Camilla Rothe
email: rothe@lrz.uni-muenchen.de

Thorbjörn Zimmer

Mirjam Schunk

Claudia Wallrauch

Kerstin Helfrich

Fatih Gültekin

Gisela Bretzel

Jean-François Allienne

Jérôme Boissier

Jean-François Allienne

J.- F Allienne

Developing Endemicity of Schistosomiasis, Corsica, France

Keywords: Developing Endemicity of Schistosomiasis, Corsica, France

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A 49-year-old man from Germany experienced macrohematuria in June 2020 and underwent cystoscopy in August 2020. Histologic analysis of a bladder biopsy specimen showed ova of Schistosoma spp. He was referred to the outpatient department for tropical medicine at LMU Hospital Munich.

The patient had never traveled outside Europe. He had, however, traveled twice to Corsica, France, in 2013 and 2019. He never swam in the Cavu River, which has been associated with cases of schistosomiasis in recent years [START_REF] Berry | Schistosomiasis haematobium, Corsica, France[END_REF][START_REF] Moné | Introgressive hybridizations of Schistosoma haematobium by Schistosoma bovis at the origin of the first case report of schistosomiasis in Corsica (France, Europe)[END_REF][START_REF] Ramalli | Persistence of schistosomal transmission linked to the Cavu River in southern Corsica since 2013[END_REF].

The patient visited Corsica during August 22-September 4, 2019. By using GPS data from his smartphone and camera, he reconstructed his bathing sites precisely. During August 22-24, he swam several times in the Solenzara River in the southeastern part of the island, near a busy campsite (Figure). Further minor water contacts might have occurred at the Gravona River in western Corsica near Ajaccio, at a turtle park and near a campsite, and at the Tavignano River (Figure). He did not bathe in either of Corsica's other rivers but could not rule out contact with water for cooling purposes. The patient reported swimming in the Restonica River in 2013 (Figure ), but he did not recall any itchy rash suggestive of cercarial dermatitis.

Physical examination was unremarkable. Full blood count did not show any eosinophilia. Urine sediment microscopy showed terminal-spined ova resembling those of S. hematobium parasites. Urine tested in-house by quantitative PCR for S. hematobium parasites was positive. A schistosoma serologic test was positive by enzyme immunoassay (64 U/L; reference <10 U/L); results of a blood immunochromatographic rapid diagnostic test were positive, but Western blot results were negative. Results of a urine circulating cathodic antigen point-of-care test was weakly positive. His son, who traveled with him in 2013, and his wife, who had joined him on both trips, had negative serologic test results and were negative for urine schistosoma by PCR.

DNA extracted from 7 ova was successfully amplified on nuclear internal transcribed spacer and mitochondrial cytochrome oxidase I markers. For the nuclear marker, DNA (995 bp) of all eggs showed a typical signature of S. hematobium parasite. The mitochondrial marker (873 bp) of all eggs had a typical signature of S. bovis parasite and displayed the same haplotype. In summary, all eggs were identified as hybrids of S. hematobium and S. bovis (ShxSb) parasites. This type of hybrid (nuclear Sh and mitochondrial Sb) was the most frequently observed type during the 2013 outbreak (4). The mitochondrial cytochrome oxidase I sequences of all eggs had 100% homology with haplotype Sb2, the most frequent haplotype observed during the 2013 outbreak (4). Presence of this haplotype was also documented in subsequent reports on the first outbreak in Corsica (2,3).

Transmission of schistosomiasis in Corsica has been documented since 2013 [START_REF] Ramalli | Persistence of schistosomal transmission linked to the Cavu River in southern Corsica since 2013[END_REF][START_REF] Holtfreter | Schistosoma haematobium infections acquired in Corsica, France, August 2013[END_REF]. Cases have been detected in residents and in tourists [START_REF] Holtfreter | Schistosoma haematobium infections acquired in Corsica, France, August 2013[END_REF][START_REF] Berry | Schistosomiasis screening of travelers to Corsica, France[END_REF]. In spring 2014, after the first outbreak, intense screening and public health efforts led to the identification of 106 cases linked to the Cavu River [START_REF] Noël | Large outbreak of urogenital schistosomiasis acquired in Southern Corsica, France: monitoring early signs of endemicization?[END_REF]. During 2015-2018, sporadic cases were detected and linked to the Cavu [START_REF] Ramalli | Persistence of urogenital schistosomiasis in Southern Corsica since 2013[END_REF]. The cases reported in 2018 involved 2 patients who had bathed in the Cavu and Solenzara Rivers [START_REF] Ramalli | Persistence of schistosomal transmission linked to the Cavu River in southern Corsica since 2013[END_REF].

The patient we describe reported having no contact with the Cavu River. Instead, the infection was most likely acquired in the Solenzara River, near a busy campsite. The Solenzara neighbors the Cavu, but their waters do not intermingle. No confirmed cases acquired in the Solenzara River have been documented, but Bulinus truncatus snails or their DNA have been found along the river during environmental surveys [START_REF] Berry | Schistosomiasis haematobium, Corsica, France[END_REF][START_REF] Mulero | Persistent establishment of a tropical disease in Europe: the preadaptation of schistosomes to overwinter[END_REF]. Snail density close to the main bathing site of our patient appears to be high [START_REF] Mulero | Persistent establishment of a tropical disease in Europe: the preadaptation of schistosomes to overwinter[END_REF]. Neither of the other rivers mentioned was ever proven to be a source of schistosomiasis; in addition, they are all relatively far away from the Cavu region.

Genotyping revealed the S. hematobium-S.bovis hybrid parasite described in previous outbreaks, suggesting ongoing transmission rather than reintroduction. The parasite's emergence in another river cannot be explained by the persistence of infected snails (9) but could be explained by reseeding of the river by a mammalian host.

Animal reservoirs have been discussed as a possible explanation for ongoing transmission; however, evidence of a major role is lacking. No infection has been detected in livestock in the region, and the only infected animals found were 2 rats [START_REF] Oleaga | Epidemiological surveillance of schistosomiasis outbreak in Corsica (France): are animal reservoir hosts implicated in local transmission?[END_REF]. Even if we cannot rule out the influence of an undetected animal reservoir (e.g., Ovis aries musimon, wild sheep native to Corsica, have never been tested), the most likely explanation is that 1 or several infected persons continue to infest the water.

In summary, this case highlights that transmission of schistosomiasis in Corsica is ongoing and is no longer restricted to the Cavu River. The parasite appears to be of the same strain detected previously on the island. The infection was acquired at a frequented tourist site, suggesting that more persons might have been infected. Further screening of residents and tourists is urgently needed.

About the Author

Dr. Rothe is deputy head of the division of tropical medicine at the LMU Hospital Munich. Her main areas of interest are clinical tropical medicine, neglected tropical diseases, emerging infections, travel medicine, and migrant medicine.


Angela Di Cesare

Jérôme Depaquit
email: jerome.depaquit@univ-reims.fr

Leila Hadj-Henni

Thibaut De Meulemeester

Philippe Noël

Adeline Germain

Remi Helder

Denis Augot

Comparison of vertebrate cytochrome b and prepronociceptin for blood meal analyses in Culicoides

Keywords: Culicoides, blood meal, PCR, PNOC, Cyt b, France

To date, studies on host preferences and blood meal identification have been conducted for Culicoides species using molecular-based methods such as PCR techniques to amplify only a fragment from universal vertebrate mitochondrial genes such as cytochrome c oxidase subunit I or cytochrome b (Cyt b). The vertebrate prepronociceptin gene (PNOC) was also tested in this field. However, the choice of molecular marker to identify blood meal is critical. The objective of our study is to compare the ability of Cyt b and PNOC as molecular markers for blood meal identification depending on the stage of blood meal digestion. In order to determine whether these Cyt b and PNOC could provide a positive result, 565 blood-fed females of Culicoides spp were collected and morphologically identified. The samples were collected between 2012 and 2014, in two localities in France. The collection localities were near either livestock or a forest. To catch the specimens, we used UV CDC miniature light traps. PNOC sequence of donkeys (Equus asinus) was sequenced and submitted because it was missing in GenBank. Our findings emphasize that the PNOC marker is not suitable to separate closely related Equid species such as horses and donkeys. The Cyt b marker was able to identify 204 more samples when compared to PNOC (99.55% of specimens). Cyt b appears to be better able to detect the origin of blood meals from females with digested blood in their abdomens. We conclude that Cyt b is a good marker as it increases the accuracy of blood meal identification of engorged females containing digested blood in their abdomens. The host opportunist behavior of Culicoides, especially that of C. obsoletus and C. scoticus, the main vectors of BTV in Europe was also highlighted.

Introduction

The study of host preferences of the native population of hematophagous Culicoides spp is critical in assessing and taking action to prevent infectious disease outbreaks. A better knowledge of this field could help identify the animal reservoirs for arboviruses. In Europe, before BTV-8 experience, bluetongue was considered an exotic disease that could be controlled by animal movement restriction and vaccination [START_REF] Wilson | Bluetongue in Europe: past, present and future[END_REF]. Since the spread of BTV-8 in northern Europe, despite the absence of C. imicola, the main African vector of BTV, some abundant autochthonous Culicoides species have been suspected and incriminated, mainly species from Obsoletus and Pulicaris groups [START_REF] Mellor | Bluetongue virus in the Mediterranean Basin 1998-2001[END_REF].

More recently, in autumn 2011, an unidentified disease of livestock was reported in Germany and, the etiologic agent of this disease was identified as a novel orthobunyavirus and named Schmallenberg virus (SBV) [START_REF] Hoffmann | Novel orthobunyavirus in cattle, Europe, 2011[END_REF]. Midges of the group Obsoletus have been shown to be natural vectors for this virus (4).

To date, many studies on host preferences have been conducted for Culicoides species using serological assays [START_REF] Murray | The identification of blood meals in biting midges, (Culicoides: Ceratopogonidae)[END_REF][START_REF] Blackwell | Identification of blood meals of the Scottish biting midge, Culicoides impunctatus, by indirect enzyme-linked immunosorbent assay (ELISA)[END_REF][START_REF] Blackwell | The use of an enhanced ELISA method for the identification of Culicoides blood meals in host-preference studies[END_REF]. However, these techniques are time consuming and have limited sensitivity and specificity [START_REF] Önder | Tracking the sources of blood meals of parasitic arthropods using shotgun proteomics and unidentified tandem mass spectral libraries[END_REF]. Molecular-based methods were also used. PCR techniques were used to amplify only a fragment from the universal vertebrate mitochondrial genes cytochrome c oxidase subunit I (COI) or cytochrome b (Cyt b) [START_REF] Calvo | Host feeding patterns of Culicoides species (Diptera: Ceratopogonidae) within the Picos de Europa National Park in Northern Spain[END_REF][START_REF] Lassen | Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides latreille) in Denmark[END_REF][START_REF] Martínez-De La Puente | Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain[END_REF][START_REF] Pettersson | Molecular identification of blood meals and species composition in Culicoides biting midges[END_REF][START_REF] Santiago-Alarcon | Urban forests as hubs for novel zoonosis: blood meal analysis, seasonal variation in Culicoides (Diptera: Ceratopogonidae) vectors, and avian haemosporidians[END_REF]. A species-specific multiplex PCR assay was also developed [START_REF] Garros | Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on palaearctic biting midge species, vectors of orbiviruses[END_REF][START_REF] Ayllón | Feeding behaviour of Culicoides Spp. (Diptera: Ceratopogonidae) on cattle and sheep in Northeast Germany[END_REF]. The vertebrate prepronociceptin gene (PNOC) was also tested [START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF][START_REF] Slama | Blood meal analysis of Culicoides (Diptera: Ceratopogonidae) in Central Tunisia[END_REF]. However, the proportion of successful identification of blood meal origins is variable and may be affected by the quality of the samples and the laboratory protocols.

To date, there is no study to compare these molecular markers, except that of Slama et al. [START_REF] Slama | Blood meal analysis of Culicoides (Diptera: Ceratopogonidae) in Central Tunisia[END_REF] during which the authors showed that in the PNOC-PCR sequencing methods, the sensitivity of host DNA detection was lower than found by Cyt b PCR method. Here, to compare and to test the ability to identify the origin of blood meals taken from vertebrate hosts of both molecular markers, i.e., PNOC and Cyt b, we carried out our study on 565 wild-caught females of Culicoides spp trapped near livestock and forest in two localities in France. Determination of the blood meal origin was carried out through simple sequencing and Blast of the sequences in GenBank. We also compared the ability of each marker to provide interpreted sequences depending on blood digestion stage. The capacity of each marker to amplify bird DNA and to separate related animal species was also checked.

Materials and Methods

Collection and Morphological Identification of Biting Midges

In this study, 565 engorged Culicoides females were collected using UV CDC miniature light traps (John W. Hock Company, FL, USA). These traps were operated monthly in the period from 2012 to 2014, from sunset to sunrise. Three traps were positioned at a farm containing livestock (horses, sheep, cattle, and other domestic animals) as described by Lassen et al. [START_REF] Lassen | Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides latreille) in Denmark[END_REF] and Ninio et al. [START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF]. The fourth trap was set in a forest area (Table 1).

All blood-fed females of Culicoides were preserved in a 95% ethanol solution until their processing. To avoid crosscontamination, every dissection was performed with single-use sterile equipment. Head, genitalia, and wings were mounted between slide and cover slip to identify the specimens morphologically according to wing pattern [START_REF] Delécolle | Nouvelle contribution à l' étude systématique et iconographique des espèces du genre Culicoides[END_REF]. The insect identification was conducted twice by two different persons. The species as C. obsoletus and C. scoticus are difficult to distinguish morphologically and were therefore identified according to specific morphological and morphometrical characters [START_REF] Augot | Discrimination of Culicoides obsoletus and Culicoides scoticus, potential bluetongue vectors, by morphometrical and mitochondrial cytochrome oxidase subunit I analysis[END_REF]. In addition, their identification was confirmed by molecular sequencing of the mitochondrial COI gene using the primers C1-J-1718 and C1-N-2191 (20) (Table 2).

Extraction of DNA for Blood Meal Analyses

Using a conservative approach, we included all available females showing any remainder of blood in their abdomen in order to detect the maximum number of potential hosts. The color of the abdomen was observed under a magnifying glass and, if necessary, under a microscope, in order to evaluate the stage of digestion of the blood meal a visual estimation of the digestion status of Culicoides blood meals). We thus included: (i) fully engorged females (n = 220) with red, brownish-red, and darkred abdomens (the color of the blood meal could depend on the time that passed before the midges were transferred in ethanol, and/or the length of storage in ethanol before dissection); (ii) abdomens showing advanced signs of digestion (n = 264); and finally (iii) females showing any remainder of blood in their abdomen (n = 81) (Figure 1).

Abdomens were transferred in individual, sterilized, 1.5 ml vials, and stored at -20°C. After an initial manual crushing step, the extraction was carried out using the QIamp DNA mini Kit (Qiagen, GmbH, Hilden, Germany) following the manufacturer's instructions. The DNA was eluted in a final volume of 120 µl of AE buffer.

Blood Meal Molecular Analyses

The blood meal analyses were performed by sequencing the vertebrate's PNOC using the primers PNOC (F) and PNOC (R) following the PCR conditions described by Haouas et al. [START_REF] Haouas | Development of a molecular tool for the identification of Leishmania reservoir hosts by blood meal analysis in the insect vectors[END_REF]. We have also used the degenerate and universal vertebrate primers from the Cyt b gene modified by Kocher et al. [START_REF] Kocher | Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers[END_REF]. The amplification conditions and the primer sequences are illustrated in the Table 2. PCRs for both markers were performed in a 50 µl volume using 10 µl of extracted DNA solution, 50 pmol of the primers, and Taq polymerase (5 ′ , Germany).

Contamination was checked via the inclusion of a negative control in each run. Moreover, two positive controls were also processed in each run: DNA extracts from cattle (Bos taurus) and from Mallard ducks (Anas platyrhynchos). Mallard DNA was used to check the capacity of each marker to amplify bird DNA. The PCR products were examined using electrophoresis on a 2% agarose gel with ethidium bromide staining. PCR products were sequenced bi-directionally using the primers typically used for PCR.

Sequencing and Data Analyses

Sequences of at least 200 bp for PNOC and 300 bp for Cyt b were compared to homologous sequences deposited in Gen-Bank using BLAST. Blood meals were considered as identified to the species level if their sequence showed ≥98% homology with a sequence deposited into GenBank. Sequence editing and analyses were performed using BioEdit Sequence Alignment Editor V7.2.3 software.

Statistical Analyses

To test the efficiency of each marker to provide a positive result and to check if the two markers react in the same way at different stages of digestion (PNOC vs. Cytb), a chi-square analyses were carried out.

Results

Culicoides Species Identification

A total of 59,704 Culicoides biting midges [females (nulliparous, blood-fed, gravid, parous) and males] were collected by the four UV CDC miniature light traps used in this study during the period 2012-2014. The total sample included the following groups: 33,470 were nulliparous (56.05%), 565 were blood-fed (0.95%), 6173 were gravid (10.34%), 17,988 were parous (30.13%), and 1508 were male (2.53%).

In the present study, the most abundantly sampled species was C. obsoletus (34.64%), followed by C. achrayi (24.54%), C. scoticus (14.43%), and C. punctatus (13.2%). Although C. lupicaris (2.06%), C. newsteadi (1.44%), C. furcillatus (5.77%), C. brunnicans (0.44%), C. pulicaris (1.44%), C. subfasciipennis (1.88%), C. vexans (0.2%), and C. chiopterus (0.4%) were less numerous in our sampling (Table 3).

Blood Meals Analyses

From the 565 Culicoides identified as blood-fed, at least 11 different species were found to have fed on horses (73.92% for Cyt b and 95.28% for PNOC), 5 different species on donkeys (Equus asinus), 4 different species on cattle, only 1 on a cat (Felis silvestris), and 1 on a chicken (Gallus gallus). Finally, Cyt b analyses show that 19.20% of Culicoides fed on humans (Homo sapiens) vs. 4.81%, the result established with PNOC (Table 3).

Analyses from Cyt b show that all C. punctatus, which were caught in Boult-aux-Bois (forest) (n = 13) (Table 3), are the main species that took blood meals from donkeys. Further, we observed in GenBank two haplotypes of horse sequences with a PNOC gene differing by a single base (e.g., sequences under the accession numbers XM_001493120 and AY011855) (Figure 2). We noted that the sequence of donkeys was missing. Consequently, we provided it from donkey blood following the protocol previously described (GenBank accession number KM521860). We compared the donkey blood and found it to be identical to the first haplotype of Equus caballus (accession number XM_001493120). This led us to the conclusion that the PNOC gene is unable to distinguish E. caballus sequences from those of E. asinus (99% homology) whereas Cyt b is able to distinguish both species.

Equally, no sequences were reported from PNOC using the Mallard DNA as opposed to the successful amplification that was reported using Cyt b. Evidence of mixed blood meals was not observed in the present study.

In our study, analyses from PNOC and Cyt b genes detected blood meals of all 220 fully engorged females, 184 (83.63%) and 219 (99.55%), respectively. During the analyses from females showing advanced signs of blood digestion (n = 264), PNOC provided only seven sequences (2.65%) whereas Cyt b identified 176 blood meals (66.67%). No identification was reported from either marker on abdomens containing only traces of blood.

Statistical analyses from Cytb (χ 2 = 281.3717, df = 2, p < 2.2e-16) and PNOC (χ 2 = 399.9959, df = 2, p < 2.2e-16) show a very low p-value for both markers, meaning that the success of the two markers to provide a positive result is different depending the stage of digestion. Comparison of the difference in success between the two markers (PNOC vs. Cytb) provided a zero value of p-value (χ 2 = 1717.429, df = 2, p = 0) meaning that markers do not react the same way facing the stage of digestion.

Discussion

In the present study, the PCR technique based on the Cyt b gene of mitochondrial DNA (mtDNA) and the PNOC was selected for blood meal identification: (i) fully engorged Culicoides, (ii) Culicoides showing an advanced sign of digestion, and (iii) females showing any remainder of blood in their abdomen. The objective was to determine the ability of each marker to detect DNA from vertebrate hosts in the abdomens of Culicoides depending on the stage of blood meal digestion. Our results show that the Cyt b marker was more efficient (66%) than PNOC to detect the origin of blood meals in advanced stages of digestion; this could be explained by the fact that immature erythrocytes have lost their mitochondria during the final stages of maturation, which are found in abdomens of engorged arthropods [START_REF] Tyler | The Mitochondrion in Health and Disease[END_REF]. Several studies have also highlighted that there is a significant negative relationship between the time since ingestion and the success of analyses [START_REF] Haouas | Development of a molecular tool for the identification of Leishmania reservoir hosts by blood meal analysis in the insect vectors[END_REF][START_REF] Mukabana | Analysis of arthropod blood meals using molecular genetic markers[END_REF][START_REF] Oshaghi | Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes[END_REF][START_REF] Martínez-De La Puente | Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus[END_REF]. It is interesting to note that most studies in this field include only fully engorged females. The lack of identification of blood meal origin by both markers in digested blood and in abdomens with some traces of blood can be explained by the fact that host DNA was extensively degraded during digestion. Kirsten and Gray [START_REF] Kirstein | A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus[END_REF] in their study on engorged Ixodes ricinus ticks, a vector of Lyme disease, explain that nested PCR amplification of PCR product could increase detection sensitivity and the limit of detection to 40 days postdigestion [START_REF] Kirstein | A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus[END_REF].

In the present study, no interpretable sequences were reported with both markers, perhaps due to contamination even though the samples were prepared under sterile conditions. DNA purification prior to PCR would be necessary for these samples and the use of specific-species primers could increase the yield of successful identification [START_REF] Garros | Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on palaearctic biting midge species, vectors of orbiviruses[END_REF].

More than 19% of our positive samples gave sequences that matched Homo sapiens in our study. In order to check contamination, it would be better to test Culicoides provided sequences matching Homo sapiens consistently with humanspecific primers that amplify a segment of the hyper variable mitochondrial control region [START_REF] Morin | Kin selection, social structure, gene flow, and the evolution of chimpanzees[END_REF]. In fact, it was not expected that primers amplifying specific segments of human mtDNA would also amplify the corresponding segments of mtDNA from other species [START_REF] Kocher | Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers[END_REF].

In our study, no identification conflicts were reported between PNOC and Cyt b markers except blood meals originating from donkeys and horses because the PNOC marker failed to separate donkey and horse DNA. The same findings were reported in the study of Ninio et al. [START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF], where no differentiation was made with PNOC between wild boars (Sus scrofa scrofa) and pigs (Sus scrofa). We conclude that PNOC is not suitable to separate closely related species such as Equidae; this marker is not a good candidate to be used alone. We confirm also the findings of Haouas et al. [START_REF] Haouas | Development of a molecular tool for the identification of Leishmania reservoir hosts by blood meal analysis in the insect vectors[END_REF] and Slama et al. [START_REF] Slama | Blood meal analysis of Culicoides (Diptera: Ceratopogonidae) in Central Tunisia[END_REF], in which PNOC is only suitable to detect blood meal origin from mammals but not from birds. In addition, the PNOC gene underperforms in detecting blood meal origin, especially from abdomens with digested blood. Consequently, we confirm that in this type of study, the gene of choice is the Cyt b, because it is present in numerous copies in each cell [START_REF] Townzen | Identification of mosquito blood meals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences[END_REF]. It is also known that vertebrate mtDNA can evolve faster than nuclear DNA, thus they are more polymorphic and more useful for species identification [START_REF] Kocher | Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers[END_REF]. The Cyt b marker has also been used successfully on ticks [START_REF] Kirstein | A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus[END_REF][START_REF] Wodecka | Host and pathogen DNA identification in blood meals of nymphal Ixodes ricinus ticks from forest parks and rural forests of Poland[END_REF], mosquitoes [START_REF] Lee | Identification of mosquito avian-derived blood meals by polymerase chain reactionheteroduplex analysis[END_REF][START_REF] Kent | Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome b[END_REF], tsetse flies [START_REF] Steuber | PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae)[END_REF], sandflies [START_REF] Laskay | Development of a host blood meal database: de novo sequencing of hemoglobin from nine small mammals using mass spectrometry[END_REF], and Culicoides [START_REF] Pettersson | Molecular identification of blood meals and species composition in Culicoides biting midges[END_REF][START_REF] Santiago-Alarcon | Urban forests as hubs for novel zoonosis: blood meal analysis, seasonal variation in Culicoides (Diptera: Ceratopogonidae) vectors, and avian haemosporidians[END_REF]. In most of these studies, Cyt b has proven its discriminating power although in some studies, no differentiation has been made between domestic pigs and wild boars (Sus scrofa), sheep and mouflon (Ovis gmelini musimon), and ibex (Capra ibex) and goat (Capra aegagrus) because of their close phylogenetic heritage [START_REF] Garros | Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on palaearctic biting midge species, vectors of orbiviruses[END_REF]. RFLP analysis based on Cyt b sequences has been limited to discriminating animals of the same genus, namely, C. elaphus and C. nippon. However, analyses of the available sequence data indicate that this problem could be resolved, because sufficient variability exists in the Cyt b sequence to design specific probes, which could distinguish between related species [START_REF] Kirstein | A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus[END_REF]. In the present study, our degenerate primers discriminated E. caballus and E. asinus, two closely related species from the same genus (Equus).

In the study of Pettersson et al. [START_REF] Pettersson | Molecular identification of blood meals and species composition in Culicoides biting midges[END_REF], in order to increase the opportunity for host identification, the authors added a second widely studied gene COI for blood meal analysis for samples that did not produce results with Cyt b primers. Consequently, identification of Culicoides blood meals using the Cyt b gene coupled with COI gene for vertebrates is a logical application since the COI and Cyt b DNA barcoding regions from more species are being sequenced and submitted to public databases. The capacity for identification of blood meals via both markers will become more and more precise.

In the present study, our sampling strategy was to use light traps close to livestock as suggested by Garros et al. [START_REF] Garros | Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on palaearctic biting midge species, vectors of orbiviruses[END_REF] to maximize the capture of blood-fed females on the hosts present at study sites (wild fauna for Boult-aux-Bois and domestic fauna for Louvois). Although most studies on host preferences of Culicoides found that large mammals, specifically ruminants, were the preferred host for biting midges (Culicoides spp.) [START_REF] Calvo | Host feeding patterns of Culicoides species (Diptera: Ceratopogonidae) within the Picos de Europa National Park in Northern Spain[END_REF][START_REF] Lassen | Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides latreille) in Denmark[END_REF][START_REF] Martínez-De La Puente | Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain[END_REF][START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF]. However, we observed the susceptibility of horses and ponies (=E. caballus) to the attacks of Culicoides biting midge species (more than two-thirds of blood-fed Culicoides, examined with both markers, fed on horses). This observation could be expected given that horses and ponies were the most common hosts available in the vicinity of the four trap locations. In fact, every trap had horse as the most numerous blood meal host. Even the trap in the forest caught a higher number of specimens with blood meals originating from horses than any other potential hosts even though no horse was nearer than 450 m. In addition, C. obsoletus was the most common species found to have fed on horses () suggesting that this species is prone to opportunistic feeding behavior as it is known to be attracted to horses, cattle, and sheep [START_REF] Calvo | Host feeding patterns of Culicoides species (Diptera: Ceratopogonidae) within the Picos de Europa National Park in Northern Spain[END_REF][START_REF] Lassen | Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides latreille) in Denmark[END_REF][START_REF] Townley | Preferential landing and engorging sites of Culicoides species landing on a horse in Ireland[END_REF][START_REF] Carpenter | An assessment of Culicoides surveillance techniques in Northern Europe: have we underestimated a potential bluetongue virus vector?[END_REF][START_REF] Gerry | Biting rates of Culicoides midges (Diptera: Ceratopogonidae) on sheep in Northeastern Spain in relation to midge capture using UV light and carbon dioxide-baited traps[END_REF][START_REF] Viennet | Host preferences of palaearctic Culicoides biting midges: implications for transmission of orbiviruses[END_REF]. Host-feeding behavior of Culicoides species may also be influenced by semiochemicals factors, and even shape or size of potential hosts [START_REF] Humphreys | Blood-feeding activity of female Culicoides (Diptera: Ceratopogonidae)[END_REF][START_REF] Raich | Culicoides variipennis (Diptera: Ceratopogonidae) host selection in Colorado[END_REF][START_REF] Logan | Identification of human-derived volatile chemicals that interfere with attraction of the Scottish biting midge and their potential use as repellents[END_REF]. In the present study, no mixed blood meals were detected. Our results also suggest that other species than the putative vectors can feed on horses, including C. furcillatus, C. subfasciipennis, C. brunnicans, and C. achrayi. There are poor studies on those species maybe because is not predominant in light traps' catches [START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF]. In our study, C. achrayi was abundant. It would also be interesting in further studies to assess the variety of hosts through diversification of trapping sites and a larger sampling. The investigation on host-feeding pattern of those species and on other species of Culicoides in general is important in understanding the epidemiology of Culicoides transmitted diseases, which affect domestic as horses and wild animals as well.

Analyses from Cyt b show that some blood-fed females (C. punctatus) that were caught on Boult-aux-Bois took blood meals from donkeys; note that a donkey was closer than ∼1.3 km from where the blood meal was collected (trap N°1, Table 1). Lassen et al. [START_REF] Lassen | Identity and diversity of blood meal hosts of biting midges (Diptera: Ceratopogonidae: Culicoides latreille) in Denmark[END_REF] collected Culicoides fed on cattle in a forest area 800 m from the nearest cattle; they also collected Culicoides fed on sheep 1.5 km from the nearest sheep. This is another example, of bloodfed biting midges traveling long distances after their blood meal. It also indicates that blood-engorged Culicoides species may engage in long-distance dispersal, engorged females were still able to cover a distance of about 200 m (42). This may have been caused by wind drift; indeed, it is known that midges are easily carried by the wind stream.

Unfortunately, no females fed on wild animals were found, the same observations were reported by Bartsch et al. (42), Garros et al. [START_REF] Garros | Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on palaearctic biting midge species, vectors of orbiviruses[END_REF], and Ninio et al. [START_REF] Ninio | Contribution to the knowledge of Culicoides (Diptera: Ceratopogonidae) host preferences in France[END_REF]. It can be assumed that biting midges prefer domestic livestock if available. In addition, as most species of Culicoides are displaying crepuscular or nocturnal behavior [START_REF] Mellor | Culicoides biting midges: their role as arbovirus vectors[END_REF], they may prefer to bite domestic animals resting at night instead of wild animals in movement.

Since the study of blood meals is also an important component in understanding the pathogenic role of hematophagous insects, especially Culicoides, the present study indicates that the PCR direct sequencing system using universal primer sets for vertebrate Cyt b gene is a promising technique for blood meal identification especially because: (i) Cyt b mtDNA has a multicopy loci (8000 copy per cell) ( 44) and (ii) primers used for the amplification of the PNOC gene do not recognize birds and reptiles DNA [START_REF] Slama | Blood meal analysis of Culicoides (Diptera: Ceratopogonidae) in Central Tunisia[END_REF][START_REF] Murphy | Resolution of the early placental mammal radiation using Bayesian phylogenetics[END_REF]. It is more judicious then to concentrate efforts to provide a single homogeneous protocol to render studies comparable. Supplementary studies on the diurnal activity of Culicoides and their breeding sites and capturing methods could contribute to the understanding of their feeding behavior and provide additional information on their role in the transmission of pathogens.

′ 6 TABLE 2 |FIGURE 1 |

 621 FIGURE 1 | Culicoides females with different stages of blood meal digestion. (1) Fully engorged females; (2) abdomen showing advanced signs of digestion; (3) abdomen showing any remainder of blood in their abdomen.

a

  digestion (n = 264) C. obsoletus (83/85/20) Equus caballus (n = 52) Bos taurus (n = 1) Equus caballus (n = 80) Equus caballus (n = 23) Bos taurus (n = 3) Homo sapiens (n = 2) Homo sapiens (n = 2) Equus asinus (n = 1) Homo sapiens (n = 38) Equus caballus (n = 1) Homo sapiens (n = 2) Felis silvestris (n = 1) C. scoticus (27/43/25) Equus caballus (n = 23) -Equus caballus (n = 26) Equus caballus (n = 1) Homo sapiens (n = 1) Homo sapiens (n = 1) Homo sapiens (n = 2) C. chiopterus (1/1/5) Equus caballus (n = 1) Bos taurus (n = 1) Equus caballus (n = 1) Bos taurus (n = 1) C. lupicaris (7/3/9) Equus caballus (n = 7) -Equus caballus (n = 6) Equus caballus (n = 1) Equus asinus (n = 1) Equus asinus (n = 1) Homo sapiens (n = 1) C. pulicaris (1/6/11) Equus caballus (n = 1) Bos taurus (n = 1) Equus caballus (n = 1) Equus caballus (n = 3) Bos taurus (n = 1) Homo sapiens (n = 2) C. newstead (2//5/0) Equus caballus (n = 2) -Equus caballus (n = 2) Equus caballus (n = 2) Equus asinus (n = 1) Homo sapiens (n = 1) C. punctatus (26/38/1) Equus caballus (n = 25) Equus caballus (n = 1) Equus caballus (n = 20) Equus caballus (n = 13) Homo sapiens (n = 1) Equus asinus (n = 5) Equus asinus (n = 8) Homo sapiens (n = 1) Homo sapiens (n = 4) C. achray (63/55/7) Equus caballus (n = 62) -Equus caballus (n = 61) Equus caballus (n = 38) Equus asinus (n = 1) Equus asinus (n = 2) Gallus gallus (n = 1) Homo sapiens (n = 9) C. furcillatus (5/23/3) Equus caballus (n = 5) -Equus caballus (n = 5) Equus caballus (n = 8) Homo sapiens (n = 8) C. subfasciipennis (4/3/0) Equus caballus (n = 2) -Equus caballus (n = 4) Homo sapiens (n = 3) The same data set was tested for both markers. b Sample with any remainder of blood (n = 81) are not represented in the table because they provide all a negative or not interpretable results with both markers.

FIGURE 2 |

 2 FIGURE 2 | Sequencing chromatograms obtained by sequencing of the PNOC-PCR products for two different samples. (A) A sample identified as Equus asinus by Cytb sequencing. (B) A sample identified as Equus caballus by PNOC and Cytb sequencing. Arrow shows the difference between the two samples.

TABLE 1 | Sample of blood-fed Culicoides in this study.

 1

TABLE 3 | Origin of blood meals from blood-fed Culicoides spp.

 3

Frontiers in Veterinary Science | www.frontiersin.org May 2015 | Volume 2 | Article 15

Acknowledgments

This work is funded by the CPER project "Culichamp" and by ANSES. The authors thank Stavana Strutz for proofreading this manuscript. TM was supported in part by "STEP -Status and Trends of European Pollinators" (EC FP7 grant no. 244090) and by SUPER-B COST ACTION FA 1305. The study was also funded by an internal Applied-Oriented Research Grant from Naturalis Biodiversity Center. We are grateful to Modeste De Meulemeester and Véronique Dubois for help in donkey blood acquisition. The authors would like to thank the reviewers for their constructive comments which helped to improve the manuscript.

Author Contributions

LH-H, JD, TM, and DA designed this study, carried out data collection, data analysis, data interpretation, and composed the paper. PN, RH, and AG carried out data collection and data analysis.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Carole Kerdelhué

Franck Dorkeld

Rejane Streiff

Mylène Ogliastro
email: helene.ogliastro@inrae.fr.

Réjane Streiff

Kerdelhué

Coding-Complete Genome Sequence of a Partitivirus Isolated from Pine Processionary Moth Eggs

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT Two coding-complete nucleotide sequences of a partitivirus (family Partitiviridae) were discovered in transcriptomic data sets obtained from eggs of the Lepidoptera Thaumetopoea pityocampa. Each segment encodes a single open reading frame, and these two segments are predicted to encode an RNA-dependent RNA polymerase and a coat protein, respectively.

P ine processionary moths (PPMs; Thaumetopoea pityocampa) are aggressive defoliators of pine trees in the Mediterranean basin [START_REF] Kerdelhué | Quaternary history and contemporary patterns in a currently expanding species[END_REF]. PPMs reproduce in summer and lay their eggs on needles at the tip of pine tree branches, where the caterpillars hatch and weave a large protective silken nest for the colony. Large transcriptome sequencing (RNA-Seq) data sets corresponding to several PPMs at all developmental stages (eggs to adults) were mined to determine the viral diversity in different populations from southern Europe. We describe here the full-length coding-complete genome sequence of a betapartitivirus (family Partitiviridae), associated with the eggs of a unique PPM population from Portugal that lays eggs in the spring [START_REF] Santos | Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)[END_REF].

Viruses from the family Partitiviridae have a small and nonenveloped capsid protecting their bisegmented double-stranded RNA (dsRNA) genomes that are believed to be separately encapsidated [START_REF] Vainio | ICTV Virus Taxonomy Profile: Partitiviridae[END_REF]. These viruses group into five genera infecting plants, fungi, and protozoa, with host-based clades and clusters within each genus (3), including viruses infecting mildew species, fungal causal agents of diseases, and major threats to agriculture (4). Eggs from Thaumetopoea pityocampa were collected in the region of Leiria, Portugal, in June 2012 [START_REF] Santos | Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepidoptera, Notodontidae)[END_REF][START_REF] Burban | From sympatry to parapatry: a rapid change in the spatial context of incipient allochronic speciation[END_REF]. The eggs were crushed, and total RNAs were extracted with TRIzol reagent. A library was constructed using the TruSeq stranded mRNA prep kit (Illumina) with an average insert size of 450 bp and sequenced in a lane of paired-end (PE) 2 Â 100-bp reads on the HiSeq 2000 platform [START_REF] Gschloessl | Draft genome and reference transcriptomic resources for the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera: Notodontidae)[END_REF]. We obtained 34,478,275 paired-end reads that were trimmed (Trimmomatic v.0.33) [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] and assembled into contigs (Trinity v.2.0.2) [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF]. Default parameters were used for all software unless otherwise specified. Viral contigs were identified by subjecting the contigs consecutively to BLASTX (E value, 1.e-10) and BLASTN searches against the viral RefSeq and nonredundant (nr) databases (NCBI), respectively. We identified two contigs of similar sizes (2,210 and 2,164 bp) made of the assembly of 754 and 830 reads, respectively, which represent 4.5% of the total viral reads in the sample. Both genetic elements are monocistronic; each is predicted to contain a single open reading frame (ORF). The first contig (2,210 bp; GC content, 39%) encodes a putative 710-amino-acid RNA-dependent RNA polymerase (RdRP); the second contig (2,164 bp; GC content, 40%) encodes a 654-amino-acid capsid protein (CP). Taxonomic sequence assignment showed that both contigs share the same closest relative, i.e., the Plasmopara viticola lesion-associated Partitivirus 7 (GenBank accession numbers MN556979.1 and MN556980.1), displaying 46% and 30% identity with the RdRP and CP, respectively. The contigs were named RNA1 and RNA2, according to the bisegmented genomic organization of this virus family (Fig. 1). According to the ICTV, taxonomic assignation in the family Partitiviridae is based on the RdRP homology, with a genus threshold of ,24%. With 46% identity with the RdRP of the closest relative, two monocistronic contigs, and predicted proteins within the size range of this virus family, this PPM-associated partitivirus is likely a novel species in the genus Betapartitivirus (2). This virus was tentatively named Thaumetopoea pityocampaassociated partitivirus.

Members of the genus Betapartitivirus can infect plants and fungi, and the Plasmopara viticola lesion-associated Partitivirus 7 is an unclassified virus that infects the fungal causal agent of grape downy mildew (GenBank accession numbers MN556979.1 and MN556980.1 for RNA1 and RNA2, respectively). Although no fungal host can be associated with the novel partitivirus described here, mildew species are potential candidates, as they are commonly found in conifer forests, such as the powdery mildew that makes a white powdery substance on tree leaf surfaces. The most likely hypothesis is that infected fungi associated with pine trees have contaminated the surface of T. pityocampa eggs laid on the branches.

Data availability. The GenBank accession numbers are MT799182 and MT799183. The reads were deposited in the NCBI Sequence Read Archive (SRA) database under BioProject accession number PRJNA663237. The reads from the specific metagenome used here for virus reconstruction are registered with the SRA under accession number SRX9125299.

FIG 1

 1 FIG 1 Genomic organization of the PPM-associated partitivirus. The complete major open reading frames (ORFs) predicted are represented by the arrows labeled RdRP (RNA-dependent RNA polymerase) and CP (capsid protein) and corresponding to RNA1 and RNA2, respectively. Matching reads mapped to the virus genomes are represented by a gray area with a scale that represents the coverage. The solid line at the bottom represents the contigs corresponding to the segments RNA1 and RNA2.

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ACKNOWLEDGMENTS

This work was supported by INRAE, departments ECODIV (project EVIL) and SPE. M.O. is supported by the European Union's Horizon 2020 research and innovation program under grant agreement number 773567.