05) but not by the collection year (df=1, dev=0.571, p>0.05) and the Wolbachia group (df=1, dev=0.162, p>0.05 ). C. Correlation between Wolbachia and CpDV loads. CpDV load significantly increased with the increased Wolbachia load (r 2 =0.11, p<0.001).

Figure 4 .

 4 Figure 4. Sequence Alignment of CpDV genomes. To infer the diversity of CpDV from geographically close locations we have sequenced a divergent part of the CpDV genome including NS3 coding region. Subscript under each nucleotide represents how many variant had a given nucleotide. N0.5 indicates double peaks. A total of 42 sequences are shown after clonal sequences were removed. Numbers on the right side represents the nucleotide position within the sequenced fragment. Figure generated using classic.datamonkey.org

Figure 5

 5 Figure 5 CpDV diversification in northern Tunisia. Maximum likelihood tree based on the sequences of CpDVs NS3 coding region. CpDV samples found in the area grouped in two well-supported clades, CpDV-Ia and CpDV-Ib (highlighted in purple and pink). CpDV variants from same populations generally clustered together with high bootstrap values (e.g. samples from Oued Melah, El Battan and Tabarka). Some other populations also grouped together while their bootstrap values (100 iterations) were not as strong (e.g. Utique, Pompe). While both CpDV-Ia and CpDV-Ib clades could be found in both wPip-I and wPip-IV infected mosquitoes, CpDV-Ib found more in wPip-IV infected mosquitoes.

Figure 6

 6 Figure 6 Similar spatial distribution of Wolbachia groups (A) and CpDV clades (B) (Mantel, r= 0.34, p<0.05). There was no spatial correlation between the distribution of the CpDV clades and geographical distance between their collection location (Mantel test, r= 0.17 ,p>0.05).

  demonstrated a persistent infection by CpDV in apparently healthy laboratory Cx. pipiens lines (Chapter 3). Although the mechanisms of this persistence and the putative mutualistic side of CpDV have to be investigated in further experiments, other densoviruses have been shown to have mutualistic interactions with their hosts. For instance, an ambidensovirus genetically close to

  ! Wolbachia-CpDV interactions in Tunisian natural populations of Cx. pipiens (Chapter 5): o! An influence of wPip groups on CpDV prevalence o! A positive correlation between wPip density and CpDV density in infected mosquitoes suggesting their mutualistic interactions •! Microevolution of CpDV in geographically close populations and putative influence of wPip group variations on CpDV diversification in Tunisia (Chapter 5)

  role of recombination and purifying selection on the evolution of CpDV as expected in ssDNA viruses in general (Chapter 2). General incongruence of different genes (NS and VP) in CpDV possibly caused by recombination made it difficult to infer their real tMRCA with certainty (Chapter 2).The current phylodynamics methods cannot take recombination into account hence we had to exclude the recombinants from the datasets (Chapter 2). Such exclusions of the recombination seemed to change tMRCA estimates a lot (Chapter 2). Being one of the major forces of virus evolution recombination constitutes one of the challenges that phylodynamics methods are facing today, new phylogenetic methods might improve this current situation(Frost et al., 2014). As our study was limited to already extracted DNA samples (Chapter 2) new sampling of fresh material could allow to sequence more samples, and/or the whole genome sequencing could be a way to circumvent this problem and possibly can give better estimates. Additionally, while our approach using Sanger sequencing allowed us to use older samples, we were not able quantify within host diversity even though we have observed double and sometimes triple peaks in some electropherograms (Chapter 2, Chapter 3). Future studies employing next generation sequencing methods could also be useful to understand the within host diversity, especially the effects of this diversity on the host-virus interactions.Discovery of the worldwide prevalence and persistence of CpDV brought an important question in mind: how do they transmit from host to host? To answer this question we have tried first to test their horizontal transmission by using infected crushed larvae and cell cultures transinfected with synthetic CpDV plasmid (Chapter 3).

  variant could have arrived to the insectary around 1997, and the co-infection by reference variant (CpDV-2 clade) and the new variant (CpDV-1 clade) might have resulted in the recombination event we have detected in Slab 1997 sample (Chapter 2). After this, either the arriving variant might have out competed the reference variant or newly emerged recombinant variant could have evolved further with their hosts. To gain more insights on how this change occurred in the insectary populations, we now are sequencing samples that have been collected from our Another way to understand the evolution of the CpDV in the insectary lines would be to continue comparing a natural isolate from the insectary with a synthetic CpDV having the same genome, in addition to synthetic CpDV with the reference genome. This would help us to compare the infectivity of both genomes and synthetic vs. natural isolates. However, it is crucial to first establish CpDV free lines to separate the effects of the virus from the effects of a co-infection by different variants. Once established this model system could also be used to test both the evolution of CpDV, their interactions with their hosts and Wolbachia more directly. For instance, How the mosquito is affected by CpDV infection? What is the role of the immune system? If immune system is activated by a CpDV infection does this affect the rest of the microbiota? How the changes in microbiota affect the Culex and hence CpDV? What is the route for the CpDV horizontal infection of Cx. pipiens (ingestion, anal papillae)? Easy manipulation of the CpDV genome by simple cloning techniques could also make functional studies possible. Last part of this PhD thesis focused on Wolbachia and CpDV interactions in natural populations (Chapter 4-Chapter 5). We have first identified both Wolbachia and CpDV presence and diversity in natural populations in Turkey (Chapter 4). Turkey can provide ideal conditions for both the amplification of already circulating viruses and the emergence of new viruses due to its geographical location, its climate and the biodiversity of arthropods vectors. Previous studies have shown the diversity of mosquitoes and the RNA viruses they carry (arboviruses and insect-specific viruses), although Wolbachia diversity in Cx. pipiens mos-

  pipiens populations, particularly where only wPip-I or wPip-II harbouring females were present. In this context, local studies of wPip diversity and CI patterns in finer scale could be useful for Wolbachia based vector control in the region. We have also revealed CpDV diversity especially in the Thrace region (North-western Turkey). This proved that Turkey could be a great place to study virus-virus, virus-Wolbachia interactions in nature, while our study so far stayed more in explorative nature due to the lack of previous studies on wPip in the region. Indeed, adding the investigation of diversity and prevalence of insect-specific viruses to the existing arbo-

  understand their interactions in natural populations better.By sequencing NS3 coding region in CpDV genome, we revealed the microevolution of geographically close populations in Northern Tunisia. All belonging to CpDV1 clade (Chapter 1), variants from the region were closely related as expected.

  ............................................................................................................

  mission verticale dans des lignées de laboratoire naturellement infectées par le CpDV et Wolbachia, nous avons montré que i) la transmission verticale avec des faibles charges virales, ii) la présence des CpDV dans les ovocytes (où se trouve aussi le Wolbachia) et pas seulement à la surface des oeufs, iii) par des traitements antibiotiques que le microbiote, en particulier Wolbachia semble influencer positivement la transmission verticale du CpDV (Chapitre 3). Bien que nous n'ayons pas pu la quantifier de manière robuste la transmission horizontale en raison de l'absence de lignées de moustiques sans CpDV, celle-ci semble être élevée étant donnée la transmission verticale aux larves du premier stade larvaire (~20%) et la prévalence élevée chez les femelles adultes émergées (~100%) (Chapitre 3).Les modes de transmission des endosymbiontes sont fondamentaux dans l'évolution de leur virulence à savoir leur trajectoire vers le mutualisme ou l'antagonisme. Dans les lignées de laboratoire, il a été observé que le CpDV pouvait causer de la densonucléose et qu'il était probablement la cause d'une mortalité larvaire élevée lorsqu'il a été isolé pour la première fois dans la fin des années 1990(Jousset et al., 2000). Deux décennies plus tard, le virus CpDV semble persister en faibles quantités et se transmettre verticalement dans nos lignées de laboratoire dont les individus ne semblent pas malades. Ce changement de virulence suggère un changement dans le virus au fil des années. Le séquençage des variants de CpDV présents au laboratoire pendant les 30 dernières années a en effet indiqué un changement dans les variants présents. Dans les années 90, les variants appartenaient au clade CpDV 2 alors qu'à l'heure actuelle ils appartiennent au clade CpDV1. Ce « shift » a eu lieu avant les années 2000 (Chapitre 2). Nous pensons que ce remplacement d'un variant par un autre est dû à l'importation de nouveaux échantillons dans nos élevages dont les nouveaux variants de CpDV ont exclu les variants résidants. En effet, un échantillon de l'année 1997, zone de transition entre les deux clades, montre une séquence composite, issue de recombinaison, entre un CpDV 1 et un CpDV 2 suggérant qu'ils ont coinfectés les mêmes hôtes pendant une période restreinte. Nous devrons dans les années à venir tester la différence de virulence entre les virus actuels et passés (CpDV 2 versus CpDV 1). Pour cela, il nous faudra améliorer les méthodes de transfection des virus en cellule et l'isolement des CpDV à partir de larves afin de comparer de nombreux CpDV synthétiques ou provenant d'isolat naturel en réalisant des infections expérimentales comparatives. Pour cela, nous sommes en train de mettre en place une lignée cellulaire de Cx. pipiens qui puisse optimiser le succès de la transfection. Bien que les résultats déjà acquis en termes de virulence des CpDV soient préliminaires (un seul réplicat), ils suggèrent que le CpDV synthétique (qui est conçu à partir du génome de référence qui est un CpDV 2) était pathogène tandis que l'isolat actuellement présent dans le laboratoire (CpDV 1) est au contraire plutôt mutualiste. C'est résultats sont de première importance et représentent le chainon manquant de cette thèse. Cependant, pour mener ces expériences de manière rigoureuse, il est crucial d'établir d'abord des lignées exemptes de CpDV pour séparer les effets confondant d'un variant testé avec les effets du variant déjà présent dans la lignée. Une fois établi, un système d'infection expérimental pourrait être utilisé pour réaliser des manipulations d'évolution expérimentales des CpDV et mieux étudier ses interactions avec ses hôtes et Wolbachia. Nous pourrions, par exemple, répondre aux questions suivantes, comment les traits de vie du moustique sont-ils modifiés (positivement ou négativement) par l'infection par le CpDV ? Quel est le rôle du système immunitaire dans cette interaction ? Si le système immunitaire est activé par une infection par le VCDPC, est-ce que cela affecte le reste du microbiote ? Quelle est la voie d'infection horizontale des Cx. pipiens par les CpDV (ingestion, papilles anales) ? Les manipulations qui semblent aisée du génome du CpDV par de simples techniques de clonage pourraient également permettre des approches fonctionnelles. La dernière partie de cette thèse portait sur les interactions entre Wolbachia et CpDV dans les populations naturelles (Chapitre 4-Chapitre 5). Nous avons d'abord révélé la présence et la diversité des Wolbachia et des CpDV dans les populations naturelles de Cx. pipiens en Turquie (Chapitre 4). La Turquie offre des conditions propices tant pour l'amplification des virus déjà en circulation que pour l'émergence de nouveaux virus en raison de sa situation géographique, de son climat et de la biodiversité des arthropodes vecteurs. Des études antérieures ont montré la diversité des moustiques et des virus à ARN qui circulent dans cette région (arbovirus et virus spécifiques aux insectes)

Wolbachia

  et des CpDV chez les moustiques Cx. pipiens était jusqu'à lors inconnue. J'ai pu montrer que la diversité des Wolbachia (et de leur patrons d'incompatibilité cytoplasmique) mais aussi des CpDV, en particulier dans la région de Thrace (nord-ouest de la Turquie), était très importante. Cela démontre une nouvelle fois que la Turquie, une zone au carrefour entre l'Europe, l'Asie et l'Afrique est une région pertinente pour étudier les interactions virus-virus et les interactions virus-Wolbachia dans les populations naturelles de vecteurs. D'autre part, dans le nord de la Tunisie, la diversité des wPip et de leurs patrons d'IC avaient déjà été étudiés de manière approfondie par notre équipe EVAS à l'ISEM (Atyame et al., 2015). Ainsi, les auteurs avaient découvert une zone de contact où des moustiques hébergeant deux groupes différents de wPip, les groupes wPip I et IV, coexistaient. Leurs patrons d'IC respectifs permettaient de prédire l'invasion de la zone de contact par la souche induisant un IC unidirectionnel, à savoir wPipI, en quelques générations seulement. Cependant, la zone de contact a été montrée comme stable pendant plus de sept ans. En utilisant une partie des échantillons, nous avons étudié la prévalence du CpDV dans la même région et l'effet des différents groupes de wPip sur sa prévalence (Chapitre 5). Notre hypothèse initiale était qu'une protection contre les pathogènes (ici CpDV) conférée par l'un des groupes de wPip, en l'occurrence wPip IV, pourrait expliquer pourquoi le groupe wPip I ne pourrait pas envahir l'ensemble des populations hôtes dans la zone de contact. De manière surprenante, nous avons démontré que ce sont les individus infectés par le groupe wPip IV qui étaient plus susceptibles d'héberger le CpDV. Bien qu'il soit difficile d'interpréter ces résultats sans avoir pu étudier la nature des interactions CpDV-hôte, la prévalence plus élevée de CpDV chez les moustiques porteurs du wPip-IV pourrait résulter d'une transmission verticale plus efficace du CpDV en interaction avec ce groupe de Wolbachia. En appui de cette hypothèse, nous avons observé, en laboratoire (Chapitre 3), que les moustiques porteurs de wPip IV avaient dans leurs ovaires une quantité de CpDV supérieure aux moustiques porteurs de wPip I. Dans cette même zone, bien que la prévalence soit affectée par les groupes de wPip, il n'y a aucun effet des différents groupes sur les variations de charges virales du CpDV. Cependant, nous avons montré que la charge virale de CpDV est positivement corrélée aux densités des wPip. Cela suggère que les interactions entre Wolbachia et les virus pourraient ne pas toujours être en accord avec l'emblématique « protection » en général accordée à Wolbachia. S'il est vrai que dans le cas des arbovirus (virus à ARN) des interactions de nature antagoniste ont été principalement démontrées ce n'est pas le cas pour les virus à ADN (Teixeira et al., 2008; Graham et al., 2012). Il possible que ce ne soit pas le fait que les virus soit à ADN ou à ARN qui soit la cause des différences mais plutôt la nature de leur interaction avec l'hôte (hôte principal versus vecteur) qui change la relation entre Wolbachia et virus. En effet, les arbovirus vectorisés par les moustiques ont a priori moins d'impact sur la biologie des moustiques, donc sur leur valeur sélective, que les virus spécifiques aux moustiques. Il a été récemment signalé que la présence de Wolbachia renforçait la multiplication d'un virus à ARN spécifique d'insecte dans des cellules d'Aedes aegypti. De plus, les populations naturelles d'Ae. aegypti transinfectées de façon stable par Wolbachia présentaient des taux d'infection et des charges d'ARN spécifiques aux insectes plus élevés que leurs homologues sans Wolbachia (Amuzu et al., 2018). Ces résultats démontrent que la relation entre Wolbachia et les virus n'est pas toujours antagoniste et que nous pourrions bien démontrer dans les années à venir que CpDV est un mutualiste de Cx. pipiens. Par ailleurs, en séquençant la région codante NS3, très variable dans le génome du CpDV, nous avons pu étudier la microévolution des CpDV au sein de populations de Cx. pipiens géographiquement proches dans le Nord de la Tunisie. Tous les variants de Tunisie appartiennent au clade CpDV1 (Chapitre 1), mais se regroupent en deux clades distincts et bien soutenus statistiquement (Chapitre 6). Nous montrons une forte structuration spatiale des variants de CpDV. De plus, l'étude de la distribution de ces deux groupes distincts a révélé un lien potentiel avec la distribution des deux groupes de wPip. Compte tenu de leur co-transmission verticale, il est possible que ces deux endosymbiontes co-divergent et que leurs distributions résultent de cette co-transmission. Bien que mon doctorat ait principalement porté sur les interactions CpDV-Wolbachia, les interactions entre endosymbiontes sont par essence de nature beaucoup plus complexe que de « simples » interactions tripartites. Il est probable que le reste du microbiote affecte également les interactions CpDV-Wolbachia et réciproquement. Ainsi, la diversité et la forte prévalence de virus à ARN spécifiques d'insectes dans les populations naturelles de moustiques ont été démontrées au cours des dernières années. Néanmoins, les recherches sur leurs effets sur leurs hôtes et le microbiote hôte n'ont pas encore étaient menées. Un tel virus à ARN associé à Cx. pipiens a récemment été découvert dans le nord de la Tunisie (Bigot et al., 2018) offrant la possibilité d'étudier les interactions entre virus à ADN et à ARN spécifiques des insectes, l'endosymbiote Wolbachia et Cx. pipiens dans cette zone géographique. Les études futures devront tenir compte de ces interactions virales multipartenaires comme c'est le cas actuellement dans l'étude des endosymbiotes bactériens en regard du reste du microbiote. Les MDV ont longtemps été étudiés en raison de leur pathogénicité et de leur spectre d'hôtes étroit en tant qu'agents potentiellement stratégiques en lutte antivectorielle. Deux méthodes ont été envisagées pour leur utilisation : i) leur utilisation directe pour tuer les larves de moustiques en association ou non avec d'autres traitements; ii) leur utilisation comme agents transducteurs (Bu-

  blement pathogènes, utilisés dans cette technique, ne causeraient pas une pression sélective aussi élevée que les MDV très pathogènes et permettraient ainsi aux larves de survivre et aux jeunes adultes de se reproduire mais empêcheraient la transmission des arbovirus(Ren et Rasgon, 2010). Bien que les deux méthodes soient prometteuses en termes de lutte contre les maladies vectorielles, elles sont encore entravées par le manque de connaissances sur la biologie et de l'écologie de base des MDV et par le manque de MDV spécifiques aux moustiques d'importance médicale(Johnson et Rasgon, 2018). Dans ce contexte, nos études sur le CpDV pourraient trouver un sens appliqué, en particulier comme agent transducteur en raison de sa persistance dans les populations de Cx. pipiens et de sa faible pathogénicité. Pour mieux comprendre la biologie du CpDV, il serait utile d'étudier sa spécificité en termes d'hôtes. Comme ils sont génétiquement plus proches des ambidensovirus lépidoptères (Baquerizo-Audiot et al., 2009 ; Cotmore et al., 2014), il est possible qu'ils soient infectant pour ce groupe d'insecte. Très récemment, un densovirus génétiquement similaire au CpDV a été découvert dans des populations naturelles de Drosophiles proches de Paris montrant que ce virus n'est peutêtre pas spécifique des Culex au sein des Diptères (Kapun et al., 2018). Les transfections de cultures cellulaires par le CpDV avaient par ailleurs montré que ce virus étaient capables de s'amplifier dans les cellules d'Anophèles suggérant également que leur spécificité n'était pas stricte (Jousset et al., 2000). Nos résultats préliminaires d'infection expérimentale ont également montré que les CpDV (clade 1 ou 2) n'entrainent pas de mortalité chez les larves d'Ae. albopictus. Des études futures sur Anophèles, certains lépidoptères ravageurs et la drosophile pourraient permettre de préciser le spectre d'hôtes de CpDV. Les récent progrès de la technologie de séquençage et des techniques de biologie moléculaire ont permis aux chercheurs de révéler l'ubiquité et la diversité des virus. Malgré ces progrès, la découverte de virus s'est surtout concentrée sur les virus de vertébrés. Les arthropodes avec leur histoire évolutive ancienne représentent 80% de la diversité animale sur notre planète. En supposant une corrélation entre la diversité des hôtes et la diversité des virus, cela pourrait signifier que nous sommes encore inconscients de la plupart de la diversité des virus existants. De plus, même les virus connus ne sont que peu étudiés en termes d'écologie virale. L'omniprésence de l'endosymbiose bactérienne chez les arthropodes et la capacité des arthropodes à soutenir des symbiotes (parfois multiples) et de leur « déléguer des fonctions » suggèrent que leurs interactions avec leurs virus pourraient également être de nature symbiotique, allant des interactions durables de type mutualiste à celles de type antagoniste. Cette nouvelle approche conceptuelle des virus, tout en présentant de nombreux défis, pourrait répondre à un large éventail de questions sur l'écologie et l'évolution des interactions hôte-microbiote. Les densovirus, aussi anciens et divers que leurs hôtes arthropodes, sont omniprésents dans la nature et ont des petits génomes faciles à manipuler. Ils représentent donc de bons modèles pour étudier les interactions hôte-virus-bactéries. À l'intersection de nombreux domaines, nos résultats, bien qu'ils aient pu soulever plus de questions qu'ils n'en aient répondu, ont suggéré que le système CpDV-Cx. pipiens-Wolbachia pourrait être un système modèle pertinent pour étudier les interactions écologiques entre ces partenaires et leur évolution conjointe tant dans la nature qu'au laboratoire.

  3.3.1 INTRODUCTION .................................................................................................................................................. 59! 3.3.2 RESULTS ............................................................................................................................................................... 61!

2.1 DIVERSITY AND DISPERSAL OF CULEX PIPIENS DENSOVIRUS A WORLDWIDE HIGHLY PREVALENT AMBIDENSOVIRUS ...................................................................................... 28 2

  .1.1 INTRODUCTION .............................................................................................................29 2.1.2 METHODS .....................................................................................................................30 Phylodynamics and dispersal of CpDV ................................................................ 32 2.1.3 RESULTS .......................................................................................................................33

	2
	2.1.2.1 Sample collection and DNA extractions ............................................................... 30
	2.1.2.2 CpDV genomes partial sequencing ...................................................................... 31
	2.1.2.3 Selection, recombination and probabilistic phylogenetic analyses ............................ 32
	2.1.2.4 2.1.3.1 CpDV prevalence in Culex pipiens populations worldwide ...................................... 33
	2.1.3.2 Worldwide diversity and evolution of CpDV......................................................... 33
	2.1.4	DISCUSSION ...................................................................................................................38
	2.1.5	ACKNOWLEDGEMENTS ..................................................................................................40
	2.1.6	REFERENCES .................................................................................................................41
	2.1.7	APPENDIX .....................................................................................................................43

.1! Diversity and dispersal of Culex pipiens densovirus a worldwide highly prevalent ambidensovirus Altinli M., Courcelle

	M., François S., Justy F.,
	Gosselin-Grenet A.S., Ogliastro M., Weill M., Si-
	card M., in prep., invited for the special issue 'New

Table 1 . Nucleotide substitution rates, time to most recent common ancestor (tMRCA), population dynamics of CpDV estimated using concatenated data set and separate NS1 (ORF2) and VP gene fragments.

 1

		Mean Substi-tution Rate (substitu-tion/site/year)	HPD Rate (95%CI) Substitution	tMRCA (Years)	HPD tMRCA (95%CI)	Growth Rate	HPD Rate (95%CI) Growth	Sequence Length
	NS1	9.3x10 -5	1.27x10 -5 -2.16x10 -4	245	50-540	2.36x10 -3	-0.01-0.02	750
						-
	VP	3.33x10 -5	1.92x10 -6 -6.59x10 -5	1103	133-2391	5.15x10 -4	-0.01-0.003	1326
						-
	Concatenated	6.18x10 -4	2.22x10 -4 -1.41x10 -3	71	40-108	1.25x10 -2	-0.05-0.02	2076

Table . 1. Samples that have been used in the study.

 . Collection country, population, coordinates, collection year, CpDV presence, total sample size(N) for a given population and the reference study that the samples were taken from are given.

												Chapter 2 -Prevalence and Evolution of CpDV Chapter 2 -Prevalence and Evolution of CpDV
	67	32 101		Mtsangamouji Fontaine 36.32 m33		-12.80 9.24 41.83	45.20 32.71	2009	2013 2016	0	1 0	Poquet et al. 2013 Atyame et al 2015 Altinli et al 2018
		33 102			Sada m34	36.32		-12.80 9.24 41.99	45.20 35.09	2010	2013 2016	29	0 6	Poquet et al. 2013 Atyame et al 2015 Altinli et al 2018
		34 103			Tsounozou m35	36.32		-12.80 9.24 41.88	45.20 32.99	2011	2013 2016	52	2 5	Poquet et al. 2013 Atyame et al 2015 Altinli et al 2018
	68	Country 35 New Caledonia Poindimie Location Goubellat 104 m37	Latitude -20.93 Longitude 165.34 Collection 2012 CpDV+ 36.31 9.40 2011 24 41.74 32.38 2016	6 0	N	Reference Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018
	69	36 105	Pakistan	Hamra	Lahore m4	37.02		31.54 40.64	9.39	74.34 29.09	Year 2011	1988 2016	19	0 19	Beyssat-Arnaouty et Atyame et al 2015 Altinli et al 2018
	1 70	Angola 106		Lobito Ichkeul	m42	-12.36 37.07	41.93	13.53 9.44	34.58	2011 2011	2016	0 24	6	Dumas et al.2013 al. 1989 Atyame et al 2015 Altinli et al 2018
	2 71	Armenia 37 Portugal 107	Am109VR Bodes Jedaida m45	41.22 36.48		38.56 41.41	44.85 9.56	2016 -27.91 2011 32.08	1994 2016	9 21	0 9	This study Duron et al. 2005 Atyame et al 2015 Altinli et al 2018
	3 72	38 108		Am51VR Ferreira Khetmine m46	39.20 37.08		41.26 41.35	46.44 9.59	-8.39 32.09	2016 2010	1993 2016	10 0	0 15	This study Duron et al. 2005 Atyame et al 2015 Altinli et al 2018
	4 73	Benin 39 109		Ouidah Mateur	Mitra m47	6.36 37.02		38.73 41.45	2.08 9.38	-9.14 31.82	2016 2010	1993 2016	4 0	4 7	This study Duron et al. 2005 Atyame et al 2015 Altinli et al 2018
	5 74	Brazil 40 110		Brasilia Mjez_el_bab Praias m5	-15.77 36.39	38.73 40.94	-47.92 9.36	-9.14 29.30	1993 2011	1993 2016	1 23	2 19	Guillemaud et al. 1997 Duron et al. 2005 Atyame et al 2015 Altinli et al 2018
	6 75	41 111	Recife Porto Rico Nofrancaoui Rico m52	-8.05 36.25		18.46 41.28	-34.88 9.19	1995 -66.10 2011 31.42	1992 2016	0 8	17 6	Duron et al. 2005 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018
	7 76	BurkinaFaso 42 Russia 112	Bobo Oued_Melah Krasnodar m54	11.17 36.35		55.74 41.07	-4.29 9.30	37.60 30.96	2011 2011	2006 2016	0 22	2 10	Dumas et al. 2013 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018
	77	43 113		Dioulassabo Moscou Pompe m55	36.54		55.74 41.07	10.13	37.60 30.84	2010	2006 2016	43	1 5	Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018
	8 78	Cameroon 44 114	Yaounde Volgograd Ras_Jebel m6	3.86 37.13		48.65 40.94	11.51 10.07	44.49 29.30	2008 2011	2006 2016	15 20	1 29	Dumas et al. 2013 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018
	9 79 10 80 11 12 81 14 82 15 83 16 84 17 85 18 86 19 87 88	Central 45 115 African Rep. Seychelles China 46 Southafrica Bangui Ras_Rajel Mahe m61 Karaoke Bed Riadh 116 m64 Taiqui 47 BSQ 117 m65 Zhuchang 48 Killcare Si_Thabet 118 m66 BJBJT 49 Wellington Sloughia 119 m67 Comoros Anjouan 50 Tanzania Arusha Tabarka 120 m69 Cotedivoire BouA_1 51 Thailand Bangkok Tebourba 121 m7 Cyprus Nene 52 Tunisia Ariana Utique 122 m70 France m73 53 Azib Utique_pont 123 m9 French Polynesia Moorea 54 Beja_Gare Zerga 124 tr501 55 Beja_Oued Turkey m1 125 UK Liv	4.36 36.57 27.29 36.50 34.10 36.50 26.8 36.57 35.98 36.32 40.11 36.56 7.68 36.52 34.75 37.04 43.69 37.02 -17.52 36.4014 40.62	18.55 8.52 40.688217 -4.68 119.92 -32.31 9.58 39.795018 116.29 -32.31 9.58 41.613471 105.11 -33.16 10.02 41.673115 119.39 -33.64 9.24 41.673115 -8.26 -3.36 8.45 41.663512 -5.01 13.75 9.5187 2011 2011 55.48 2010 30.279797 2003 27.65 2009 30.497299 2003 27.65 2010 26.965631 2003 151.10 2010 26.980916 2003 19.00 2008 26.980916 2011 36.68 2010 26.507806 2016 100.50 40.979638 29.055797 32.40 2004 36.54 10.13 10.00 2011 41.845866 27.806536 3.88 2016 37.13 9.56 10.02 2011 39.873036 32.737012 -149.82 1992 36.43 9.11 9.26 2011 39.7098832 30.403559 36.43 9.12 29.17 2016 53.40964 -2.907825	2011 2016 1993 2016 1993 2016 1993 2016 2011 2016 2011 2016 2012 2016 2011 2016 2011 2016 2009 2016 2011 2007	9 39 12 0 0 17 3 20 8 0 0 13 1 20 2 14 30 19 3 23 19	6 7 1 14 1 8 1 1 5 10 0 9 12 14 18 10 21 19 0 0 0 1	Dumas et al. 2013 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Duron et al. 2005 Weill et al. 2003 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013 Guillemaud et al. 1997 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Duron et al. 2005 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013 Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 This study Atyame et al 2015 Atyame et al 2015 Altinli et al 2018 Duron et al. 2005 Atyame et al 2015 Atyame et al 2015 Altinli et al 2018 Altinli et al 2018 Atyame et al 2015 Atyame et al 2015 Altinli et al 2018 Pasteur et al. 2005 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013
	20 89	Greece 56 126		Thessaloniki Bizerte m11 Willow	40.64 41.38		22.93 41.43 53.372494 37.16	2010 2016 -2.578072 9.50	2011 2001	1 19	20 0	Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Duron et al. 2005
	21 90	Haiti 57 127	USA	Gonaives Boussalem2 m12 NewYork_FT 19.44 41.49		-72.68 41.53 40.797478 36.36	1991 2016 -73.776042 2002 8.58 2011	6 0	18 4	Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013
	22 91	Italy 58 128		Latina m16	41.46 41.38 NewYork_Sewer Briss		12.90 41.57 40.797478 37.02	2011 2008 -73.776042 2002 9.53 2016	4 2	20 0	Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013
	23 92	59 129		Roma m17	Buses Madeira	41.89 41.36		12.51 41.68 39.189836 36.22	2011 2009 -84.364155 2010 9.24 2016	0 15	0 1	Dumas et al. 2013 Atyame et al 2015 Altinli et al 2018 Dumas et al. 2013
	24 93	60 130		Rome m18	Chardon EDIT	41.89 41.39		12.51 41.69 36.778259 36.36	2017 2016 -119.417931 2005 9.38 2011	55 19	3 0	This study Atyame et al 2015 Altinli et al 2018 Guillemaud et al. 1999
	25 94	Mayotte 61 131	Acoua m19	Dougga Selax	-12.80 41.36	45.20 41.66 36.778259 36.23	2013 2016 -119.417931 2005 9.14 2011	2 0	4 0	Poquet et al. 2013 Atyame et al 2015 Altinli et al 2018 Duron et al. 2005
	26 95	62 132		Bouyouni El_Alia m21 Transp	41.37	-	45.20 41.62 36.778259 37.09	2013 2016 -119.417931 1975 10.02 2010	2 16	1 0	Poquet et al. 2013 Atyame et al 2015 Altinli et al 2018 Priester & Georghiou
		63			El_Battan	12.8063889 36.48		9.53		2009		0	Atyame et al 2015 1978
	96			m25		41.31			41.35		2016		12		Altinli et al 2018
	27	133	Venezuela	Dembeni CimetierePrintemps 10.4679018 -67.4509141 1997 -12.80 45.20 2013 36.48 9.53 2011	0	24 17	Poquet et al. 2013 Atyame et al 2015 Dumas et al. 2013
	97			m26		41.31			41.34		2016		5		Altinli et al 2018
	28	64 134	Vietnam	Kahani	El_Manar HotToc	-12.80	45.20 37.017 10.830359	2013 106.691428 1995 9.52 2010	1	61 6	Poquet et al. 2013 Atyame et al 2015 Pasteur et al. 2001
	98			m27		41.32			41.30		2016		34		Altinli et al 2018
	29	135		Kawen	Saigon	-12.80	45.20 10.76262 37.01	2013 106.660172 1995 9.52 2011	4	24 7	Poquet et al. 2013 Atyame et al 2015 Pasteur et al. 2001
	99			m3		40.60			29.20		2016		6		Altinli et al 2018
	30	65 136	Zimbabwe	Mramadodou Elevage Harare	-12.80	45.20 -17.827047 31.067834 2013 36.33 9.00	2009 2001	5	0 1	Poquet et al. 2013 Atyame et al 2015 Weill et al. 2003
	100			m32		40.89			39.71		2016		33		Altinli et al 2018
	31	66		Mtsamoudou Font_Mjez	-12.80	36.39	45.20	9.28	2013	2010	0	33	Poquet et al. 2013 Atyame et al 2015
															51 53

Table . 2

 . . Primers and PCR protocols for CpDV detection (NS2) and the sequencing for partial NS1, VP codons

				CHAPTER 3:
		NS1'		diagnostic test-NS2
	step1	step2
	primer F	primer R	primer F	primer R	primer F	primer R
	CpDV_1681F	CpDV_3087R	CpDV_1681F	CpDV_2648R	Nsq1F	CpdvR2
	CACCACCACGGA	TTGGGGAGCACAA	CACCACCACGGA	TAAGACGGGAGT	CATTGGAGGAGAAAGT	TCCCCATTATCTTGCTG
	CTGGTA	GATCC	CTGGTA	TCTGGTTACATG	TGGAA	TCG
	PCR program		PCR program		PCR program
		95C 5min		95C 5min		95C 5min
	35 cycles	94C 30sec	35 cycles	94C 30sec	33 cycles	94C 30sec
		60C 30sec		58C 30sec		56C 30sec
		72C 2min		72C 2min		72C 45sec
		72C 10 min		72C 10min		72C 5min
		sequencing
		CpDV_1681F & CpDV_2648R
		VP
	step1	step2
	primer F	primer R	primer F	primer R
	CpDV_3570F	CpDV_5281R	CpDV_3570F	CpDV_4959R
	ATACCCAACCTCC	TAGCTAATGCTGG	ATACCCAACCTCC	GTATGTCTTCAAC
	CCAGGAA	TGCAAAGTGG	CCAGGAA	ACTTCGTCGTC
	PCR program		PCR program
		95C 5min		95C 5min
	35 cycles	94C 30sec	35 cycles	94C 30sec
		57C 30sec		58C 30sec
		72C 2min 45sec		72C 2min
		72C 10min		72C 10min
		sequencing

CpDV horizontal and vertical transmission 3.1 CPDV PERSISTENCE IN THE LABORATORY LINES

.......................................................... 58 3.2 EXPERIMENTAL INFECTIONS ........................................................................................

. 58 3.3 VERTICAL TRANSMISSION OF CULEX PIPIENS

  INTRODUCTION ..............................................................................................................59 3.3.2 RESULTS........................................................................................................................61 3.3.2.1 CpDV presence in ovaries and eggs ..................................................................... 61

	!
	!
	!
	!
	!
	!

DENSOVIRUS .......................................... 58 3.3.1

3.3.2.2 Effect of infection with distinct Wolbachia strains on CpDV amount in ovaries ........ 65 3.3.2.3 Effect of infection with distinct Wolbachia strains on CpDV vertical transmission ..... 66 3.3.2.4 Effect of Wolbachia curing by tetracycline on the amount of CpDV in ovaries .......... 66 3.3.2.5 Effect of Wolbachia curing by tetracycline on CpDV vertical transmission rate ......... 66 3.3.3 DISCUSSION ...................................................................................................................67 3.3.4 METHODS ......................................................................................................................70 3.3.4.1 Mosquito Lines .................................................................................................. 70 3.3.4.2 Immunolabelling ................................................................................................ 70 3.3.4.3 Electron microscopy ........................................................................................... 71 3.3.4.4 Vertical transmission assays ............................................................................... 71 3.3.4.5 Quantification of CpDV and Wolbachia................................................................ 71 3.3.4.6 Data Analysis.................................................................................................... 72

3.4 ACKNOWLEDGMENTS..................................................................................................... 72 3.5 REFERENCES .................................................................................................................. 73 3.6 APPENDIX ...................................................................................................................... 75 3.6.1 APPENDIX A ..................................................................................................................75

3.6.1.1 CpDV prevalence in Laboratory lines ................................................................... 75 3.6.1.2

Experimental Infections ...................................................................................... 75 3.6.2 APPENDIX B ..................................................................................................................78 3.6.3 REFERENCES .................................................................................................................80

#=#=! )+*7/.2<!7*2-,:/,,/0-!2,,2?,!

		water to hatch, to prevent any possible contamina-
		tion from females to the water. Five egg-rafts were
		randomly chosen per line whenever possible and 10
		L1 stage larvae from each female have been col-
		lected right after they hatched. This was repeated
		twice adding up to about 10 females and 100 L1
		stage larvae per line with a total of 500 L1 tested.
		Each L1 stage larva was individually collected
		to a 1.5 µl Eppendorf tube and crushed in 10" µl of
	sections were prepared from ovaries of 6 days old females and dechorionated eggs. Dis-sected ovaries and eggs were first fixed in 2.5% glu-taraldehyde in PBS for 2 h at 4°C, and postfixed in 2% osmium tetroxide in the same buffer for 1 h at room temperature. Samples were then thor-oughly rinced in distilled water, stained en bloc in 2% aqueous uranyl acetate solution for 2 h at room temperature, dehydrated in increasing-concentra-tion ethanol baths, embedded overnight in Embed	STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to obtain a crude DNA extraction. Two PCR reactions were conducted using these crude extractions, 1) to check the presence of CpDV using CpDVquantiF and CpDVquantiR pri-mers and 2) to check the amplifiability of DNA us-ing ace-2 gene primers, acequantidir and acequan-tirev (see above for sequences). The larvae crude extraction that did not allow any amplification in the latter were excluded from further analysis.
	812 resin (Electron Microscopy Sciences), and fur-
	ther polymerized for 2 days at 60°C. Then, ul-
	trathin sections of approximately 100 nm, per-
	formed using an LKB Ultrotome III on copper
	grids, were counterstained with uranyless (Delta
	Instruments) and lead citrate and examined using
	a JEOL 1200 EX II transmission electron micro-
	scope operating at 100 kV and equipped with an
	Olympus camera (Quemesa model, 4008 x 2664
	pixels) (MEA Platform, University of Montpellier,
	France).
	"#"Adult mosquitoes were collected from population
	cages with a mouth aspirator after 5 days following
	blood meal. CO2 was used to immobilize the mos-
	quitoes. Ten females from each line (to test the ef-
	fect of distinct Wolbachia strains: Slab, Harash,
	Tunis, BcTunis, BcHarash; effect of curing
	Wolbachia with tetracycline: SlabTC, BcTunisTC,
	BcHarashTC, Table 1) were isolated in individual
	autoclaved glass containers covered with a net.
	Once females were awake, one third of the glass
	containers were filled with bottled water that has
	been tested negative for CpDV prior to use and
	females were left to lay eggs. Egg-rafts were then
	transferred to a new sterile glass container and

3.6.2! Appendix B Supplementary Table.1. Mosquito lines, their origin, genetic background and their Wolbachia group

			Host Genetic
	Lines	Origin	Background	Wolbachia group
		USA
	Slab		Slab	wPip III
		(Georghiou et al., 1966)
		Algeria
	Harash		Harash	wPipIV
		(Alout et al., 2009)
		Tunisia
	Tunis		Tunis	wPipI
		(Duron et al., 2005)
	BcHarash	SlabTC x Harash	Slab	wPipIV
	BcTunis	SlabTC x Tunis	Slab	wPipI
	SlabTC	USA	Slab	None
	BcHarashTC	SlabTC x Harash	Slab	None
	BcTunisTC	SlabTC x Tunis	Slab	None
		Brazil (Capurro Margareth la-
	Brasil		Brasil	wPipI
		boratory)
	Ichkeul09	Tunisia (Bonneau et al., 2018)	Ichkeul09	wPipIV
	Ichkeul13	Tunisia (Bonneau et al., 2018)	Ichkeul13	wPipIV
	Istanbul	Turkey (Duron et al., 2005)	Istanbul	wPipIV
	Lavar	France (Duron et al., 2005)	Lavar	wPipII
	Utique	Tunisia (Atyame et al., 2014)	Utique	wPipI

CpDV and Wolbachia Prevalence and Diversity in Turkey 4.1 INTRODUCTION

  Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: Evidence of genetic diversity, superinfection and recombination.Mol. Ecol. 14: 1561-1573. Georghiou, G.P., Metcalf, R.L., and Gidden, F.E. (1966) Carbamate-resistance in mosquitos.

	CHAPTER 4:

....................................................................................................... 84 4.2 WOLBACHIA DIVERSITY AND INCOMPATIBILITY PATTERNS IN TURKEY ................. 85 4.3 CPDV PREVALENCE AND DIVERSITY IN TURKEY .................................................. 95 4.3.1 METHODS ......................................................................................................................95 4.3.1.1 Sample Collection. ............................................................................................. 95 4.3.1.2 PCR diagnostic test ........................................................................................... 95 4.3.1.3 Data Analysis.................................................................................................... 96 4.3.2 RESULTS........................................................................................................................96 4.3.2.1 Prevalence of CpDV. ......................................................................................... 96 4.3.2.2 Diversity of CpDV ............................................................................................. 97

Table 1

 1 Sampling sites, year, life stage and wPip groups of Culex pipiens (s.l.) individuals collected from Turkey

	Province	Sampling site	Latitude (°N)	Longitude (°E)	Breeding site type	Stage	Year	wPip-I	wPip-II	wPip-IV
	Adana	1	36.9475	35.485	Rural	A		-	5	-
	Ankara	2	39.8730	32.7370	Suburban	L		-	27	-
		3	39.8716	32.7356	Suburban	A		2	3	-
	Artvin	4	41.3884	41.4335	Suburban	L		1	35	-
		5	41.4919	41.5367	Suburban	L		-	5	-
		6	41.3833	41.5716	Rural	L		-	5	-
		7	41.3651	41.6835	Suburban	L		1	20	-
		8	41.3911	41.6933	Rural	L		7	17	-
		9	41.3646	41.6686	Suburban	L		-	3	-
		10	41.3742	41.6235	Rural	L		21	4	-
		11	41.3192	41.3534	Rural	L		12	-	-
		12	41.3178	41.3412	Rural	L		10	-	-
		13	41.3274	41.3022	Rural	L		36	-	-
		14	40.7823	41.4991	Rural	A		-	1	-
		15	41.3928	41.6937	Rural	A		4	1	-
	Aydin	16	37.4123	27.3612	Rural	A		3	-	-
	Bartin	17	41.8383	32.7115	Rural	L		10	-	-
		18	41.7411	32.3827	Suburban	L		5	-	-
	Bursa	19	40.0948	29.4912	Urban	A		1	9	-
	Duzce	20	41.0708	30.9645	Rural	L		7	3	-
	Edirne	21	41.6134	26.9656	Rural	L		-	16	11
		22	41.6731	26.9809	Rural	L		11	-	-
		23	41.6635	26.5078	Suburban	L		-	17	-
		24	40.8548	26.6897	Suburban	A		-	4	-
		25	40.9404	26.4382	Rural	A		-	1	-
	Erzincan	26	39.2476	38.5050	Rural	A		-	3	-
	Eskisehir	27	39.7950	30.4972	Urban	L		-	27	-
		28	39.2051	30.7145	Rural	A		-	2	-
		29	39.7098	30.4035	Rural	L		-	5	-
	Hatay	30	36.2516	36.3166	Suburban	A		1	-	-
	Istanbul	31	40.9481	29.3050	Urban	L		-	38	-
		32	40.9418	29.3016	Urban	L		-	44	-
		33	40.9796	29.0557	Urban	L		8	24	-
		34	41.0783	29.0136	Urban	A		-	-	1
	Kahramanmaras	35	37.5588	36.9737	Urban	A		3	-	-
	Karadeniz Ereglisi	36	41.2824	31.4241	Urban	L		6	-	-
	Kastamonu	37	41.8886	32.9995	Suburban	L		5	-	-
	Kirklareli	38	41.8458	27.8065	Rural	L		-	10	-
		39	41.5239	27.0258	Rural	A		-	6	-
		40	41.8300	27.0638	Rural	A		-	2	-
	Kocaeli	41	40.6882	30.2797	Urban	L		-	15	-
	Malatya	42	38.8180	37.9769	Rural	A		-	1	-
	Mardin	43	37.5607	40.8865	Rural	A		5	-	-
		44	37.5477	40.9588	Rural	A		5	-	-

Table 2

 2 Crossing relationships between lines from Turkey (Tek wPip-I & Tek wPip-II) and reference laboratory Wolbachia strains

		Males	Tek	Tek	Istanbul	Slab	LaVar	MaClo
			wPip-I	wPip-II	wPip-IV	wPip-III	wPip-II	wPip-III
		Mod	vi	ix	viii
	Females	Resc
	Tek I	2		C (

Les objectives de la thèse

  7.1.1 LES OBJECTIVES DE LA THESE ......................................................................................

	7.2 LES Près de trois décennies après la révolution concep-tuelle autour des interactions hôtes-bactéries mon-trant que ces dernières ne représentaient pas seule-ment une menace mais sont impliquées dans des interactions très variables avec leurs hôtes, la mé-tagénomique virale a récemment bouleversé notre façon de voir les virus. Tout a commencé par l'étude des communautés virales marines au début des années 2000. Depuis, le nombre de métaviromes publiés a augmenté de façon significative et, avec eux, notre appréhension de la diversité des virus, de leur écologie et de l'étendue des conditions en-vironnementales dans lesquelles ils interagissent avec leurs hôtes (Breitbart et Rohwer, 2005 ; Ed-wards et Rohwer, 2005 ; Rosario et Breitbart, 2011). Des études récentes ont permis de mettre en évidence de façon claire que la plupart des virus ne causent pas de dommages détectables à leurs hôtes contrairement à ce que l'on pensait (Roossinck et Bazán, 2017). Ainsi, la meilleure façon de décrire la grande majorité des virus, étant données leurs relations intimes et parfois bénéfiques avec leurs hôtes, serait de les qualifier d'endosymbiotes. En effet, tout comme les endosymbiotes bactériens, leurs interactions avec leurs hôtes peuvent se situer n'importe où -du mutualisme à l'antagonisme dans le continuum des interactions durables (Roossinck, 2015a). Si les relations antagonistes, de type para-sitaire, sont les plus étudiées, des virus mutualistes ont également été caractérisés notamment chez les plantes (Roossinck, 2015b). Par exemple, il a été du riz » de se transmettre verticalement à la pro-géniture de leur vecteur commun. Le virus nain du 7.2! Les résultats 7.3! Discussion 7.1.1! Dans ce document de thèse, je présente comment sur l'écologie et l'évolution de leurs hôtes n'est pas seulement celui d'une pression parasitaire. Il est donc à présent nécessaire de conduire des études intégratives considérant les virus comme étant en-gagés dans des interactions durables symbiotiques le long d'un gradient allant du mutualisme au pa-synergétiques entre les endosymbiote bactérien et viral (Chapitre 5) L'objectif de ma thèse de doctorat était tout riz fait de l'autostop grâce à une interaction spéci-nous avons étudié la prévalence et la diversification Au cours de mon travail de doctorat, nous avons -car la prévalence des CpDV est influencée d'abord d'étudier la prévalence et la diversité d'un fique entre une protéine de sa capside virale et une du CpDV à travers le monde (Chapitre 2). Puis montré : par les différents groupes de wPip dans la région densovirus original de moustique, le CpDV, dans protéine de la membrane externe de Sulcia (Jia et comment, nous avons décrypté leur cycle de vie et !! Une prévalence élevée et une grande diversité (Chapitre 5) les populations naturelles du moustique Cx. pi-al., 2017). leur dynamique dans nos colonies de laboratoire en d'un densovirus de moustique, le CpDV, à -car la diversification des CpDV est peut-piens. Une fois sa répartition et les relations phylo-Dans ce contexte théorique, au cours de mon particulier en étudiant leur transmission verticale l'échelle mondiale dans les populations hôtes être influencée par les différents groupes de génétiques étables, nous avons étudié plus en avant doctorat, j'ai étudié ces interactions multiples dans (Chapitre 3). Enfin, comment nous avons exploré naturelles (Chapitre 2) wPip (Chapitre 5) les interactions avec leurs hôtes et l'endosymbionte un système naturel tripartite hôte-bactéries-virus, rasitisme (Roossinck et Bazán, 2017). les interactions Wolbachia-CpDV dans les popula-!! Que l'évolution du CpDV a été façonnée par la Wolbachia dans les lignées de laboratoire et les po-entre les moustiques Culex pipiens-les endosym-Un autre changement de paradigme, égale-tions naturelles de Cx. pipiens en étudiant leur pré-recombinaison et la sélection purificatrice pulations naturelles. biotes bactériens Wolbachia-et le virus CpDV, à la ment lié au progrès des techniques de séquençage, valence et leur évolution à l'échelle locale (Cha-(Chapitre 2) Les densovirus de moustiques (MDVs) qui ont fois dans les populations naturelles et les élevages. a été de montrer que la virulence d'un micro-orga-nisme n'étaient pas la seule résultante d'une inte-raction entre un micro-organisme (" le " patho-gène) et un hôte mais plutôt la conséquence d'inte-ractions multiples entre micro-organismes au sein de l'organisme hôte (Vayssier-Taussat et al., 2014). Pourtant, et c'est le cas même dans le contexte de teurs-insectes. Ainsi, il a été démontré qu'un sym-génétique et structurelle à partir de moustique pipiens : et verticale des virus des végétaux par leurs vec-avant cette thèse limitées à leur caractérisation les populations naturelles tunisiennes de Cx. rectes semblent affecter la transmission horizontale Les connaissances sur ce virus CpDV étaient la charge virale, et la diversité des CpDV dans par voie orale. D'autres types d'interactions di-évolutive des interactions hôte-virus-bactéries. !! Une influence des Wolbachia sur la prévalence, biote lorsqu'elles sont infectées par un poliovirus virus pertinents pour étudier l'écologie (Chapitre 4) deux fois moins que celles qui n'ont pas de micro-hôtes arthropodes, semblent également être des gion critique dans la lutte antivectorielle les souris ayant un microbiote normal meurent !! Les densovirus, aussi anciens et divers que leurs wPip ainsi que des CpDV en Turquie, une ré-(Almand et al., 2017 ; Douglas, 2016). Par exemple, toutes origines évolutives. !! La forte prévalence et diversité des Wolbachia indirectement par le biais de leur phénotype étendu relations symbiotiques avec des organismes de pitre 3) biotes peuvent interagir entre eux directement ou très diversifiés et capables d'établir des sur la transmission verticale du CpDV (Cha-glas, 2016). Il est cependant clair que les endosym-pertinents dans l'endosymbioses car ils sont chia, sur la charge de CpDV dans les ovaires et sur les interactions binaires hôte-symbiote (Dou-!! Les insectes représentent des modèles d'étude !! L'effet du microbiote-hôte, y compris Wolba-diées et les recherches se sont surtout concentrées et médicales. intime avec Wolbachia (Chapitre 3) symbiotes au sein d'un hôte n'ont été que peu étu-de la science, y compris les sciences appliquées les ovocytes où ils peuvent être en interaction la symbiose mutualiste, les interactions entre les pitre 4 et 5). été comparativement plus étudiés que de nombreux !! L'absence d'une structure géographique dans la !! Les symbiotes bactériens, dont Wolbachia, ont diversité du CpDV qui suggère leur dispersion autres virus arthropodes, le sont encore rarement été caractérisés depuis de nombreuses années efficace dans le monde entier (Chapitre 2) dans un contexte écologique et évolutif. En effet, comme étant des partenaires intimes des les recherches conduites sur les MDVs se sont prin-!! Infection persistante par le CpDV dans des li-organismes multicellulaires. Le fait que les gnées de laboratoire apparemment en « bonne cipalement concentrées sur leur pathogénicité micro-organismes soient fondamentaux dans santé » (Chapitre 3) et/ou leur application comme outil de lutte anti-l'édification du phénotype des animaux et des végétaux, a révolutionné de nombreux aspects !! La transmission verticale du CpDV en utilisant vectorielle
	démontré que plusieurs virus végétaux confèrent à issus des élevages de l'institut des sciences de -du fait de la corrélation positive entre les	biote bactérien obligatoire commun chez les cica-
	leurs hôtes une tolérance à la sécheresse et au froid l'évolution de l'Université de Montpellier. densités des wPip et celle des CpDV chez les	delles, nommé Sulcia, permettait au « virus nain
	(Xu et al., 2008), soulignant que l'impact des virus moustiques infectés suggérant des interactions

RESULTATS ............................................................................................................ 7.3 DISCUSSION .................................................................................................................

7.1! Introduction

  Les récents progrès des technologies de séquençage et des techniques de biologie moléculaire ont permis aux chercheurs de révéler l'ubiquité et la diversité des virus. Malgré ces progrès, la découverte des virus s'est surtout concentrée sur les virus de vertébrés. De plus, les virus connus sont très peu étudiés en terme d'écologie virale. Les arthropodes de par leur histoire évolutive ancienne représentent 80% de la diversité animale sur notre planète. L'omniprésence de l'endosymbiose chez les arthropodes et leur capacité d'héberger des symbiotes (parfois multiples) suggèrent que leurs interactions avec leurs virus pourraient également être de nature symbiotique, allant des interactions durables de type mutualiste à celles de type antagoniste. Dans ce contexte, mon travail de thèse s'est concentré sur un densovirus de moustiques, le Culex pipiens densovirus (CpDV), en étudiant leur prévalence et leur diversité dans les populations naturelles de moustiques Culex pipiens et leurs interactions avec les bactéries endosymbiotiques Wolbachia dans un système naturel hôte-bactérie-virus (Cx. pipiens -Wolbachia -CpDV), tant dans des populations naturelles que des colonies de laboratoire. Nous avons révélé une forte prévalence et une grande diversité de CpDV dans les populations mondiales de Cx. pipiens. De plus, le CpDV infecte de façon persistante les lignées de laboratoire et est transmis verticalement avec l'endosymbiote Wolbachia. Nous montrons une prévalence et une diversité élevées du CpDV en Turquie et en Tunisie, et nous nous sommes concentrés davantage sur ces deux pays pour étudier les interactions Wolbachia-CpDV dans les populations naturelles. En Tunisie, dans une zone de contact étroite où deux groupes de Wolbachia génétiquement distincts coexistent, ces différents groupes de Wolbachia influencent la prévalence et la diversité du CpDV. Nous montrons également une corrélation positive entre la densité de Wolbachia et la densité de CpDV. D'une manière globale, nos résultats suggèrent que le système CpDV-Cx.pipiens -Wolbachia et plus généralement les densovirus sont de bons modèles pour étudier les interactions écologiques entre ces partenaires et leur évolution dans la nature et au laboratoire.

Chapter 6 -Discussion

Acknowledgements

I would like to start by warmly thanking my supervisor Mathieu Sicard, for believing in me when no one else did and for all his efforts to make this PhD project possible! Thank you Mylene Weill for believing in Mathieu (and later on in me-I hope) and accepting me to the team EVAS and supporting me through my PhD. I also thank my co-supervisor Bülent Alten from Hacettepe University for accepting to collaborate with me and for his support, and to Filiz Gunay for being my co-co-supervisor, friend and inspiration with her invincible optimism.

Acknowledgements

We are thankful to Dr Hilal Bedir (Kafkas University, Kars, Turkey), Dr Berna Demirci (Kafkas University, Kars, Turkey) and Dr Mustafa Akiner (RTE University, Rize, Turkey) for their guidance during fieldwork, to Dr Sırrı Kar (Namık Kemal University, Tekirdağ, Turkey) for the collection of Cx. pipiens from Tekirdağ (Turkey), to Patrick Makoundou, Sandra Unal and Marco Perriat-Sanguinet from team EVAS (ISEM) for technical support.

5.1.5! Acknowledgements

We thank Sandra Unal and Patrick Makoundou for technical support, Dr Philippe Clair for his help in the Real Time Quantitative PCR experiments which were performed at the "qPCR Haut Debit (qPHD) Montpellier génomiX" platform. Sequencing data were produced at the LabEX "Centre Méditerranéen de l'Environnement et de la Biodiversité" in the GENSEQ platform. We also thank Emmanuel Douzery for helpful discussions.

Embassy in Turkey and Project "ISEM Sud", 2016 for her PhD.

3.5! References

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Funding

This work was funded by the French ANR program (project ANR-16-CE02-0006-01 "CIAWOL", 2017-2020). Mine Altinli received support from Ministère des affaires étrangères (Campus France) via the French Embassy in Turkey and Project "ISEM Sud", 2016 for her PhD.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

iii Ethics approval and consent to participate Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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