Nichole A Broderick

Jennifer K Upfold
email: jennifer.upfold@inrae.fr

Carlotta Savio
email: carlotta.savio@inrae.fr

Loretta Mugo-Kamiri

Bugs in Bugs: The role of probiotics and prebiotics in maintenance of health in mass-reared insects

Keywords: probiotics, prebiotics, mass-reared insects, insect diseases, microbiota, performance, health

AMP -antimicrobial peptide BHiA -brain-heart infusion agar Bt -Bacillus thuringiensis Btg -Bacillus thuringiensis galleriae CO2 -carbon dioxide Cry -crystal proteins of Bacillus thuringiensis Cyt -cytotoxin proteins of Bacillus thuringiensis ddH2O -double distilled sterile water DNA -deoxyribonucleic acid DOPA -dihydroxyphenylalanine EPF -entomopathogen fungi et al. -et alia HCT -hydrolysat de caséine trypton Imd -immune deficiency JAK/STAT -Janus kinase/Signal Transducer and Activator of Transcription JNK -c-Jun N-terminal kinase LAB -lactic acid bacteria LBA -lactic acid bacteria LPS -lipopolysaccharide LTA -lipoteichoic acid Mr -Metarhizium robertsii MRS -de-man rogosa -sharp agar mRNA -messenger ribonucleic acid NF-κB -Nuclear Factor kappa-light-chain-enhancer of activated B cells PAMPs -pathogen associated molecular patterns PGN -peptidoglycan PO -phenoloxidase proPO -prophenoloxidase PRR -pattern recognition receptors PCR -polymerase chain reaction qRT -PCR -quantitative reverse transcription polymerase chain reaction RNA -ribonucleic acid rRNA -ribosomal ribonucleic acid RNS -reactive nitrogen species ROS -reactive oxygen species ITN -integrated training network UV -ultraviolet Vip

Titre : Analyse de l'interaction entre le microbiote intestinal et l'immunité intestinale chez Galleria mellonella au cours de l'infection par Bacillus thuringiensis et Metarhizium robertsii Mots clés : Bacillus thuringiensis, Metarhizium robertsii, expression génétique, microbiote intestinal, interactions tripartites, axénique. Résumé Les agents pathogènes peuvent fortement impacter la santé des insectes, entraînant souvent leur mort et la proliferation de l'agent pathogène. Ceci est particulièrement problématique dans un environnement d'élevage de masse où un grand nombre d'insectes vivent à proximité les uns des autres. De tels environnements sont propices à l'émergence d'agents pathogènes la propagation de la maladie. Parallèlement, les insectes hébergent dans leur intestin des microorganismes symbiotiques bénéfiques dont on sait qu'ils contribuent à des fonctions importantes telles que l'assimilation des nutriments ou la résistance de l'hôte aux agents pathogènes.

Des modifications de cette communauté microbienne peuvent survenir à la suite d'une infection par un agent pathogène, ce qui peut soit accroître, soit amoindrir la résistance de l'hôte aux agents pathogènes. Il est, par conséquent, essentiel de mieux comprendre le rôle du microbiote intestinal de l'insecte et ses interactions avec le système immunitaire de son hôte ainsi qu'avec les pathogènes, tant pour renforcer la lutte et le contrôle des insectes nuisibles que pour améliorer la santé des insectes utiles. Dans ce projet, les larves de Galleria mellonella (fausse teigne des ruches) ont été utilisées comme insecte hôte modèle pour étudier le rôle du microbiote dans la santé et la résistance à la colonisation par différents entomopathogènes pénétrant par voie orale ou topique. Bacillus thuringiensis ssp. galleriae et Metarhizium robertsii (Mr) ont été utilisés pour provoquer une infection de l'hôte, par voie orale et topique, respectivement. Notre approche a consisté à réaliser des tests sur des larves de Galleria mellonella élevées avec ou sans microbiote (larves conventionnelles vs larves stériles "axéniques") et à étudier la réponse de l'hôte au cours de l'infection. Nous avons émis l'hypothèse que les isolats bactériens symbiotiques résidant dans l'intestin de l'insecte pouvaient contribuer à renforçer la réponse immunitaire de l'hôte, l'aidant ainsi à lutter contre l'infection par des pathogènes. Cependant, l'efficacité de cette réponse immunitaire renforcée par la symbiose peut varier en fonction du pathogène impliqué ainsi que par sa voie d'entrée dans l'organisme hôte. Nous avons tout d'abord montré que les larves sans microbiote présentaient plus de variations dans leur temps de développement, mais sans que leur valeur selective générale (fitness) ou leur capacité à accomplir leur cycle de vie ne soient affectées. Nous avons ensuite effectué des tests de survie sur des larves de 250 à 300 mg en utilisant des doses léthales 50 (DL50) de Btg et de Mr, et nous avons constaté que les larves axéniques étaient plus sensibles à une infection orale par Btg ; la DL50 pour le groupe conventionnel entraînait une mortalité de 90 à 100 % dans le groupe axénique, démontrant que les larves axéniques étaient significativement plus sensibles à une infection orale par Btg. Par contre, des taux de mortalité similaires chez les larves axéniques et conventionnelles ont été observés lors de l'infection par Mr. Afin d'étudier le lien entre le microbiote et la réponse immunitaire, nous avons ensuite analysé simultanément, dans le même groupe de larve, au cours de l'infection par Btg et Mr, la composition et la dynamique du microbiote, par séquençage du gène de l'ARNr 16S, ainsi que le niveau d'expression, par qRT-PCR, de plusieurs gènes codant pour des effecteurs de l'immunité intestinale. Cette étude a révélé que le microbiote de notre population de larves conventionnelles est dominé par quatre espèces d'Enterococcus, et que, les niveaux d'expression de base des gènes codant pour la Gallérimycine, la Glovérine et l'IMPI étaient significativement plus élevés dans le groupe de larves conventionnelles que dans le groupe de larves axéniques. Ce résultat indique que les espèces d'Enterococcus, dominantes au sein du microbiote, contribuent à maintenir une expression basale plus élevée de ces gènes de la réponse immunitaire dans l'intestin. En outre, nous avons montré qu'une souche d'Enterococcus mundtii, isolée de l'intestin des larves conventionnelles était capable d'inhiber la croissance de Btg in vitro. Nous avons ensuite recolonisé des larves de G. mellonella axéniques de dernier stade larvaire avec cette souche afin d'obtenir des larves gnotobiotiques mono-colonisées par E. mundtii et montré que ces larves présentaient une survie légèrement accrue à une infection par Btg 96 heures après l'infection par rapport aux larves axéniques. Ces résultats mettent en avant le rôle protecteur et immuno-modulateur du microbiote intestinal dominé par un petit nombre d'espèces d'Enterococcus, un assemblage de microbiote souvent observé chez des populations de lépidoptères vivant en laboratoire, et son influence sur la réponse de l'hôte aux infections orales et topiques.

Title : An analysis of the interaction between the gut microbiota and host gut immunity in Galleria mellonella during infection by Bacillus thuringiensis and Metarhizium robertsii Keywords : Bacillus thuringiensis, Metarhizium robertsii, gene expression, gut microbiota, tripartite interactions, axenic Abstract : Pathogens can severely affect insect fitness, often resulting in death and the further spread of the pathogen. This is particularly problematic in a mass rearing environment where there are large numbers of insects living in close proximity. Such environments are susceptible to the emergence of pathogens and vulnerable to the rapid proliferation of disease. At the same time, insects host mutually beneficial symbionts in their gut which are known to help mediate important functions such as nutrient assimilation or host resistance to pathogens when this microbial community is balanced. Changes to this community can occur following pathogen infection which can either contribute to-or decrease host resistance to pathogens. Due to this potential, more research is required to study the tripartite interaction of the gut microbiota, pathogen and host response. In this project the larvae of Galleria mellonella (wax moth) was used as a model insect host to study the role of the microbiota in the health and resistance against colonization by entomopathogens entering via oral or topical routes. Bacillus thuringiensis ssp. galleriae and Metarhizium robertsii (Mr), were used to induce immune responses via an oral-and topical infectious route, respectively. Our strategy was to conduct fitness assays on Galleria mellonella larvae reared with a different microbiota status (sterile -"axenic" vs conventional larvae) and to investigate the host response following infection. We hypothesized that symbiotic bacterial isolates residing within the insect gut can enhance the host's immune response, aiding in the fight against pathogen infection. However, the efficiency of this symbiotically-boosted immune response may vary depending on the specific pathogen involved as well as its route of entry into the host organism. We observed that larvae without a microbiota exhibited more variation in their developmental time, but the overall fitness and ability to fulfill their lifecycle is not impeded.We then conducted survival assays on 250-300 mg larvae using halflethal doses of Btg and Mr, and found axenic larvae were more susceptible to an oral infection of Btg where the half-lethal dose for the conventional group led to 90-100% mortality in the axenic group. We however did not detect any difference in mortality rates between axenic and conventional larvae after the topical Mr infection. Through a dual-analysis experimental design, We determined the gut microbiota by 16S rRNA sequencing, and assessed the level of immunerelated gene expression (qRT-PCR) in the same group of larvae during infection by Btg and Mr. This analysis revealed that the microbiota of our conventionally reared population is dominated by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two AMPs, Gallerimycin and Gloverin, in the conventional larvae compared to the axenic. Furthermore, Enterococcus mundtii isolated from the gut of conventional larvae showed inhibition activity against Btg in vitro, and when axenic larvae were re-inoculated with this isolate, an improved resistance to Btg was observed by 96 hours post infection. These findings highlight the immune-modulatory role of the Enterococcusdominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

List of Figures and Tables in literature review

Prelude:

Context of the work

Insects, being one of the earliest and most diverse animal groups on Earth, maintain a plethora of roles. Insects provide vital ecosystem services, such as pollination in agriculture and biological control of pest populations [START_REF] Engel | Insect Evolution[END_REF], However, certain insects can be problematic as pests and disease vectors, posing risks to human and animal health. They occupy most environments on land, where they share most of the ecological niches with numerous microorganisms, including viruses, bacteria, fungi, and protozoa. Many of these microorganisms are harmless, but some are highly infectious, often leading to the insect's death. The ability of insects to adapt to these environments could be due in part to their particularly powerful resistance to microbial aggression.

Insects have innate immune systems that enable them to protect themselves against various invading pathogens. The first lines of defense for insects are physical barriers, including the cuticle, which protects the host's cells and tissues from the external environment. This is often the route of entry for entomopathogenic fungi, such as Metarhizium species or Beauveria bassiana. For entomopathogenic bacteria, such as Bacillus thuringiensis, which constitutes one the most studied bacteria in insect pathology due to its use as a biopesticide, the ingestion of contaminated food appears to be the most frequent route of entry of the bacteria into larvae or adult insects. The ingested bacteria are transported passively by the food to the midgut. The epithelial cells of the midgut constitute an important immuno-reactive barrier where various effectors of innate immunity are secreted, namely reactive oxygen species (ROS) and antimicrobial peptides (AMPs), which are strongly induced following recognition of different infectious agents (Lemaitre and Hoffmann 2007).

Insects maintain a dynamic microbiota that can vary depending on factors such as diet, habitat, life stage, sex, and social interactions. This microbiota plays a crucial role in many aspects of the host's physiology, metabolism, development, and behavior.

Recent studies suggest that microbial communities associated with insects can confer protection or vulnerability to insects against different pathogens, depending on the context and specificity of the interactions. However, the current knowledge on the role of the microbiota in host resistance and immunity from pathogens in insects is still limited and fragmented. In addition, insects also serve as valuable model organisms in scientific research and are increasingly explored for sustainable food production (van Huis, 2013;Payne & Van Itterbeeck, 2017;Jankielsohn, 2018). Nevertheless, by deepening our knowledge of insect health and into the role of the microbiota in host resistance and immunity from pathogens in insects, we can grow our expertise in both insect control and insect health in order to effectively manage and optimize their positive contributions while mitigating the negative impacts they may have.

At the same time, the forecasted population growth has increased the demand for animal proteins worldwide, coupled with the limited land area available for agriculture this has led to an urgent need for additional supplies of protein from new sustainable sources, such as insect-derived products. Aside from human consumption, insects are a natural component of the diets of animals such as carnivorous fish, poultry and pigs.

Therefore, insects can provide a valuable, locally-produced, source of proteins and nutrients to livestock and aquaculture producers. Moreover, substituting insect-based animal feed for current plant feed (especially soybean) would have the great advantage of reducing global deforestation, land use and water consumption. For all these purposes, mass-rearing insects for food and feed is rapidly becoming a valuable industry for its potential contributions to improving global food security, particularly as a source of protein (EIMA 2020). Since 2018 more than $300 million has been invested into large-scale production facilities in efforts to create a mainstream protein source from insects ( van Huis, 2013;[START_REF] Joosten | Review of Insect Pathogen Risks for the Black Soldier Fly ( Hermetia Illucens ) and Guidelines for Reliable Production[END_REF]. The industry itself is expected to grow by 47% from 2019 to 2026 which will see production volumes reaching 730, 000 tons (Alliedmarketresearch). Their potential to be produced in large-scale production facilities holds promise for addressing various environmental and nutritional challenges confronting our world. However, inherent risks arise when handling large numbers of animals, such as disease outbreaks which commonly occur amongst mass-reared animals. Indeed, pathogens can severely affect insect health and well-being, often resulting in death and the further spread of the pathogen (Eilenberg et al., 2015b). This is particularly problematic in a mass-rearing environment where there are large numbers of insects living in close proximity. Such environments are susceptible to the emergence of pathogens and vulnerable to the rapid proliferation of disease (Eilenberg et al., 2015b;[START_REF] Joosten | Review of Insect Pathogen Risks for the Black Soldier Fly ( Hermetia Illucens ) and Guidelines for Reliable Production[END_REF]. This could create consequences for public health as well as raise concerns for animal welfare if diseases are not adequately managed (Eilenberg et al., 2015a;Eilenberg & Maciel, 2018).

Successful insect mass production for food or feed relies on continuous rearing of large insect colonies, under high density monocultures, in artificial environments. In these mass rearing facilities, preserving insect health is critical because for a company rearing these organisms, pathogen emergence can lead to extensive economic losses.

Therefore, the increase in mass-rearing insects for food and feed production has brought to light some challenges associated with farming insects. To remain sustainable, achievable and profitable, insect production needs to become more resilient. The lack of knowledge on pathogens affecting insects reared for these purposes, in particular the lack of diagnostic tools and processes, the lack of understanding of how to optimally rear insects, and of course a better knowledge on insect physiology and pathology is therefore essential to achieve insect health under mass rearing conditions. This gap of knowledge and skills led to the development of a European Joint Doctoral Programme, INSECT DOCTORS, funded in the framework of the H2020 Marie Sklodowska-Curie ITN program (Fig. 1). The program is based on close interaction between academic and industrial sectors and aims to train 15 young researchers to develop the expertise on how to diagnose and manage diseases in commercial insect production systems. The PhD research projects (numbered 1 -15) are divided into three work packages, each with its own objectives under the umbrella of understanding insect health. The first work package focuses on pathogen-host interactions and determines abiotic and biotic triggers for disease outbreaks. The second work package looks at covert infections and pathogen detection while the third work package is focused on increasing insect resistance against pathogens, by looking at the interactions between pathogens and the insect microbiome and how nutritional adaptations can increase insect fitness. The figure to the right places each project into a Venn diagram describing which type of insect pathogen will be evaluated as part of the work.

The following thesis (project 12 in Fig. 1) aims to expand our knowledge of insect health by exploring the role of gut microbiota in host resistance to different naturally occurring pathogens in the readily used insect model, Galleria mellonella larvae. I present firstly an introduction to all the elements explored in the research, ending with a discussion on what is understood of the relationship between the gut microbiota and host immune response during infection by oral and topical pathogens, highlighting the gaps in our knowledge. The aims and objectives of the thesis are then presented before an introduction to the main results that are then presented as a manuscript, along with follow-up experiments on the subject. Finally there is an overall discussion and conclusion with future recommendations.

CHAPTER 1

Literature review

Chapter 1

CHAPTER 1

Literature review

Then there were three… the host, the microbiome and the pathogen The principle of 'self' and 'non-self' is crucial in immunology, where 'self' reflects an organism's distinct and intrinsic characteristics, while 'non-self' includes elements outside the organism's inherent identity (Pradeu 2012). One of the fundamental functions of the immune system is to differentiate between self-components, that are integral parts of the host organism, while selectively rejecting that is non-self, especially harmful pathogens, thereby ensuring the body's defense and protection. To separate self and non-self, invertebrates such as insects, only feature innate immunity. The innate immune system can be defined as the host's non-specific defense mechanisms that rapidly respond to microbes entering the body (Wojda, 2017). There is a dilemma here however, as the microbiota challenges our concept of 'self'. Although of foreign origin, the microbiota also often referred to as the "forgotten organ" due to its importance, could be treated as "honorary self" [START_REF] Rees | How the Microbiome Challenges Our Concept of Self[END_REF]. The composition of the microbiota can vary independently of insect genotype, both among individuals and over time within a single individual. This fluidity in host-microbiota relations also has profound consequences for the role the microbiota plays in supporting the insect's overall health and abilities. The microbiota confers several important functions to its host, including supporting the immune system functioning, however in certain conditions, such as during pathogenic infections, it may play a role in contributing to host death. Therefore, the interactions between the host, the microbiota and pathogens should be approached with a more tripartite-relational point of view.

Introduction to the tripartite model

Interactions occur between hosts, their microbiota, and pathogens. These interactions between the three entities can be bipartite, as has been well-explored between the host and microbiota, and/or the host and pathogens [START_REF] Armitage | Tripartite Interactions: How Immunity, Microbiota and Pathogens Interact and Affect Pathogen Virulence Evolution[END_REF]. But direct and indirect tripartite interactions likely occur between all three groups, resulting in different levels of pathogen virulence as well as host susceptibility and fitness (Fig. 2).

Historical focus on these interactions has mostly been binary, between a pathogen and host, or microbiota and host. However, more attention is now being given to the interactions between microbes within the microbiota and how these interactions impact the host. The balance of these interactions depends on factors such as the specific microorganisms involved, their functions, the host's immune response, and the environment. Further exploration of the multitude of interactions is crucial in order to better understand the role microbes have in driving insect health and development and for developing strategies to manipulate insect-microbiota-pathogen dynamics and potentially control disease transmission.

Chapter 1

Figure 2. The tripartite interactions between the host, its resident gut microbiota, and invading pathogens

Interactions can be binary but also influence each other in a tripartite manner, thus showcasing the intricate balance of health and disease within the host organism. Several factors influence each actor independently, for The Host these are factors such as the phenotype, genotype, species or developmental stage; for The Microbiota, this involves the acquisition of the microbiota which can occur vertically (from parent to offspring, when bacteria is deposited onto eggs) or horizontally (via the environment which is different for laboratory reared vs wild populations, and of course from the diet); for the Pathogen this is the type of microbe, either Gram-positive (G+), Gram-negative (G-), fungi, virus or nematode. Microbe-microbe interactions will be factors such as competition, exclusion or facilitation, or other colonization resistance mechanisms; The 'wings' of the model highlight the similar factors influencing Host -microbiota interactions and Host-pathogen interactions. Both interactions will be influenced by environmental conditions, such as temperature, CO2, oxygen and pH (specific to the gut environment). The graph in essence denotes how independent influences drive interactions which themselves can be binary or tripartite, for example, the age of the insect can drive microbiota changes which in turn influences the insects susceptibility to disease by pathogens, but this tripartite interaction is also influenced by environmental factors and competition in the gut.

Host and Immune System

Insect: Galleria mellonella 'La fausse teigne de cire' in French, and 'voksmøl' in Danish, Galleria mellonella are a cosmopolitan insect species with a global distribution [START_REF] Ellis | Standard Methods for Wax Moth Research[END_REF]. It is also commonly referred to as the greater wax moth in English, due to its pest status in honeybee combs. The moth belongs to the Subfamily Galleriinae of the Pyralidae Family in the Order Lepidoptera (Kwadha et al., 2017a). Galleria mellonella is a typical holometabolous insect with four distinct life stages: egg, larva, pupa and adult (Fig. 3).

Depending on food availability and quality as well as room temperature and relative humidity, the life cycle of the moth can vary between a few weeks (4-5 weeks) to a few months [START_REF] Kwadha | The Biology and Control of the Greater Wax Moth, Galleria Mellonella[END_REF].

Morphology of life stages

The egg

The eggs are small, spherical, creamy white in appearance and are laid in clusters of several hundred eggs. They are approximately 0.55mm in diameter, and found on the surface of food substrate, or in a rearing chamber, eggs would be laid between two sheets of filter paper. The eggs typically hatch after 5-10 days in optimal conditions however the exact time for hatching can depend on factors such as temperature, humidity and quality of diet (Kwadha et al., 2017a;[START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF].

The larva

Once the eggs hatch, the larvae emerge and burrow into their diet. In the wild, the larvae would be found in honeycombs of beehives, consuming wax, debris, honey, dead bees and the cast-off honeybee pupal skins (Kwadha et al., 2017a;[START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF]. In general, the larvae are voracious feeders and can also be found in stored products (especially of nuts and seeds) but they have also been found to chew and degrade other materials such as plastics and polystyrene [START_REF] Bombelli | Polyethylene Bio-Degradation by Caterpillars of the Wax Moth Galleria Mellonella[END_REF]Cassone et al., 2020). Laboratory cultures of G. mellonella are typically fed an artificial diet consisting of cereals, milk powder, yeast, honey and glycerol [START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF].

The hatchlings appear white, however the larvae will develop into a light or dark brown with a bright ecdysal line visible along the middle of the dorsal side (Kwadha et al., 2017a). There are some discrepancies in the reporting of the number of larval instars, with some authors reporting 5 [START_REF] Fasasi | Life Cycle and Impact of Greater Waxmoth, Galleria Mellonella L. (Lepidoptera: Pyralidae) Feeding on Stored Beeswax[END_REF] others reporting 6 [START_REF] Kwadha | The Biology and Control of the Greater Wax Moth, Galleria Mellonella[END_REF]Jorjão et al., 2018;[START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF][START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF] and others reporting 7 [START_REF] Opoosun | Life Cycle Stages of Greater Wax Moth, Galleria Mellonella (L.) [Lepidoptera: Pyralidae], in Ibadan, Oyo State, Nigeria[END_REF][START_REF] Serrano | The Virtuous Galleria Mellonella Model for Scientific Experimentation[END_REF]. These differences could be due to the quality of diets and rearing conditions [START_REF] Kwadha | The Biology and Control of the Greater Wax Moth, Galleria Mellonella[END_REF]. Typically, the larva will pause feeding between each larval moult, and during the moult the cuticles are shed separately from the head capsule and the rest of the body. The duration of the larval stage at the average optimal development temperature (29-33°C) is around 45 days [START_REF] Pastagia | Biology of Galleria Mellonella L. on Brood Comb of Apis Cerana F[END_REF][START_REF] Swamy | Bionomics and Biometrics of Greater Wax Moth Galleria Mellonella Linnaeus[END_REF][START_REF] Desai | Biology of Greater Wax Moth, Galleria Mellonella L. on Artificial Diet[END_REF]. However, Chapter 1 20 this can vary drastically, depending on temperature, humidity and quality of diet. The larvae are also capable of spinning protective silk cocoons, to aid with avoiding detection and injury by bees (and other parasites) in the hive [START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF]. The pupal sac is also an important tool for the larvae to remain protected as they enter the pupal phase.

The pupa Fully grown larvae will stop feeding and search for a place to pupate, in the wild this could be the outer surfaces of the beehive and in rearing conditions they usually migrate to the top of the chamber [START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF]personally observed behaviour).

The pupae are brownish red to dark brown as they age, usually between 14-16mm in size [START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF]. There is well defined sexual dimorphism of the pupae, where the males have a pair of rounded knobs on the 9 th segment [START_REF] Desai | Biology of Greater Wax Moth, Galleria Mellonella L. on Artificial Diet[END_REF].

The length of pupal stage can be the most various, with a reported range of between 7 -60 days [START_REF] Pastagia | Biology of Galleria Mellonella L. on Brood Comb of Apis Cerana F[END_REF][START_REF] Swamy | Bionomics and Biometrics of Greater Wax Moth Galleria Mellonella Linnaeus[END_REF][START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF][START_REF] Desai | Biology of Greater Wax Moth, Galleria Mellonella L. on Artificial Diet[END_REF][START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF].

The adult

Adult moths eclose from the pupae, and have a greyish to purple-brown appearance.

The adults are rather small, 10-18mm in length, with the females being slightly larger and heavier than males [START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF]. They do not consume food at this stage of life, as their mouth parts are degenerate. Therefore they do not live long, between 7-30 days depending on conditions [START_REF] Kumar | Study of the Life Cycle of Greater Wax Moth (Galleria Mellonella) under Storage Conditions in Relation to Different Weather Conditions[END_REF][START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF]. The adults mate rapidly, with oviposition being observed around 24 hours after eclosion and continuing for 4 consecutive nights [START_REF] Wojda | The Greater Wax Moth Galleria Mellonella: Biology and Use in Immune Studies[END_REF].

Chapter 1

21

Model organism and status as lab-reared species

A variety of insects are used as in vivo models to test the immune response to different pathogens including Drosophila melanogaster, Manduca Sexta and Bombyx mori [START_REF] Kavanagh | The Use of Galleria Mellonella Larvae to Identify Novel Antimicrobial Agents against Fungal Species of Medical Interest[END_REF]. The larvae of Galleria mellonella L. (Lepidoptera: Pyralidae) have gained popularity as a model organism due to the similarity of its innate immune response to aspects of the mammalian immune system, as well as being easy to handle, the ease and accuracy of inoculation, the low cost of rearing and ability to generate rapid results [START_REF] Trevijano-Contador | Immune Response of Galleria Mellonella against Human Fungal Pathogens[END_REF]. It can also be reared at a wide variety of temperatures, including the human temperature of 37°C, adding to its advantage of being used as a model in research of host-pathogen interactions (Fig. 4).

Figure 4.

Galleria mellonella as a model organism for different infection assays including force-feeding, or by infection using a hypodermic needle into the hemocoel. The larvae used for bioassays should be between 250-300mg, or in the final larval instar, free from melanisation spots. There are many reasons making G. mellonella an effective model organisms including A. its relatively short life-cycle and ease of rearing, where B. assays can be conducted on large groups of individuals resulting in statistically significant data, C. the range at which the insect can be reared (20-42°C) but optimally between 29-33°C, makes it especially interesting as an alternative model to study microbial infections and antimicrobial drug testing, D. the larvae is easy to manipulate due to its larger size and slow moving nature at this life stage, E. which also makes it easier to extract different tissues, as up to 15ul of hemolymph can be collected, and tissues such as the gut or the fat body can easily be dissected, F. the insect also has a comparative immune response to mammals which has been increasingly studied leading to the publishing of a large gene repertoire and understanding of immune system functioning which can then be used to predict a similar mammalian response. Finally, even in death, the insect exhibits extreme 'disease indicators' as seen by the difference in cadavers post death by an entomopathogenic fungi, Metarhizium robertsii and bacteria, Bacillus thuringiensis.

A broad overview of the Lepidopteran immune system

The lepidopteran immune system, as with all insects, exhibits an innate response to foreign entities, which is most classically described by characterizing the responses into two main branches: cellular and humoral defenses. The cellular defenses involve the action of immune cells, primarily modulated by hemocytes, which can be understood as insect blood cells (Wojda, 2017). The humoral response, which involves the synthesis of antimicrobial peptides (AMPs), the pro-phenol oxidase (proPO) cascade, and toxic reactive oxygen species (ROS/RNS), occurs more slowly than the initial cellular response by the hemocytes (Jiang et al., 2010). The production of AMPs is a major and well characterized aspect of defense mechanisms in insects [START_REF] Jeyashree | Antimicrobial Peptides from Insects: An Overview[END_REF][START_REF] Yi | Insect Antimicrobial Peptides and Their Applications[END_REF][START_REF] Eleftherianos | Diversity of Insect Antimicrobial Peptides and Proteins -A Functional Perspective: A Review[END_REF][START_REF] Azmiera | Antimicrobial Peptides Isolated from Insects and Their Potential Applications[END_REF]. Their expression is regulated mainly at transcriptional level through the activation of NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) type signaling pathways. Fat body cells, which are analogous to mammalian liver, are a major regulatory organ for the systemic humoral response in insects [START_REF] Yu | Drosophila Innate Immunity Involves Multiple Signaling Pathways and Coordinated Communication Between Different Tissues[END_REF].

Additionally, the gut is another site where the expression of AMPs is regulated, given its exposure to ingested pathogens. The Toll, Imd (Immune Deficiency) and JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathways are three major signaling pathways that mediate these insect immune responses to different types of pathogens, and the activation of these pathways will be explained further in the next section. In addition to cellular and humoral immunity, an action known as "priming" has also been observed in some Lepidoptera, and other insect Orders, whereby following a previous exposure to a pathogen or parasite, a faster and more effective immune response occurs upon reinfection. There is evidence that the presence of a microbiota can also "prime" the immune response for certain future pathogenic challenge [START_REF] Sheehan | Immune Priming: The Secret Weapon of the Insect World[END_REF]. This can potentially result in reduced pathogen virulence with lower host susceptibility and improve the survival rate of the insect [START_REF] Sułek | Immune Priming of Galleria Mellonella Larvae with Bacillus Thuringiensis Affects Coagulation and Phenoloxidase Activity upon Subsequent Infection[END_REF][START_REF] Sheehan | Immune Priming: The Secret Weapon of the Insect World[END_REF][START_REF] Prakash | Why Do Insects Evolve Immune Priming? A Search for Crossroads[END_REF].

Recognition of microorganisms and immune pathways

An important role of the innate immune system is to rapidly recognize microorganisms after they enter the host. Pattern recognition receptors (PRRs) are a crucial part of the immune system found in various cells, including fat body cells, hemocytes and epithelial cells [START_REF] Hillyer | Insect Immunology and Hematopoiesis[END_REF]. PRR's act as 'sensors' and play a key role in the immune response by recognizing the specific pathogen-associated molecular patterns (PAMPs) present on microorganisms. Bacteria, fungi, viruses and other pathogenic microorganisms have specific structural components which are completely different from the host body, these PAMPs are typically the conserved components of microbes that are often necessary for their survival or growth, and are recognized by the host's innate immune system [START_REF] Thomma | Of PAMPs and Effectors: The Blurred PTI-ETI Dichotomy[OA][END_REF]Zhao et al., 2023). Essentially, it is the job of the PRRs to recognize the conserved components (PAMPs) that are present in microbes but absent in insects own cells [START_REF] Hillyer | Insect Immunology and Hematopoiesis[END_REF], hence an important aspect of distinguishing from the 'self' and 'nonself'. These conserved structural components are mostly located on the pathogen's cell surface, with a few located inside the cells. In brief, examples of PAMPs include bacterial cell wall components like peptidoglycans (PGNs), lipopolysaccharides (LPS), lipoteichoic acids (LTAs), fungal cell wall components like ß-1,3, glucans, and viral nucleic acids (e.g., double-stranded RNA) (Jiang et al., 2010;[START_REF] Tsakas | Insect Immunity and Its Signalling: An Overview[END_REF][START_REF] Bang | JAK/STAT Signaling in Insect Innate Immunity[END_REF]. Lepidopteran PRRs include peptidoglycan recognition protein (PGRP), gram-negative binding protein (GNBP), ß-1,3-glucan reconition proeiten (ßGRP), C-type lectin (CTL), and scavenger Chapter 1 25 receptor (SR) (Zhao et al., 2023). These PRRs activate distinct signaling pathways including the Toll, Imd, JAK/STAT, and c-Jun N-terminal kinase (JNK) pathways upon recognition of the different PAMPs [START_REF] Wojda | JNK MAP Kinase Is Involved in the Humoral Immune Response of the Greater Wax Moth Larvae Galleria Mellonella[END_REF][START_REF] Mak | A Different Repertoire of Galleria Mellonella Antimicrobial Peptides in Larvae Challenged with Bacteria and Fungi[END_REF][START_REF] Bang | JAK/STAT Signaling in Insect Innate Immunity[END_REF].

Additionally, the differences in the structure of PGNs can be exploited by the insect immune system to discriminate between different types of bacteria, leading to the activation of specific immune response pathways. The Toll pathway becomes activated due to the Lys-type PGN present in the cell walls of most Gram-positive bacteria, but is also activated by yeast and filamentous fungi, whereas the Imd pathway is activated by DAP-type PGN present in Gram-negative bacteria [START_REF] Troha | Nephrocytes Remove Microbiota-Derived Peptidoglycan from Systemic Circulation to Maintain Immune Homeostasis[END_REF]Zhao et al., 2023). The Toll pathway is also involved in insect development along with immunity while the Imd pathway functions solely in immunity [START_REF] Viljakainen | Evolutionary Genetics of Insect Innate Immunity[END_REF]. The activation of these pathways leads to pathogen killing via broad-spectrum cellular and humoral host responses. By differentiating between PAMPs, insects are able to better tailor their immune responses to the specific type of pathogen infection they encounter while minimizing unnecessary immune activation.

The cellular immune response branch: modulated by hemocytes

Hemocytes are immune cells derived from mesodermal stem cells that differentiate into distinct lineages based on their morphology, function, and molecular markers [START_REF] Lavine | Insect Hemocytes and Their Role in Immunity[END_REF]. The most abundant circulating hemocytes in the Lepidoptera are the granulocytes and plasmatocytes followed by spherule cells and oenocytes [START_REF] Ribeiro | Insect Haemocytes: What Type of Cell Is That?[END_REF]Wojda, 2017). Further physical description of the hemocytes present in G. mellonella is discussed in Figure 5. The primary immune activity of the hemocytes is understood by three activities, nodulation, encapsulation, and phagocytosis, with each type of hemocyte involved in different activities (Lavine & Chapter 1 Strand, 2002;Wojda, 2017). Nodulation occurs when the hemocytes, usually plasmatocytes, form a layer of cells around masses of microorganisms, resulting in the formation of a nodule. The nodule formations can appear identical to the capsule which is formed during encapsulation, with the primary difference being the target of the microorganism (Wojda, 2017;[START_REF] Eleftherianos | Diversity of Insect Antimicrobial Peptides and Proteins -A Functional Perspective: A Review[END_REF]. Encapsulation is formed by plasmatocytes and granulocytes, more often around larger pathogens than would be targeted by nodules, such as protozoa, nematodes, and parasitic eggs or larvae. The formation of nodules and capsules by the hemocytes causes a decrease in the number of total circulating hemocytes, as they are used in the process [START_REF] Pereira | Recent Advances in the Use of Galleria Mellonella Model to Study Immune Responses against Human Pathogens[END_REF].

Phagocytosis can occur when nodulation and encapsulation have failed to remove the foreign bodies, although the action of hemocyte activity can also be determined by pathogen characteristics and concentration (Jiang et al., 2010;Wojda, 2017;[START_REF] Pereira | Recent Advances in the Use of Galleria Mellonella Model to Study Immune Responses against Human Pathogens[END_REF]. Phagocytosis involves the production of enzymes (again by plasmatocytes and granulocytes) to destroy pathogens. Hemocytes release antimicrobial enzymes via a process called degranulation, which is facilitated by complement-like proteins that opsonize 1 pathogens, thereby activating the enzymatic system. These opsonins, once bound to microorganism surfaces, trigger the activation of phagocytosis and other proteinase signaling cascades. 1 Opsonize/ opsonization -an immune process that uses opsonins to tag foreign pathogens for elimination by phagocytes

The humoral immune response branch

The humoral immune response involves the production and circulation of antimicrobial proteins and molecules in the hemolymph, to help neutralize and eliminate pathogens.

One of these components is the production of AMPs most of which are small amphiphatic cationic molecules, mostly produced in the fat body, hemocytes, and epithelial cells, and secreted into the insect hemolymph (Jiang et al., 2010;Wojda, 2017). AMPs possess potent antimicrobial activity against a broad range of pathogens, where they can disrupt the integrity of microbial cell membranes, thereby inhibiting cell wall synthesis, and interfering with essential cellular processes [START_REF] Eleftherianos | Diversity of Insect Antimicrobial Peptides and Proteins -A Functional Perspective: A Review[END_REF].

Antimicrobial peptides (AMPs)

More than 150 AMPs have been discovered and characterized in insects. AMPs are small cationic molecules, usually made of less than 100 amino acids but typically between 15-50 amino acids [START_REF] Tsakas | Insect Immunity and Its Signalling: An Overview[END_REF][START_REF] Jeyashree | Antimicrobial Peptides from Insects: An Overview[END_REF].

These cationic peptides bind anionic bacterial or fungal membranes, leading to disruption and cell death, but may also suppress intracellular processes such as protein and DNA synthesis [START_REF] Yi | Insect Antimicrobial Peptides and Their Applications[END_REF]. The AMPs have different structures and targets, depending on the aggressor agent (bacteria: Gram+/Gram-or fungi, or virus) making them a central focus in the study of invertebrate pathology [START_REF] Jeyashree | Antimicrobial Peptides from Insects: An Overview[END_REF][START_REF] Pereira | Recent Advances in the Use of Galleria Mellonella Model to Study Immune Responses against Human Pathogens[END_REF]. They are mainly produced in the digestive tract, fat body, hemocytes, reproductive tract, and salivary glands of insects [START_REF] Tsai | Galleria Mellonella Infection Models for the Study of Bacterial Diseases and for Antimicrobial Drug Testing[END_REF]. Most AMPs can be assigned into families, such as cecropins, attacins, defensins and cysteine-rich peptides, proline-rich peptides, and glycine-rich peptides. Cecropins were the first purified from the hemolymph of immunized Hyalophora cecropia (Lepidoptera: Saturniidae) [START_REF] Boman | Cell-Free Immunity in Insects[END_REF]. For Galleria mellonella, a wide variety of AMPs (and other immune-response genes) have been identified and used in studies to understand the response of the immune system to diverse pathogens (Table 1.), mostly for the intended purpose of using as a model to study the immune response against human pathogens [START_REF] Mak | A Different Repertoire of Galleria Mellonella Antimicrobial Peptides in Larvae Challenged with Bacteria and Fungi[END_REF][START_REF] Tsai | Galleria Mellonella Infection Models for the Study of Bacterial Diseases and for Antimicrobial Drug Testing[END_REF][START_REF] Lange | Galleria Mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts[END_REF][START_REF] Pereira | Recent Advances in the Use of Galleria Mellonella Model to Study Immune Responses against Human Pathogens[END_REF].

Chapter 1

29

Table 1. The most studied AMPs and immune-response genes in Galleria mellonella.

Gene

Type Function Ref

Cecropins

AMP

Active against Gram-positive, Gram-negative and filamentous fungi 1,2

Gallerimycin

Active against filamentous fungi 1,2,3

Galiomycin

Active against filamentous fungi and two yeasts 1,2,3

Moricins

Antibacterial activity against Gram-positive and Gram-negative bacteria; targets cytoplasmic membrane; increases membrane permeability 1,2,3

Gloverin

Antimicrobial activity against fungi, and Gram-negative and Gram-positive bacteria; targets outer membrane; inhibition of outer membrane proteins 1

Cobatoxin cationic peptide

Strongly induced in following bacterial challenge but AMP activity is not yet characterized. 1

Lysozyme

Is constitutively present, degrades cell wall peptidoglycans and shows non-enzymatic activity against yeast and fungi 6.

apoLp-lll (Apolipophorin lll)

Is constitutively present but is active against Gram-postitive and Gram-negative bacteria. Has been shown to work in synergistic action with lysozyme against E. coli cells by increasing the permeabilizing activity of lysozyme. 5.

IMPI (Insect metalloproteinase inhibitor)

Inhibits microbial metalloproteinases such as bacterial thermolysin, induction of IMPI can induce other AMPs 7.

Nox

Phenoloxidase and melanization

Phenoloxidase (PO) is a copper-containing enzyme that plays a vital role in melanin formation, sclerotization, opsonization, and wound healing and has been detected in prokaryotic and eukaryotic organisms (Dubovskiy et al., 2013;[START_REF] Pereira | Recent Advances in the Use of Galleria Mellonella Model to Study Immune Responses against Human Pathogens[END_REF]Marieshwari et al., 2023) The inactive form, or the proenzyme, prophenoloxidase (proPO), is usually present in a zymogen form, and in Galleria mellonella is mainly in the oenocytoids (Wojda, 2017;Marieshwari et al., 2023). Upon activation of the prophenoloxidase (proPO) system, also called the PPO cascade, which occurs after the recognition of infection, the enzyme is released from the hemocytes and activated by limited proteolysis by cascades of serine proteases. When activated, PO converts tyrosine to dihydroxyphenylalanine (DOPA), which oxidizes phenolic substances to quinones and melanin, resulting in the darkening of infected insects (Fig. 6) [START_REF] Ashida | The Prophenoloxidase Cascade in Insect Immunity[END_REF]. Phenoloxidase activity is tightly controlled due to the high cytotoxicity of intermediate products such as DOPA melanin, quinones and free radicals (such as reactive oxygen species/reactive nitrogen species) which could damage host proteins, cells, and DNA (Dubovskiy et al., 2016;Marieshwari et al., 2023). Melanins are blackbrown pigments that contribute to insect immune defense and wound repair.

Melanization produces oxidative species and cytotoxic intermediates that are thought to result in the death of the invading microbe [START_REF] Nappi | Melanogenesis and Associated Cytotoxic Reactions: Applications to Insect Innate Immunity[END_REF]. Another function of melanin is to act as a physical barrier, restricting nutrient uptake and gas exchange, this can limit pathogen replication and spread to other tissues.

Chapter 1

Reactive Oxygen Species

The secretion of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is part of the primary intestinal innate immune defense, and plays a critical role in both prophylactic mechanisms (preventing infection) and the response to infection in insects [START_REF] Grasberger | Increased Expression of DUOX2 Is an Epithelial Response to Mucosal Dysbiosis Required for Immune Homeostasis in Mouse Intestine[END_REF][START_REF] Lange | Galleria Mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts[END_REF]. These mechanisms help insects defend themselves against pathogens like bacteria, viruses, fungi, and parasites. In the gut, ROS are produced as a part of the digestive process, and as such they can limit or prevent pathogen invasion prior to any recognition process by directly damaging bacterial membranes. Intestinal ROS is mainly produced by NADPH oxidases (NOX), and sometimes by nitric oxide synthase 2 (NOS2), which primarily generates RNS [START_REF] Fang | Antimicrobial Reactive Oxygen and Nitrogen Species: Concepts and Controversies[END_REF][START_REF] Nathan | Beyond Oxidative Stress: An Immunologist's Guide to Reactive Oxygen Species[END_REF]. Both ROS and RNS are generated by phagocytes and epithelial cells during infections to combat microbes [START_REF] Fang | Antimicrobial Reactive Oxygen and Nitrogen Species: Concepts and Controversies[END_REF][START_REF] Lange | Galleria Mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts[END_REF]. ROS are also involved in cell signaling pathways, including those regulating growth, differentiation, and apoptosis. While ROS and RNS are crucial for immune defense, maintaining their levels is equally important. Excessive production of ROS/RNS can lead to collateral damage to host tissues affecting DNA, RNA, or proteins, even causing apoptosis [START_REF] Bae | Regulation of Reactive Oxygen Species Generation in Cell Signaling[END_REF]. This process is known as oxidative stress [START_REF] Fahmy | Impact of Two Insect Growth Regulators on the Enhancement of Oxidative Stress and Antioxidant Efficiency of the Cotton Leaf Worm, Spodoptera Littoralis (Biosd.)[END_REF]. Insects have mechanisms in place to regulate and detoxify ROS/RNS to prevent self-harm. It has previously been found in D. melanogaster that DUOX-related signaling (dual oxidative) was involved in balancing the intestinal microbiota load [START_REF] Xiao | A Mesh-Duox Pathway Regulates Homeostasis in the Insect Gut[END_REF]. Similarly, in G. mellonella, the induction of ROS and RNS related responses contributed to a decrease of bacterial load. ROS and RNS contribute to the suppression of microbial growth and intestinal homeostasis in mammals [START_REF] Nathan | Beyond Oxidative Stress: An Immunologist's Guide to Reactive Oxygen Species[END_REF]. Due to their versatility in the immune response of insects, both as a preventive measure and as a reactive defense against invading pathogens, it is clear that further investigation into the exact roles played by ROS/RNS is needed for understanding how various organisms respond to pathogens.

Overall, the immune system of Lepidoptera is a complex network of cellular and humoral defense mechanisms that work together to protect these insects from pathogens, and while much research has been conducted on their immune responses, there are still many aspects that remain mysterious, in particular the relationship between the immune response and the gut microbiota. Questions remain about how these gut microbes interact with the insect's immune system, how they influence the response to pathogenic infections, and how changes in the gut microbiota might affect the insect's health and survival.

Pathogens

Insects interact with a wide array of pathogens, many of which are entomopathogenic.

Entomopathogens are specifically arthropod-infecting pathogens which can be bacteria, fungi (EPF), nematodes, protozoa, and viruses [START_REF] Vega | Insect Pathology[END_REF][START_REF] Hillyer | Insect Immunology and Hematopoiesis[END_REF]. The use of entomopathogens in managing pest populations has become increasingly popular over broad-spectrum chemical insecticides, due to its more targeted nature and its capability of naturally regulating arthropod populations. Insect pathogens can also maintain covert infections (non-productive latency or sublethal infections) where only low-level production of the pathogen occurs, such infections are often associated with virus and virus progeny [START_REF] Williams | Covert Infection of Insects by Baculoviruses[END_REF]. For the most part, in a covert state, the insect can carry out normal biological functioning however if exposed to different stresses, the covert infection may be able to overcome the host's defences and lead to outbreaks, or overt infections.

Rearing insects away from their natural environment can expose them to varying stresses, such as living in abnormally high densities, fluctuating temperatures, and humidity as well as elevated CO2 [START_REF] Herren | Environment-Host-Parasite Interactions in Mass-Reared Insects[END_REF]. They may also not be able to carry out 'wild' behaviours contributing to their stress. There are also indications, that the use of entomopathogens as biopesticides is becoming problematic for companies mass-rearing insects for food and feed, due to the risk of the insects' diet being exposed to entomopathogenic biopesticides and then re-infecting the mass-reared insects feeding on wheat or bran-based diet (knowledge from industry discussions).

As the insects are living in different conditions to that of their natural environment, outbreaks, and diseases may persist. Two commonly used entomopathogens for biocontrol purposes are the bacterium Bacillus thuringiensis and species of the entomopathogenic fungi Metarhizium.

Bacillus thuringiensis

Diversity and Taxonomy

Bacillus is one of the most diverse genera in the class Bacilli, of the Phylum Firmicutes, which includes both aerobic and facultatively anaerobic bacteria [START_REF] Garbeva | Predominant Bacillus Spp. in Agricultural Soil under Different Management Regimes Detected via PCR-DGGE[END_REF].

Biology and ecology

The life cycle of Bt inside of the insect can be divided into three stages, which successively are virulence, necrotrophism and sporulation stages (Ben Rejeb, Lereclus & Slamti 2017 ;Slamti et al. 2014 ;[START_REF] Verplaetse | Cell Differentiation in a Bacillus Thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection[END_REF]. The vegetative cells are characterized as large stout rods, usually straight or slightly curved with rounded ends (Fig. 7B.) During the sporulation cycle, crystal proteins are produced along with the endospore (Fig. 7 A-C.) [START_REF] Ibrahim | Bacillus Thuringiensis A Genomics and Proteomics Perspective[END_REF]. The toxic action by the crystal proteins is an additional survival advantage for Bt. Through the lethal action of the toxins, enough host nutrients are available to allow the dormant Bt spore to germinate and return to vegetative growth. As mentioned, Bt is known to produce deltaendotoxins, or crystalline inclusions containing Cry and Cyt proteins, with entomopathogenic properties. When ingested, the crystals first dissolve in the intestinal tract dependent on gut pH. For the majority of Cry toxins, gut conditions must be strongly alkaline in order to achieve dissolution. After solubilization, the inactive Cry protoxins must be activated through proteolytic cleavage by the insect midgut Chapter 1

38

proteases (Lecadet and Dedonder, 1967;Hofmann et al., 1988a) to generate mature toxins consisting of the amino-terminal half of the protoxin. Activated Cry proteins then bind to specific receptors to specific receptors on the surface of the midgut epithelial cells. Following binding, the toxin rapidly and irreversibly inserts into the cell membrane, resulting in the formation of pores leading to the disruption of the midgut epithelial cells, which can lead to septicemia when bacteria breach and proliferate in the hemocoel, resulting in death of the insect (Bravo et al., 2007;Adang et al., 2014).

After the death of the insect host, NprR, a quorum sensor is active in Bt which allows the bacteria to survive in the cadaver as vegetative cells [START_REF] Dubois | Necrotrophism Is a Quorum-Sensing-Regulated Lifestyle in Bacillus Thuringiensis[END_REF]. Bt has been observed as surviving in the host cadaver in a nonsporulated form for at least 14 days [START_REF] Toukabri | A Sporulation-Independent Way of Life for Bacillus Thuringiensis in the Late Stages of an Infection[END_REF]. Finally, once all the nutrients of the cadaver are used, the bacteria will sporulate, occurring concomitantly to necrotrophism, in part of the population [START_REF] Verplaetse | Cell Differentiation in a Bacillus Thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection[END_REF]. This gives Bt an advantage and contributes to spore spreading.

Chapter 1

Metarhizium robertsii

Diversity and Taxonomy

Metarhizium (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae), commonly known as green muscardine fungi, are ubiquitous soil saprophytes occurring mostly in disturbed soil environments such as agricultural fields (Meyling & Eilenberg, 2007). It was first named Entomophtora anisopliae by Elie Metschnikoff (1879) when he found the fungus in its asexual morph during his work on stored grain beetles [START_REF] Driver | A Taxonomic Revision of Metarhizium Based on a Phylogenetic Analysis of rDNA Sequence Data[END_REF]. The classification of Metarhizium has been revisited in recent years, and before the work of [START_REF] Driver | A Taxonomic Revision of Metarhizium Based on a Phylogenetic Analysis of rDNA Sequence Data[END_REF] the classification was mainly based on morphological characters. Work by [START_REF] Bischoff | A Multilocus Phylogeny of the Metarhizium Anisopliae Lineage[END_REF]

Biology and ecology

Metarhizium conidia 2 are distinguishable by their green, elongated shape (Fig. 8.)

which are produced on chains. These chains are compacted, forming columns of conidia arising from phialides. Fungi within the genus Metarhizium are known for their ability to infect and kill a wide variety of different arthropods, including caterpillars. Most

Metarhizium fungi also have an association to plants, as rhizosphere colonizers and beneficial root endophytes [START_REF] St | Metarhizium: Jack of All Trades, Master of Many[END_REF] [START_REF] Fang | Protein Kinase A Regulates Production of Virulence Determinants by the Entomopathogenic Fungus, Metarhizium Anisopliae[END_REF]). An appressorium grows on the host cuticle to find an ideal penetration site. Narrow penetration pegs are then formed and using a combination of cuticle degrading enzymes and turgor pressure, the peg penetrates into the host. An osmosensor signals the penetrating peg that they have reached the hemocoel and the fungi will start to produce hyphal bodies [START_REF] Butt | Chapter Nine -Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions[END_REF]. The host eventually dies from the release of toxins and invasion of host tissues by the fungi, with more conidial spores released from the cadaver back into the environment (Meyling & Eilenberg, 2007;Mora et al., 2018). As stated, fungi mainly infect the insect via the cuticle, termed a topical infection. This is because if the conidia are ingested, there are many factors about the gut environment that can influence its viability, such as the pH, oxygen, nutrient and digestive enzymes which can reduce conidial germination and mycelial growth. Grown on SDA/4 medium for three weeks. The strain was collected from Danish soils and is part of the Section for Organismal Biology library of isolated entomopathogenic fungi. Galleria mellonella was used as a bait insect to collect the isolate, by the method described by [START_REF] Meyling | Occurrence and Distribution of Soil Borne Entomopathogenic Fungi within a Single Organic Agroecosystem[END_REF].

Oral and topical routes of infection for pathogens infecting insects

Pathogens that infect insects can utilize both oral and topical routes to establish infections. Insects can ingest pathogens while consuming contaminated food, water, or other organic matter. Pathogens that traditionally invade the host through ingestion may target the insect's gut, causing infections in the digestive tract. They can disrupt nutrient absorption and/or can breach the gut barrier and enter the insect's hemocoel leading to systemic infections affecting various internal organs. For example, Bt strains commonly used as a biological insecticide have a battery of virulence factors in their genomes which aids the pathogen to invade the insect causing gut damage and death Chapter 1

(Figure 9A). The insect's exoskeleton, known as the cuticle, can be a barrier to pathogens. However, fungal pathogens often infect insects topically via the cuticle.

Entomopathogenic fungi, have evolved mechanisms to penetrate the cuticle and establish infections directly in the insect's hemocoel leading to internal infections and eventual death (figure 9B). For M. robertsii, however, entomopathogenicity is a more recently acquired adaptation that evolved from a plant associate to a broader host range [START_REF] Zhang | Horizontal Gene Transfer Allowed the Emergence of Broad Host Range Entomopathogens[END_REF]. This shift occurred through the horizontal gene transfer of 18 genes involved with cuticle degradation, penetration, lipid-carrier genes involved in utilizing epicuticular lipids, and genes facilitating hemocoel colonization [START_REF] Zhang | Horizontal Gene Transfer Allowed the Emergence of Broad Host Range Entomopathogens[END_REF]. The insect cuticle is not the only entry gate for fungal infection, however. When infection is by ingestion, the microbe is usually a virus, bacteria, or protozoa; but it has also been proposed that the oral and respiratory routes could be an alternative to cuticle penetration for entrance into the host by fungi. A few studies have looked into the oral route of infection for fungi however it has mostly been observed that the conidia adhere to the mouthparts, as there is little evidence for conidial germination in the gut of insects [START_REF] Ishii | Proboscis Infection Route of Beauveria Bassiana Triggers Early Death of Anopheles Mosquito[END_REF][START_REF] Mannino | Is the Insect Cuticle the Only Entry Gate for Fungal Infection? Insights into Alternative Modes of Action of Entomopathogenic Fungi[END_REF].

Recently, mining into the available genomes of entomopathogenic fungi, some homologous genes that would allow fungi to possess oral toxicity have been found.

For example, M. roberstii has six heat-labile bacteria-like enterotoxins, and Beauveria bassiana has eight cry-like delta enterotoxins and three bacteria-like zeta toxins proteins3 [START_REF] Mannino | Is the Insect Cuticle the Only Entry Gate for Fungal Infection? Insights into Alternative Modes of Action of Entomopathogenic Fungi[END_REF], whereas other entomopathogenic fungi have one or none, suggesting M. robertsii and B. bassiana would have a greater oral toxicity than other entomopathogenic fungi. A study on Spodoptera litura (Lepidoptera: Noctuidae) larvae found that when the vegetative insecticidal protein Vip3A from Bt was integrated into the B. bassiana genome, the engineered strain was significantly more virulent than the wild strain when the larvae were orally infected, but virulence did not differ between the two strains when infection occurred via the cuticular route [START_REF] Qin | Integration of Insecticidal Protein Vip3Aa1 into Beauveria Bassiana Enhances Fungal Virulence to Spodoptera Litura Larvae by Cuticle and Per Os Infection[END_REF].

However, more histological studies, and information on the physiological and molecular changes when entomopathogenic fungal conidia are ingested needs to be conducted to fully understand the mechanisms that ingested conidia use to kill the host. The Bt spore-crystals are ingested during the intake of food which triggers peristalsis (the abrupt halting of feeding), 2. the toxins can then break down the peritrophic matrix (pm) and further destroy epithelial cells, 3. leading to pore formations in the gut where the spore and crystals can enter the hemocoel. 4. In the hemocoel the spores germinate into a vegetative state, where more spores and crystals are produced, overloading the hemocoel eventually causing death to its host. Upon death, 5. the insect cuticle melanizes and softens, allowing the spores to be released back into the environment. B. As for the topical infection by M. robertsii, 1. the conidia adheres to the surface of the cuticle and starts to secrete cuticle degrading enzymes before it 2. forms a penetration peg to enter the hemocoel and 3. form blastospores which are released into the circulating hemocoel allowing the fungi to spread throughout the host body until it 4. overwhelms the host tissues and can 5. sporulate and penetrate back out of the host by the production of hyphal bodies which will further prepare conidia to infect the next host.

The microbiota

Definition and significance of the gut microbiota

Also referred to as the gut flora, or symbionts, the gut microbiota can be described as a complex community of microorganisms including bacteria, fungi, viruses, and archaea which inhabit the digestive tract of animals. There are a lot of definitions and concepts involved when discussing the microbiota and the relationship to its host, but it is important to first define the term 'symbiosis,' which can be defined as a long-term biological interaction between two organisms of different species, usually referred to as 'symbionts.' Bacterial symbionts are traditionally classified into two categories, obligate or primary symbionts and facultative or secondary symbionts [START_REF] Ferrari | Bacterial Symbionts in Insects or the Story of Communities Affecting Communities[END_REF]). An obligate symbiont refers to a microorganism that has a long-term, essential and mutually beneficial relationship with its host and typically plays an important role in the host's survival and normal functioning. A primary symbiont is typically an obligate symbiont and a key microorganism in the host's nutrition or other vital physiological processes. These are often inherited vertically from one generation to the next via parent to offspring. A well-known example of this relationship is between aphids and the bacterium Buchnera aphidicola [START_REF] Baumann | Buchnera Aphidicola: The Endosymbiont of Aphids[END_REF]. Aphids feed on the phloem sap of plants which is rich in sugars but poor in essential amino acids. To overcome the dietary limitation B. aphidicola have formed a symbiotic relationship by living within specialized cells called bacteriocytes, inside of the aphid's body [START_REF] Baumann | Buchnera Aphidicola: The Endosymbiont of Aphids[END_REF]. It is an obligate relationship because the bacterium cannot survive outside of the aphid host, and the bacteria produce essential amino acids that are missing in the diet, thereby supplying the nutrients that the insect needs to survive. In return, the aphids provide B. aphidicola with a protected environment and the necessary resources it needs to reproduce. Facultative or secondary symbionts are not strictly Chapter 1

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required for the host's survival. Instead, they can provide various advantages, such as, protection against pathogens and parasites, nutritional supplementation, enhanced stress tolerance, adaptation to new environments or increased reproductive success, that can enhance the fitness and adaptability of the host insect under specific environmental conditions (Gupta & Nair, 2020). While these symbionts can provide advantages, they can also have costs associated with their maintenance, resource utilization, and potential negative interactions with their host's own biology. The balance between these advantages and costs determines the overall impact of facultative symbionts on their host insects' fitness. A description of the several different types of symbiotic relationships which can occur, namely, commensalism, mutualism, opportunistic and neutral amongst others are described further in figure 10.

Figure 10. The multitude of interactions between two organisms

with '+' denoting a positive, '-' a negative, and '0' a neutral effect. Where the first symbol is the response of one organism, and the second is the response of the other, so for a mutualistic relationship both the host and symbiont experience a positive effect, but for a commensal relationship, either the host benefits and the symbiont remains neutral, or vice versa. Image taken from [START_REF] Zélé | Ecology and Evolution of Facilitation among Symbionts[END_REF], originally published in Nature Communications.

Structure and function of the gut

Before the function, acquisition and members of the gut microbiota is described further, it is beneficial to understand its environment. The gut of Lepidopteran larvae is a complex environment consisting of three distinct parts. The foregut, which includes the crop and esophagus, serves as the initial site for breaking down, filtering, and sorting ingested food [START_REF] Chapman | The Insects: Structure and Function[END_REF]Engel & Moran, 2013). Consequently, it also serves as the initial site for microbes obtained through the intake of food (Zhang et al., 2022b).

The midgut, the largest section of the gut, plays a crucial role in digestion and nutrient absorption by secreting enzymes [START_REF] Chapman | The Insects: Structure and Function[END_REF]Engel & Moran, 2013). Within the midgut, there is a semipermeable barrier called the peritrophic matrix (PM), which separates the gut space into two distinct sections: the endo-peritrophic, and ectoperitrophic regions (Fig. 11). The PM is present in invertebrates and serves as a protective lining for the midgut [START_REF] Erlandson | Role of the Peritrophic Matrix in Insect-Pathogen Interactions[END_REF]Zhang et al., 2022b). Gut microorganisms normally cannot cross the endo-peritrophic space, which limits the direct contact between microbiota members or food content and the gut epithelial cells [START_REF] Liu | Biosynthesis, Modifications and Degradation of Chitin in the Formation and Turnover of Peritrophic Matrix in Insects[END_REF]. The last section of the gut in Lepidoptera is the hindgut, which is formed from three regions, the ileum, the rectum, and the posterior rectum [START_REF] Chapman | The Insects: Structure and Function[END_REF]Engel & Moran, 2013). The important function of the hindgut is to mediate the uptake of uric acid, water, and salts [START_REF] Johnson | Oxygen Levels in the Gut Lumens of Herbivorous Insects[END_REF]Zhang et al., 2022b). There can be varying physicochemical conditions across these three gut sections which can influence the microbiota community, including digestive enzymes, oxygen availability, pH, redox conditions and ion concentrations [START_REF] Johnson | Oxygen Levels in the Gut Lumens of Herbivorous Insects[END_REF]Engel & Moran, 2013;Schmidt & Engel, 2021). The midgut of lepidopteran larvae usually has a high alkalinity, with a pH of between 11-12, however

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physical conditions can vary among different insect species; due to the influence of diet, leading to variations in gut microbiota among populations of the same species.

Influences contributing to gut colonization

Microbes need to be able to colonize and establish in the gut in order for there to be a symbiosis between gut microbes and their host. There are a number of factors facilitating gut colonization, including diet, environment, transmission from parent to offspring and physicochemical characteristics of the gut (Schmidt & Engel, 2021).

Therefore, microbial communities are highly variable between species as well as at different life stages. The different dietary habits of insects create varied nutritional niches in the gut, which results in microbial symbionts with distinct metabolic capabilities and physiologies [START_REF] Dietrich | The Cockroach Origin of the Termite Gut Microbiota: Patterns in Bacterial Community Structure Reflect Major Evolutionary Events[END_REF][START_REF] Kešnerová | Gut Microbiota Structure Differs between Honeybees in Winter and Summer[END_REF]. Bacteria can also be transferred vertically from parent to offspring, when mothers deposit bacteria on the eggs during laying. When hatchlings emerge, they eat through the chorion or egg shell, ingesting bacteria which will colonize their gut. The gut physicochemical properties such as pH, available nutrients, redox potential and immune effectors also influence which bacteria can colonize (Schmidt & Engel, 2021).

Many microbes can be transient 4 and passage through the gut, however microbes that persist and replicate in the gut are effectively colonizers. The digestive tract of Lepidopteran larvae is a considerably more hostile environment to that of many other insects, given the relatively low microbial biomass [START_REF] Broderick | Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods[END_REF][START_REF] Hammer | Caterpillars Lack a Resident Gut Microbiome[END_REF]. The tube-like morphology of the gut potentially hampers colonization as this lacks spatially structured niches for microbes. Furthermore the Lepidopteran larvae gut is known to have higher pH, especially in the midgut, a high concentration of immune effectors and a fast transit time of luminal content [START_REF] Dow | Extremely High pH in Biological Systems: A Model for Carbonate Transport[END_REF][START_REF] Johnson | Potential Influence of Midgut pH and Redox Potential on Protein Utilization in Insect Herbivores[END_REF][START_REF] Brinkmann | Leaf-Feeding Larvae of Manduca Sexta (Insecta, Lepidoptera) Drastically Reduce Copy Numbers of aadA Antibiotic Resistance Genes from Transplastomic Tobacco but Maintain Intact aadA Genes in Their Feces[END_REF]Jiang et al., 2010). Once the microbiota is established, it Chapter 1

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Common bacterial members in the Lepidoptera microbiota

The microbiota of the gut environment in Lepidoptera is comprised of microbial members including bacteria, fungi, archaea, and yeasts. As molecular technologies have developed, such as metagenome analysis and high-throughput sequencing of the 16S-and 18S-rRNA and ITS gene regions, the discovery of the species constituting the gut microbiota has advanced [START_REF] Gao | An Introduction to Next Generation Sequencing Bioinformatic Analysis in Gut Microbiome Studies[END_REF]. Despite the rise of these techniques, the bacterial members of the microbiota have received more attention than the fungal 'mycobiota' [START_REF] Gurung | The Microbiome of Pest Insects: It Is Not Just Bacteria[END_REF]. This is likely due to a few reasons, firstly being the historical focus of studies on microbial communities in animals and insects which primarily looked at bacteria due to their role in physiology, development, and health [START_REF] Muñoz-Benavent | Insects' Potential: Understanding the Functional Role of Their Gut Microbiome[END_REF]. Secondly, the methodological challenges whereby bacteria were easier to culture and study, leading to an increased body of knowledge on this microbe, which further allowed for the discovery of its functional roles (like digestion, nutrition, and immune system modulation) whereas the understudied fungi were traditionally associated with pathogenic infections in insects rather than symbiotic or commensal relationships. Research trends are starting to shift and diversify by incorporating the fungal members when analyzing the gut microbiota community. That being stated, the fungal diversity in the Lepidopteran gut is reportedly far fewer and a lot more transient than bacteria (Zhang et al., 2022b;[START_REF] Šigut | Fungi Are More Transient than Bacteria in Caterpillar Gut Microbiomes[END_REF].

Voirol and colleagues (2018) could identify certain bacteria taxa to be widespread across Lepidopterans at the genus level, Pseudomonas, Bacillus, Staphylococcus, Enterobacter, and Enterococcus each being present in >70% (of the 30 Lepidopteran species analyzed). A further 21 bacteria genera were identified across the Lepidopteran species but with greater variability. Whereas Šigut et al., (2022) found

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the fungal members were shaped more by diet than by species, highlighting that fungi are more transient in the microbiota than bacteria.

Microbiota diversity in the gut of Galleria mellonella

The gut microbiota of Galleria mellonella is becoming increasingly investigated due to the larvae's application as an important model organism in various fields of research as well as for the potential role G. mellonella could play in breaking down polyethanol and polystyrene (Grizanova et al., 2014;Dubovskiy et al., 2016a;Allonsius et al., 2019;Cassone et al., 2020;Lou et al., 2020;Gohl et al., 2022;[START_REF] Réjasse | Plastic Biodegradation: Do Galleria Mellonella Larvae Bioassimilate Polyethylene? A Spectral Histology Approach Using Isotopic Labeling and Infrared Microspectroscopy[END_REF]. Although more recent studies are now focusing on enzymes in the saliva to understand their ability to breakdown plastics [START_REF] Sanluis-Verdes | Wax Worm Saliva and the Enzymes Therein Are the Key to Polyethylene Degradation by Galleria Mellonella[END_REF]. Gohl et al. in 2022, published on the influence of diet and ontogeny on the G. mellonella microbiota, and showed that it could be described as relatively simple and homogenous, dominated by one or two bacterial phyla, namely Proteobacteria and Firmicutes. This is largely still true for laboratory-reared late instar larvae (the stage most commonly investigated) reared on artificial diet (Table 2). In the study conducted by Gohl et al. (2022), an interesting finding emerged regarding the diversity of the microbiota in larvae with a diet consisting of beeswax from hives, which also contained pupal skins and debris from the bees. This discovery emphasizes the insect's ability to harbor a significantly more diverse microbiota than what is typically observed in studies that predominantly focus on laboratory populations that are mostly dominated by Enterococcus and Enterobacter species. As a result, the key takeaway from this research is the recognition of the insect's capacity to support and interact with a broader range of microbial species than previously recognized. As with most Lepidoptera, there have been fewer attempts to identify the fungal microbiota of the insect, however, [START_REF] Barrionuevo | The Bacterial and Fungal Gut Microbiota of the Greater Wax Moth, Galleria Mellonella L. Consuming Polyethylene and Polystyrene[END_REF] found the community to be dominated by the phylum Chapter 1

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Ascomycota. Although, as already mentioned, fungi and yeasts are more transient than bacteria in the gut of Lepidopteran insects, usually being introduced via the environment or diet and therefore are not seen to be a typical or critical component of the Lepidopteran gut flora (Jan Jarosz, 1979;[START_REF] Barrionuevo | The Bacterial and Fungal Gut Microbiota of the Greater Wax Moth, Galleria Mellonella L. Consuming Polyethylene and Polystyrene[END_REF].

Table 2. The dominant bacteria and fungi found in late larval instar G. mellonella listed in order of the date of publication, by researchers using 16S rRNA techniques for the bacterial composition. Data is taken from studies in which healthy larvae were used (i.e. non-infected/non-treated) and the aim of the studies was to investigate the diversity of the microbiota. The shaded column highlights the ambiguity of the data, where it is unclear if the bacteria is from multiple tissue types (gut, silk glands, fat body).

Anaconda reptiles (Belgium)

Trularv TM (UK)

The Worm Lady (Canada) Huiyude Co.

(China)

Host -microbiota -pathogen interactions

Bt interaction with host insect gut microbiota

The microbiota plays a role in modulating the interaction between the host and pathogen, however, the pathogen can also alter the microbiota community which further leads to varying degrees of susceptibility to disease by the host (Cerf-

Bensussan & Gaboriau-Routhiau, 2010; Engel & Moran, 2013;Wu et al., 2016b;Wei et al., 2017). The commensal gut microbiota can influence the ability of a foreign microbe to colonize and potentially cause detrimental effects to the host. It has previously been shown in gypsy moth larvae that the midgut bacteria is necessary for insecticidal activity by B. thuringiensis, when it was discovered that antibiotic treated, and therefore 'microbiota-free,' larvae did not die from the same dose of Bt which killed conventional larvae (Broderick et al., 2006). However, when larvae were reintroduced with a commensal microbiota member, Enterobacter sp., mortality post-Bt infection was restored. It was concluded that in gypsy moth, Bt-induced mortality depends on enteric bacteria (Broderick et al., 2006). It has, however, been highlighted that the effect of the antibiotics on Bt was not properly examined in this study, and some Bt strains are sensitive to the antibiotic rifampicin (Raymond et al., 2009). A follow-up study used a rifampicin-resistant Bt strain and found that the presence of midgut bacteria was not required for Bt induced mortality, at least in Plutella xylostella, furthermore they argued that a suppressed intestinal microbiota would be a 'sound evolutionary strategy' for Bt as it may increase the mortality rates of hosts and also reduce the number of competitors within the cadaver (Raymond et al., 2009). Another study looked to the microbiota to understand differences between a Bt resistant strain and a susceptible strain of Galleria mellonella. The researchers observed that the gut microbiota of their G. mellonella larvae population was initially dominated by

Chapter 1 57
Firmicutes, particularly Enterococcus species. However, following a Bt infection, this microbial community underwent a significant shift, transitioning to become predominantly populated by Enterobacter species. Intriguingly, this shift marked a transition from a Gram-positive to a Gram-negative bacterial dominance within the gut.

There was also a loss in Pseudomonas Furthermore, the researchers observed an upregulation of growth factor genes, which the authors claim corroborates with other findings that repair of the midgut epithelium is one of the mechanisms insects use to resist Bt (Dubovskiy et al., 2016).

Mr interactions with host insect gut microbiota (oral and topical)

Entomopathogenic fungi have been shown to cause dysbiosis or changes in certain groups of bacteria comprising the gut microbiota, but not much is known about how EPF influences the fungal mycobiome. However, in general, the role of the microbiota during fungal infection is considerably various depending on the insect species as well as the composition of the microbiota. It is near impossible to make strong conclusions about the microbiota supporting fungal pathogenesis or not. For example, in cockroaches, Blattella germanica (Blattodea: Ectobiidae) the gut microbiota was found to play a critical role in protecting the insect from fungal invasion and colonization post oral feeding of M. anisopliae and B. bassiana. Conventional cockroaches experienced significantly lower mortality than that of germ-free cockroaches and bacterial strains with antifungal activity and siderophore 5 -producing ability could be isolated from the gut and faeces (Zhang et al., 2018). Alternatively, in Lymantria dispar asiatica (Lepidoptera: Erebidae) larvae, the gut microbiota was observed as contributing to mortality where conventional larvae experienced significantly decreased rates of survival than the axenic group. Moreover, post reintroduction of microbiota bacteria in Chapter 1

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the axenic larvae, mortality was again increased, reinforcing that the gut bacteria (in particular Enterococcus sp., Pseudomonas sp., Enterobacter sp., and Microbacterium sp.) contributed to mortality, most likely due to increased septicemia as found by the increase in CFUs in the hemolymph post fungal infection (Bai et al., 2023). As for the role of the microbiota when fungal conidia are ingested, again from the study on S. gregaria (locusts) the decreased viability and germination rate of the conidia in the gut was more significant in conventional than axenic S. gregaria. Further investigation found increased phenols (such as hydroquinone and bezoic acid) with antifungal activity in the gut lumen and feces of conventional locusts but not of axenic.

Subsequently these antifungal phenols were shown to be derived from the hindgut microbiota of locusts [START_REF] Dillon | Invasion of the Pathogenic Fungus Metarhizium Anisopliae through the Guts of Germfree Desert Locusts, Schistocerca Gregaria[END_REF] The uncertainty of the role of the microbiota can be further discussed where in some mosquito species, namely Anopheles and Aedes, certain gut bacteria was found to stimulate the midgut immune responses, and/or produce antimalarial compounds, resulting in better resistance to infection by the Plasmodium parasite as well as to the dengue virus [START_REF] Bian | The Endosymbiotic Bacterium Wolbachia Induces Resistance to Dengue Virus in Aedes Aegypti[END_REF][START_REF] Bahia | Exploring Anopheles Gut Bacteria for Plasmodium Blocking Activity[END_REF]. However, in another mosquito species, Anopheles stephensi, gut microbes were found to assist in fungal pathogenicity by accelerating death during infection by B. bassiana due to the translocation of opportunistic bacteria (notably, the gram-negative S. marcescens).

Comparatively, axenic mosquitoes had improved resistance to B. bassiana. The B.

bassiana infection triggered a downregulation of midgut immune responses, notably a decrease in the production of AMPs and dual oxidase expression, along with a decrease in diversity but increase in midgut bacterial abundance (Wei et al., 2017).

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CHAPTER 2

Aims + Objectives

Chapter 2

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CHAPTER 2

Objectives

During this PhD I have used the popular insect model Galleria mellonella to study the complex tripartite interactions and crosstalk between the gut microbiota and the host immune response to entomopathogens. The study compares the growth, host immune responses and susceptibility to infections in conventionally reared insects that maintain a cultivable gut microbiota, to germ-free, 'axenic' insects. The use of this simple insect model may help unravel conserved mechanisms underlying the impact of intestinal bacteria in their host physiology and the connections between microbiota, immune response and pathology. I focused in particular on the gut immune responses, its associated gut microbiota, and two naturally occurring commonly infecting pathogens, Bacillus thuringiensis (Gram-positive bacterium) and Metarhizium robertsii (Hypocrealean fungi). By using these two pathogens, the influence of the pathogen on the microbiota and host susceptibility to infection can be explored via two routes: oral and topical. My aim was to better understand how the gut microbiota shapes the host immune system responses and how this interplay limits pathogen colonization.

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Hypothesis

We hypothesize that symbiotic bacterial isolates residing within the insect gut potentially enhance the host's immune response, aiding in the combat against the pathogen infection. However, the efficiency of this symbiotically-boosted immune response may vary depending on the specific pathogen involved as well as its route of entry into the host organism.

To address the aim of the thesis, the objectives are as follow:

1. Investigate the effects of the host intestinal microbiome composition, or its absence, on the insect's growth parameters and susceptibility to entomopathogens infecting the insect orally (Bacillus thuringiensis) and topically (Metarhizium robertsii).

2. Assess the commensal microbiota community structure and subsequent intestinal immune system responses during infection to determine how the microbiota affects the tissue innate immunity to different pathogens.

Determine if the modulation of intestinal immunity by the microbiota is a factor

assisting tolerance to these pathogens.

4.

Determine the ability of isolated bacteria from the gut microbiota to re-establish in axenic late instar larvae and restore resistance to pathogens

Organization of the manuscript

This thesis has led to the creation of an article that has been submitted for publication.

The presentation of this article will begin with an overview of the study's context and a brief introduction to the methodology and techniques used in the work. The article itself will then be presented, which covers the first three objectives of the thesis. Subsequent to the article, the work conducted to try address the fourth objective is presented. The thesis concludes with a general discussion, the study's limitations and future perspectives. Finally, a review paper focusing on the use of prebiotics and probiotics in insects that are reared for human food and animal feed is presented.

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CHAPTER 3

Results

Chapter 3

64 CHAPTER 3

Presentation of the article

Context of the study

This study examines the interactions between the gut microbiota, immune response, and host susceptibility to various pathogens in the insect model, Galleria mellonella (wax moth) larvae. While the microbiota often supports the immune response, it can also increase susceptibility to certain pathogens. Therefore, we investigated how the gut microbiota reacts to pathogens with diverse infection strategies, and how this affects the host's immune responses and susceptibility to infections. The central hypothesis is that symbiotic bacterial isolates in the insect gut may enhance the host's immune defense against pathogens. However, the effectiveness of this symbioticallyboosted immune response could vary based on the specific pathogen and its entry route into the host.

Method and techniques employed to address the hypothesis

To understand the microbiota-immune response interaction in the larvae, we reared axenic (without microbiota) and conventional (with microbiota) larvae, and firstly examined the effects of harboring a microbiota, or not, on their growth and development. We then assessed their susceptibility to pathogens, exposing them to oral infections of Bacillus thuringiensis ssp. galleriae and topical infections of Metarhizium robertsii KVL 00-89 (Mr). The clearance rate of Btg spores from the gut was also monitored over time. A dual-analysis experiment was then conducted to simultaneously study the gut microbiota's composition during infections and immunerelated gene expression in the gut on the same group of larvae. The microbiota was analyzed in the conventional larvae using Illumina sequencing of the v3-v4 region of Chapter 3

65 the 16S rRNA gene, while the immune gene expression was conducted on both axenic and conventional groups using qRT-PCR, by investigating the expression of gallerimycin, gloverin, IMPI, and lysozyme. We also measured the production of reactive oxygen and nitrogen species (ROS/RNS) in the gut to gauge oxidative stress during infections. The phenoloxidase (PO) activity in the hemolymph was also tested to determine the extent of the immune response. Lastly, a dominant gut bacterium was isolated, and its ability to inhibit Btg was tested in vitro. This approach offered a comprehensive view of the interactions between G. mellonella, its gut microbiota, and immune responses to various pathogens.

Axenic rearing system for Galleria mellonella

Utilizing axenic larvae was critical in this work as the host fitness and immune response is compared between axenic (free from living microbes) and conventionally reared larvae. An axenic rearing system ensures that the larvae are devoid of their native microbiota, thus providing a 'clean slate' for controlled experimental procedures.

Moreover, by studying axenic organisms, we can delve deeper into understanding the intricate relationships between the host and its microbiota, shedding light on aspects like microbial colonization, symbiosis, and host immune responses. This brief section is an explanation of the axenic rearing system for Galleria mellonella, detailing the rationale, equipment, methodologies, used to maintain a sterile environment for these organisms.

Selection of sterilization method

Achieving axenic status in insects can traditionally be accomplished by means of sterilizing the eggs and administering a cocktail of antibiotics to the insect's diet.

Microorganisms can be transmitted vertically when mothers transfer bacteria to their Chapter 3

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offspring by depositing bacteria on the egg, specifically on the chorion, during the egglaying process [START_REF] Nyholm | In the Beginning: Egg-Microbe Interactions and Consequences for Animal Hosts[END_REF]. Therefore, methods to eliminate egg-surface microorganisms have been developed. This can be achieved by surface sterilizing the eggs, or by removing the chorion, often referred to as dechorionation [START_REF] Wu | Axenic and Gnotobiotic Insect Technologies in Research on Host-Microbiota Interactions[END_REF].

Surface sterilization removes microorganisms present on the surface of the egg without harming the embryo inside. To achieve this, common methods involve dipping the eggs in a chemical disinfectant solution such as alkanes (Urbán-Duarte et al., 2023), diluted bleach [START_REF] Koyle | Rearing the Fruit Fly Drosophila Melanogaster Under Axenic and Gnotobiotic Conditions[END_REF][START_REF] Hamilton | Borrelia Afzelii Infection in the Rodent Host Has Dramatic Effects on the Bacterial Microbiome of Ixodes Ricinus Ticks[END_REF][START_REF] Hurry | Infection with Borrelia Afzelii and Manipulation of the Egg Surface Microbiota Have No Effect on the Fitness of Immature Ixodes Ricinus Ticks[END_REF], or using an antibiotic/ antifungal wash (Broderick et al., 2006;Genta et al., 2006;Raymond et al., 2009;[START_REF] Correa | Generation of Axenic Aedes Aegypti Demonstrate Live Bacteria Are Not Required for Mosquito Development[END_REF]. Surface sterilization and dechorionation methods are effective but can however kill many of the eggs, and the antibiotics can interfere with the insect's growth [START_REF] Wu | Axenic and Gnotobiotic Insect Technologies in Research on Host-Microbiota Interactions[END_REF]). An advantage for the surface sterilization method is that the chorion is not always removed, and in this case, the egg remains more protected. The chorion is the waxy outer shell of the egg which provides mechanical strength to the structure of the egg, maintains moisture and protects the embryo from the external environment (Urbán- [START_REF] Urbán-Duarte | An Effective Chemical Permeabilization of Silkworm Embryos[END_REF]. Upon hatching, the young larva typically eats through the egg, often consuming the entire sac and chorion, thereby integrating the bacteria into its microbiota. However the removal of the chorion to generate axenic insects also has several advantages including the specificity of the sterilization whereby only the eggs are targeted, minimizing the risk of unintended consequences to other stages of the insect's life cycle and the method has high reproducibility of generating axenic individuals [START_REF] Henn | Dechorionation as a Tool to Improve the Fish Embryo Toxicity Test (FET) with the Zebrafish (Danio Rerio)[END_REF]. The process can however be labor-intensive and careful precision is needed to avoid physical damage of the embryo to reduce high mortality rates [START_REF] Wu | Axenic and Gnotobiotic Insect Technologies in Research on Host-Microbiota Interactions[END_REF]. There is also a species limitation whereby the effectiveness and feasibility of this method Chapter 3 67 might vary among insect species based on the characteristics of their eggs and chorion. Axenic insects can also be generated in later life stages, such as that they live most of their life as conventional insects but are later cleared of their microbiota. To achieve this, antibiotics are incorporated into the diet. The use of antibiotics can be considered an effective technique, which generally eliminates a broad range of bacteria from the gut of insects [START_REF] Heys | The Effect of Gut Microbiota Elimination in Drosophila Melanogaster: A Howto Guide for Host-Microbiota Studies[END_REF]. It is also a relatively straightforward procedure to administer antibiotics, especially when it can be incorporated into the insect's diet [START_REF] Heys | The Effect of Gut Microbiota Elimination in Drosophila Melanogaster: A Howto Guide for Host-Microbiota Studies[END_REF]Zhang et al., 2022a). However, there can be a risk of incomplete sterilization as not all bacteria may be susceptible to the antibiotics used, as well as triggering off-target effects such as affecting the insect's physiology, metabolism, and development, leading to unintended side effects. The administration of antibiotics can also shock the host system in such that certain responses cannot always be correlated to the absence of bacteria. The use of antibiotics has also been implicit in undermining the validity of some results where researchers are testing the efficacy of a pathogen on an axenic host (Broderick et al., 2006;Raymond et al., 2009).

Therefore, the use of antibiotics may need to be reconsidered depending on the type of experiment one is using the sterile insect for.

A further technique, and one employed in this study, is to surface sterilize eggs using ultraviolet (UV) radiation (Fig. 13). This sterilization procedure for producing axenic larvae is chemical-free and a versatile sterilization tool as its effects are broadspectrum, killing a variety of microorganisms, including bacteria, fungi, and viruses [START_REF] Khan | Efficacy of Ultraviolet Radiations against Coronavirus, Bacteria, Fungi, Fungal Spores and Biofilm[END_REF], without impacting the morphology (Fig. 14), fitness and lifecycle of G.melonnella. It is also a fast and efficient process, allowing for the treatment of large batches of eggs in a relatively short time. It is however important to find the right Chapter 3

68 exposure time to optimally kill microorganisms but to not harm the eggs, which if overexposed to UV could decrease the hatching rates. Once the eggs are sterilized, the diet must also be treated to eliminate the risk of microbe introductions into the insect. For this, artificial diets and all equipment are usually autoclaved at conditions ensuring subsequent sterilization of the diet [START_REF] Steven | The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions[END_REF]. For experiments presented in this thesis, the diet for rearing the larvae axenically and conventionally are the same, comprising of natural beeswax and bee collected pollen. Albeit the axenic diet, as has been done for many laboratory-reared sterile animals such as mice, rats, rabbits and chickens was sterilized using gamma radiation [START_REF] Ley | Sterilization of Laboratory Animal Diets Using Gamma Radiation[END_REF]. This renders the diet pathogen-free and maintains its nutritional properties [START_REF] Ley | Sterilization of Laboratory Animal Diets Using Gamma Radiation[END_REF]. The process begins within a sterile laminar flow hood, ensuring contamination-free handling. 24 -48 h old egg sheets are retrieved from the adult cage, and inspected for obstructions. Obstructions such as some paper covering the eggs, or eggs laid in clutches blocking one another could hinder the subsequent UV sterilization's effectiveness. These sheets, along with essential tools, undergo a thorough UV sterilization with a 10-minute exposure on either side of the sheet. The previously gamma-irradiated sterile diet of pollen and wax is then poured into the autoclaved sterilized jar. Sterilized egg sheets are then placed atop this diet, followed by a three-tier sealing process involving a meshed metal lid, a layer medical gauze, a layer of cotton wool, and a final layer of medical gauze which is taped together and sealed. The jar is then placed in an incubator maintained at 27°C with a 12/12 photoperiod cycle, allowing larvae to grow to an approximate weight of 250-300mg. Finally, their axenic status is verified via both culture-dependent and independent means, by monitoring the microbial growth of dissected gut homogenates on agar plates, or by molecular analysis targeting the v3-v4 region of the 16S gene from bacterial DNA extracted from dissected guts. This comprehensive workflow ensures the production of axenic larvae with validated sterility.

Key findings of the article

Axenic larvae could successfully complete their lifecycle in the absence of a gut microbiota. Larvae maintaining the microbiota (conventional larvae) had less variation but a similar average developmental time and weight to their axenic counterparts.

Fitness assays revealed the conventionally reared larvae were more effective in suppressing Btg infection and clearing the spores and vegetative cells from the gut than axenic larvae. No significant difference in mortality was observed between axenic and conventional larvae when infected topically with Mr.

Conclusion

The study underscores the crucial role of the gut microbiota, especially Enterococcus species, in the immune response of G. mellonella. The Gram-positive Enterococcus dominated gut microbiota provides a protective mechanism against bacterial infections caused by Btg but does not significantly influence the outcome of fungal infections by Mr. Understanding these interactions offer insights into maintaining insect health against diverse pathogens. This protection might be attributed to the enhancement of the host immune system's basal level of activation and the production of antimicrobial compounds by the microbiota.

The immunostimulatory role of an Enterococcus-dominated gut microbiota in host protection against bacterial and fungal pathogens in Galleria mellonella larvae Jennifer Upfold Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota's modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host's response in Galleria mellonella larvae reared under axenic (sterile) and conventional (nonsterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae (Btg), and topical infection induced by Metarhizium robertsii (Mr). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD 50 ) of Btg and Mr. Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%-100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)-gallerimycin and gloverin-in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii, isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro. Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the Federico Cappa, University of Florence, Italy stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus-dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

KEYWORDS

Bacillus thuringiensis, Metarhizium robertsii, gene expression, gut microbiota, tripartite interactions, axenic

Introduction

Insects are essential contributors to the global ecosystem, performing important ecosystem services in agriculture such as pollination and biological control; they are heavily used for research purposes, and, more recently, for sustainable food production (1)(2)(3). Moreover, their potential to be produced in large-scale production facilities holds promise for addressing various environmental and nutritional challenges confronting our world. However, inherent risks arise when handling large numbers of animals such as disease outbreaks, which commonly occur among mass-reared animals. This could create consequences for public health, as well as raise concerns for animal welfare, if diseases are not adequately managed (4,5).

Insects have evolved innate immune systems that allow them to defend themselves against invading pathogens and subsequent diseases. They possess an innate immune response that can be elicited via cellular or humoral means (6). Their cellular immune responses are mediated by hemocytes and include phagocytosis, nodulation, and encapsulation, which are capable of fighting against a large spectrum of pathogens (7). The humoral immune response involves processes such as melanization via the activation of the phenoloxidase system, the secretion of molecules such as antimicrobial peptides (AMPs), and the production of reactive oxygen/nitrogen species (ROS/RNS) (6,8,9). The gut of insects, which harbors a complex ecosystem of microorganisms known as the microbiota, is also recognized as playing a crucial role in maintaining the overall health of its host by modulating various activities such as nutrient absorption, digestion, and immune system development, among other important biological processes (10)(11)(12). Therefore, understanding the role of the insect gut microbiota and its interactions with its host's immune system and pathogens is essential for both insect control and insect health.

The nature and dynamic of the microbiota-immune response interaction can vary greatly across different insect groups, in part due to the differences in the bacterial species comprising the gut microbiota, but also depending on factors such as diet, habitat, life stage, sex, and social interactions, resulting in differences in the scope of benefits provided to the host (10). The gut microbiome, in a balanced state, can maintain a symbiotic relationship; however, the relationship exists on a continuum that can shift toward neutrality or pathogenicity. When functioning at its best, this immune system-microbiota partnership enables protective responses against pathogens, while avoiding excessive immune activation that can harm host tissues or disrupt the composition and diversity of the symbiotic gut microbiota, thus preventing opportunistic infections by pathogenic bacteria or fungi (6,(13)(14)(15).

In some cases, the microbiota may actually facilitate pathogen infections, particularly when there are disruptions to the community caused by dysbiosis. This can result in an elevated risk of septicemia when bacteria breach the gut barrier and enter the hemocoel (16,17). Although the role of the gut microbiota in enhancing host resistance to infections is increasingly studied, the current knowledge of its role and contribution to host tolerance by different pathogens is still limited and fragmented. Although the gut microbiota has been observed to contribute to the defense against oral infections by invading pathogens in a wide range of animals, from invertebrates to vertebrates, less is known about its role in defending against topical pathogens (18,19). The gut can be an important site of immune resistance to a variety of pathogens that enter not only orally but also topically, as is typically observed in entomopathogenic fungi, which enter their host primarily through the cuticle. Therefore, this interaction between insect gut microbiota and fungi needs to be further explored.

In this study, our aim was to investigate the partnership between the gut microbiota and the immune response during single infections by pathogens that infect Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae through different routes. G. mellonella serves as a widely utilized model organism for studying host-pathogen interactions, given its similarities to the mammalian immune system and its tolerance of temperatures ≥ 37°C. As a result, there is an increasing amount of published information available on its immune system (6,20,21). In addition, the microbiota of G. mellonella has been the subject of growing research interest, particularly due to its possible ability to break down polyethylene (22)(23)(24). To gain insights into the role of the microbiota in protecting the host against infectious agents, we specifically focused on both conventional and axenic (microbiotafree) larvae (25,26).

To test the immune response via oral infection, we used Bacillus thuringiensis subsp. galleriae 69-6 (Btg), a pathogenic strain known to infect G. mellonella (27,28). B. thuringiensis is a Gram-positive, spore-forming bacteria found in soil environments, which produces Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 02 cry and cyt d-endotoxins with entomopathogenic properties. When ingested, the toxins solubilize and interact with specific receptors on the midgut epithelial cells, leading to gut epithelium breakdown and potential septicemia (29,30). For topical infection, we utilized Metarhizium robertsii, an entomopathogenic fungus that can infect various insect species, including G. mellonella. The fungus penetrates the host from the cuticle surface via the formation of germ tubes and cuticle-degrading enzymes, where it then grows rapidly in the hemocoel and destroys important structures of the host (31). These two pathogens produce spores or conidia that can contaminate many environments, from food to water or air, and, therefore, have the potential to be introduced into insect farms through contaminated feed sources, equipment, or even via simple airborne transmission (5).

Specifically, we evaluated the effects of exposure to these two naturally occurring pathogens, entering the host orally and topically, by analyzing the size and composition of the conventional larvae gut microbiota community. We further examined how the composition and diversity of the gut bacterial microbiota, as well as the absence of microbiota, affect the severity of infection, the immune response, and growth and development (i.e., time to pupation, pupal duration, and adult duration). To achieve this, we used a combination of 16S rRNA gene amplicon sequencing to quantify the dominant taxa and subsequently evaluate the microbial dysbiosis provoked by the pathogens, and target qRT-PCR analysis of immune response genes in the gut to determine the impact of the gut composition on the expression of three target genes involved in the immune response. Our findings shed light on the complex interactions between the gut microbiota and host immunity and have implications for the development of novel strategies for controlling infectious diseases.

Materials and methods

Insect rearing

A laboratory population of G. mellonella L. (Lepidoptera: Pyralidae) larvae reared at the Micalis Institute of INRAE, Jouy en Josas, France, was used in the following study. The population was reared on a nutritionally rich, natural diet of beeswax and pollen (La Ruche Roannaise, France). The nutritional compounds in pollen included essential amino acids, carbohydrates, a few lipids, and some vitamins and bioelements; the wax served as a source of fatty acids and other complex long-chain carbohydrates that G. mellonella can metabolize (32). The rearing chamber was set at 28°C with a 12-hour photoperiod.

Preparation of axenic larvae

The production and manipulation of axenic larvae, defined as having an absence of bacteria in their gut microbiota, were done in sterile conditions. To produce the larvae, the materials and egg sheets were exposed to a 10-minute surface sterilization by ultraviolet (UV) light at 254 nm in a biosafety cabinet. The sterilized egg sheets were then placed into an autoclaved sterile glass jar that was sealed with a fine mesh (with a hole diameter of 0.596 mm) and covered with natural-fiber cotton wool. The conventional and axenic larvae were fed the same diet; however, the diet for axenic larvae was sterilized via gamma irradiation (sterilization was carried out using cobalt-60 at the intensity of 25 kGy; SAFE, Augy, France). The axenic status was verified on five larvae by plating a suspension of the crushed and homogenized larvae on BHI (brain heart infusion) agar. Further verification by PCR targeting the bacterial 16S rRNA gene using the V3-V4 region (forward: 5′-TACGGGAGGCAGCAG-3′; and reverse: 5′-CCAGGGTATCTAATCC-3′; 33,34) was conducted on DNA extracted from dissected guts (Supplementary Figure S1).

Biological assay

The various life stages of the axenic and conventional larvae were monitored from the egg to the adult stage to determine if there were any differences in the biological parameters of larvae without a microbiota. The eggs of the conventional and axenic adults were collected after 2 days (approximately 250 eggs per sheet) and placed into sterile glass jars supplemented with food. A natural sterilized diet was provided to the axenic group, and a non-treated natural diet was provided to the conventional group. This was an important step to ensure that the conventional larvae would have a colonized gut microbiota. The larvae were left for approximately 2 weeks until they were large enough to handle (40 mg). They were then individually placed in 24-well plates and provided with a nutritious artifi cial sterilized wheat-based diet (35) (Supplementary Table S1), where they were monitored until pupation. The pupae were then moved to medicine cups covered in cotton wool where the adults would eclose and remain until death. The adults did not feed due to degenerative mouthparts and were therefore not provided with food or water (36).

Pathogen infection assays 2.3.1 Oral infection-Bacillus thuringiensis

The bacterium Bacillus thuringiensis subsp. galleriae 69-6 (Btg) obtained from Dr. Ekaterina V. Grizanova, Department of Plant Protection, Novosibirsk State Agrarian University, was used to induce the immune response by oral infection. The strain was transformed with pHT315WPaphA3-gfp to express GFP (green fluorescent protein). The Btg cells were grown with shaking at 30°C for 96 h in HCT (0.7% casein hydrolysate, 0.5% tryptone, 0.68% KH 2 PO 4 , 0.012% MgSO 4 • 7H 2 O, 0.00022% MnSO 4 • 4H 2 O, 0.0014% ZnSO 4 • 7H 2 O, 0.008% ferric ammonium citrate, and 0.018% CaCl 2 • 4H 2 O, pH 7.2) + 0.5% glucose medium. After 72 h, sporulated cells and crystals were subjected to three washing (in 1M NaCl) and centrifugation (4°C, 5,000 G) cycles. After the final centrifugation, the spores/crystals were resuspended in doubledistilled water (ddH 2 O). The oral infection was undertaken on ± 250-mg larvae by force feeding with a cannula hypodermic needle (30 gauge × 25 mm) and a 1-mL syringe (Terumo Corporation). A spore and crystalline toxin mixture of 10 µL was administered at a half-lethal dose [5.2 × 10 5 colony-forming units (CFU) in 10 µl], Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 03 determined in previous assays using only conventional larvae. The uninfected control larvae were force fed with 10 µL of ddH 2 O. The inoculated larvae were then placed in Petri dishes (60 mm × 15 mm) and kept in a 30°C incubator to monitor the infection over 96 h. For the mortality assays, a total of 60 larvae were infected over three replicates.

Topical infection-Metarhizium robertsii

The fungus Metarhizium robertsii KVL 00-89 (Mr) was obtained from the culture collection at the Section for Organismal Biology at the Department of Plant and Environmental Sciences of the University of Copenhagen, Copenhagen, Denmark. To induce the immune response by a topical infection, Mr was grown on Sabouraud dextrose agar (SDAY) plates (16.25 g of SDA (Sigma-Aldrich), 2.5 g of yeast extract, and 11.25 g of agar in 1 L distilled water) at 30°C. The conidia were harvested after 3 weeks and a Fuchs-Rosenthal counting chamber (Hausser Scientific) was used to determine the concentration. The conidial suspensions of Mr were prepared at 1 × 10 7 conidia in a 3-mL solution of 0.05% Triton X. The larvae were completely submerged in the conidial suspension for 30 s, and then placed in a sterile Petri dish lined with filter paper. All conidial suspensions had a germination rate of between 95% and 100%. The control larvae were submerged in a solution of 0.05% Triton X. The larvae were then placed in a 30°C incubator to monitor the infection up to 240 h, or until the surviving larvae pupated. For the mortality assays, a total of 60 larvae were infected over three replicates.

Clearance assay of Btg

To investigate the rate of clearance of Btg in the gut of the axenic and conventional larvae, 30 larvae were infected at a half-lethal dose (calculated in the conventional larvae). Every 24 h, the CFU counts of Btg were monitored by plating the dissected guts of three surviving larvae (individually) on to lysogeny broth agar (LBA). The guts were dissected in sterile conditions and homogenized in 500 µL of ddH 2 O. This was monitored for up to 96 h.

Dual-action analysis of immune response genes in the gut tissue and microbiota assessment

To investigate the relationship between the gut microbiota and immune response, surviving larvae infected by either Btg or Mr were collected at specific time points, representing early and late stages of infection. For the Btg treatment, larvae were collected at 20 h and 40 h post infection; for Mr, the larvae were collected at 20 h and 96 h, as Mr takes a longer time to achieve mortality in its host. The collected larvae were subjected to a 30-min cooling period on ice, followed by surface sterilization using ethanol and subsequent gut dissection. The gut was homogenized in 200 mL of ddH 2 O using a sterile pestle prior to splitting the contents into two Eppendorf tubes, with one half intended for a qRT-PCR analysis of immune response genes and the other for a 16S rRNA microbiota assessment. Half guts from two larvae were mixed, with each biological replicate comprising two larvae. Therefore, we could assess the changes occurring in the microbiota and the expression of immune response genes in the same group of larvae (Supplementary Figure S2).

DNA extraction of gut samples and axenic verification

The DNA from gut samples was extracted using the DNeasy® PowerSoil® Pro Kit (Qiagen) in accordance with the manufacturer's instructions, with slight modifications. The samples were incubated at 65°C with shaking for 10 minutes prior to being lysed in a FastPrep-24 ™ (MP Biomedicals) for 2 × 40 s at 4.0 M/S with a 5-min break at 4°C. The quality of the extractions was checked on a NanoDrop ™ spectrophotometer (Thermo Fisher Scientific) and a 1.2% agarose gel. For axenic samples, a PCR targeting the bacterial 16S rRNA gene using the V3-V4 region (forward: 5′-TACGGGAGGCAGCAG-3′; and reverse: 5′-CCAGGGTATCTAATCC-3′; 33,34) was conducted to verify axenic status (Supplementary Figure S1B).

16S rRNA gene sequencing

The DNA extractions of the conventional larvae (with controls including a Zymobiomics ™ microbiome standard and two axenic samples) were further processed by Eurofins Genomics (France). This included PCR reactions, library preparation, and sequencing, which was performed on an Illumina MiSeq ™ (2 × 300 bp). The resulting sequences were further processed on RStudio using the "DADA2" package to obtain an amplicon sequence variant (ASV) table, which identifies fine-scale variations compared with the more traditional operational taxonomic unit (OTU) table (37). Based on the inspection of the quality profiles and error rates, some modifications were made to the proposed functions by the DADA2 workflow and package. Taxonomic affiliations were performed using the SILVA2 database developed by the Leibniz Institute (38), and NCBI BLAST was used on ASV sequences to further identify to species level. The relative abundance plots and alpha-and beta-diversity metrices were generated using the R package "Phyloseq" (39). The permutational multivariate analysis of variance (PERMANOVA) was conducted on beta-diversity estimates with significance at < 0.05 using the adonis() function from the R "vegan" package (40) (see Supplementary Materials and Methods).

RNA extraction, cDNA synthesis, and qRT-PCR of immune response genes in the gut

RNA extractions using TRIzol ™ reagent (Invitrogen) were performed on the other half of the dissected and homogenized gut samples. The quality and quantity of the extractions were estimated on a NanoDrop spectrophotometer and via gel electrophoresis. The RNA quality was verified prior to being transformed into cDNA with SuperScript ™ IV VILO ™ master mix (Thermo Fisher Scientific), in accordance with the manufacturer's instructions. To investigate the role of the host Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 04 gut bacteria on the expression of immune response genes in the gut tissue, we performed qRT-PCR on four immune-related genes (Supplementary Tables S2,S3): two antimicrobial peptide genes (gallerimycin and gloverin), a lysozyme-like gene (lysozyme), and an insect metalloproteinase inhibitor (IMPI). The relative expression of each target gene was normalized to a housekeeping gene that is known for its stable expression across different experimental conditions: 18S rRNA (Ivan M. 41). Therefore, the data are presented as a ratio over 18S, taking into account the reaction efficiencies of the PCR. Subsequently, a heatmap of the fold change (log2) in gene expression from basal to infected conditions by either Btg or Mr is presented. The significance was determined for the difference in the fold change in expression of each gene and the time point between the conventional and axenic larvae, by oneway ANOVA on log2-transformed values.

Inhibition assay

The Enterococcus mundtii strain isolated from the conventional larvae gut (see Supplementary Material and Methods, Supplementary Figure S3) was screened using the agar spot-on lawn technique (42), as conducted by Grau et al. (43) along with minor modifications that are further described. First, overnight cultures of the E. mundtii strain was grown in a MRS (deMan, Rogosa, and Sharpe) medium at 30°C without agitation. Approximately 7 µL was then spotted onto 0.7% MRS agar plates and incubated for a further 24 h under aerobic conditions at 30°C. After the 24-hour incubation, the plates were exposed to UV light at 254 nm for 25 min in a sterile biosafety cabinet. This action was taken to kill the E. mundtii colonies, resulting in any inhibitory affect coming from only the bacteriocins already secreted into the agar. An overnight culture of the indicator bacteria, Bacillus thuringiensis subsp. 69-6, was mixed with 1% LBA. Approximately 10 mL of the indicator bacteria was poured over the previously E. mundtii "spotted" and UV-exposed plates. Once solidified, the plates were incubated for a further 48 h at 30°C under aerobic conditions. The plates were then assessed for inhibition zones, and subsequent zones were measured using a ruler (Supplementary Figure S4).

Phenoloxidase activity assay

The phenoloxidase (PO) activity in the hemolymph of larvae was measured using a technique described by Valadez-Lira et al. (44), who adapted a method from Harizanova et al. (45). Slight modifications were made as further described. Approximately 10µL of hemolymph was individually collected via puncturing the fourth proleg with a sterile microneedle, and directly pipetting the bleeding hemolymph into an Eppendorf tube containing 190 µL of ice-cold sterile 1 × phosphate-buffered saline (PBS). This was done at 20 h and 40 h for the Btg infection, and at 20 h and 96 h for the Mr infection. The homogenates were frozen at -80°C for at least 24 h, which allowed the hemocytes to lyze and release the inner-cell plasma. The enzymatic assay was then conducted on thawed (on ice) and centrifugated (8,000 g, 4°C, 5 min) samples by preparing a flat-bottomed 96-well plate with 30 µL of the sample, mixed with 100 µL of 16 mM L-DOPA (Supplementary Figure S5) (3,4, dihyroxy-L-phenylalanine). As a negative control, PBS was used. The plates were read immediately at 490 nm every 15 s (with 3 s of shaking between reads) at 30°C on a photospectrometer (Spectra Max 190) using SoftMax ™ Pro 7.1 software. The PO activity was measured from the slope (V max ) of the reaction curve in its linear phase. The samples were run in duplicate for a total of five individual larvae tested per time point and treatment, with three independent experiments for the Btg infection and two independent experiments for the Mr infection.

ROS/RNS assay

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the total free radicals generated during cellular processes and immune responses. Measuring total ROS/RNS helps evaluate oxidative stress and its impact on host fitness during infection. To determine their levels, the total ROS/RNS was quantified in the surviving larvae gut during oral infection by Btg and topical infection by Mr. The guts from three surviving larvae were dissected, pooled, and homogenized in sterile PBS at 20 h and 40 h for Btg infection, and at 20 h and 96 h for Mr infection. Three samples were prepared for a total of nine larvae per time point and treatment (three pooled guts in three separate samples). The samples were processed using the OxiSelect ™ In Vitro ROS/RNS Assay Kit (Cell Biolabs, USA) following the manufacturer's instructions. Approximately 100-fold diluted samples and kit components were incubated on a 96-well black-bottom plate for 20 min at room temperature. Fluorescence was measured on an Infinite® M200 Pro microplate spectrofluorometer (Tecan, USA), and the results were obtained using a predetermined dichlorofluorescein (DCF) standard curve linear regression equation.

Data analysis

For mortality assays, the log-rank test was performed with Holm-S ̌ıdaḱ adjustment on RStudio using the survival package (RStudio ™ 2010). All other data were analyzed using GraphPad Prism v.9.3 (GraphPad Software Inc., USA). The data were always checked for normality using the Shapiro-Wilk W-test prior to determining the statistical tests. Based on the normality statistic, the unpaired t-test was performed at each time point of the clearance assay as well as for the various biological parameters between axenic and conventional larvae.

Results

To investigate the relationship between the gut microbiota and the host's tolerance to oral and topical pathogens in G. mellonella, axenic larvae were generated via UV sterilization of the eggs followed by sterile rearing on a germ-free natural diet. The axenic status of the Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 05 larvae was verified by dissecting the whole gut and performing CFU counts on BHI agar plates, as well as by PCR amplification of the V3-V4 region of the 16S gene. As a final confirmation, two dissected gut samples from the axenic larvae were included in the Illumina sequencing. No cultivable bacteria were present in the axenic larvae gut, no amplification of the V3-V4 region was observed, and no reads were generated from the Illumina sequencing (Supplementary Figure S1). In comparison, the conventional larva gut-maintained bacteria were at a concentration of between 1 × 10 7 and 10 8 CFU/mL per larva.

Axenic group experienced a longer and more variable larval stage

We observed some differences in the various life stages of the insect between axenic and conventional larvae. The larval developmental time, that is, from egg to pupation, was significantly longer in the axenic larvae, with an average of 65.9 days, than in the conventional larvae, which took on average 61.4 days (p = 0.0058) (Figure 1A). Six individuals from the conventional group and nine individuals from the axenic group did not emerge as adults. There was also a significant difference in the length of the adult life span (Figure 1B) (p = 0.0033).

The mean average length for the conventional adults was 8.5 days compared with 7.39 days in the axenic group, albeit the axenic larvae experienced greater variation, with the shortest life span being 2.5 days and the longest being 15 days. Comparatively, the longest adult life span in the conventional group was 12.5 days and the shortest was 5 days. There was no significant difference in the total life span (Figure 1C) and pupal weights (Figure 1D).

3.2 Axenic larvae were more susceptible to orally inoculated Btg Different rates of survival were observed between axenic and conventional larvae post-oral Btg infection (Figure 1E). The larvae reared axenically were more susceptible to a half-lethal dose of a Btg spore crystal preparation, with a significantly greater mortality from 40 h post infection (60%-c 2 > 9.7, df = 1; p = 0.002), which was maintained at 72 h (90%-c 2 > 15.2, df = 1; p = 0.0004) and 96 h (96.6%-c 2 > 11.7, df = 1; p = 0.0007). For the Mr topical infection, no significant difference was observed between the axenic and conventional larvae at a half-lethal dose (Figure 1F). However, slightly faster rates of mortality were observed in the axenic larvae in the first 72 h. To further investigate the difference in Btg pathogenicity between axenic and conventional larvae, an assay to assess the rate of clearance of Btg from the gut in surviving larvae was performed. Along with the different rate of clearance between the axenic and conventional larvae (Figure 2), we also observed considerable variation in the clearance between individual larvae. At 48 h post inoculation, some surviving conventional larvae were able to completely clear Btg, while the concentration remained above 5.2 × 10 3 CFU/mL in axenic larvae. By 72 h, the conventional larvae cleared the spores to that of a sublethal level, and at 96 h all surviving larvae tested completely cleared the spores. However, at 72 h the average concentration of spores in the axenic group remained above the lethal concentration, and at 96 h all remaining larvae died.

3.4

The bacterial gut microbiota was dominated by Enterococcus spp.

We performed bacterial taxonomic gene sequencing on the bacterial gut microbiota of 56 conventional larvae to assess the community structure during infection by oral and topical pathogens, in order to infer how changes to the community may be affecting host susceptibility during pathogenesis. Two axenic samples were also sequenced but generated no reads, verifying their sterility. The sequencing of the conventional larvae revealed that the gut microbiota of our G. mellonella population was dominated by only one bacterial genus: Enterococcus. The ASVs showed strong similarity with four different species-Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus innesii, and E. mundtii (Figure 3)-as determined using the SILVA16S and BLAST databases (Supplementary Table S4). We found variation in the proportion of the four species across different cohorts of larvae; however, E. casseliflavus and E. innessi were consistently in a minority compared with E. gallinarum and E. mundtii, which dominated the community. When larvae were infected with a median lethal dose (LD 50 ) of Btg, its presence was recovered in all infected samples at 20 h post infection (n = 6 larvae), with > 50% dominance of the overall community in two of the three samples. At 40 h post infection, the presence of Btg again varied across the samples (n = 6 larvae), with two samples showing a reduction in Btg to that of what was observed at 20 h, and one sample containing > 80% Btg reads (Supplementary Figure S6). Mr did not have an effect on the bacterial community structure at either 20 h (n = 8 larvae) or 96 h (n = 8 larvae), with similar relative abundances observed in the controls (n = 8 larvae at each time point). The alpha-diversity matrices also showed no significant differences between Btg-or Mr-infected samples and their control samples in both the Observed and the Simpson Index (Supplementary Figure S7). For the beta-diversity analysis, all ASVs assigned to Btg were subsetted and discarded using the "subset_taxa" function on phyloseq. This analysis was done to assess the impact of Btg on the rest of the community without the influence of the introduced Btg affecting the results (Supplementary Figure S8). The principal component analysis (PCoA) of the data subsets revealed a small grouping of three Btg-infected samples: two from 20 h and one from 40 h post infection. However, further analysis by PERMANOVA revealed no significance between the time or treatments (Supplementary Table S5). This suggests that the microbiota community remained relatively stable (i.e., no shifts at the genus or species level) during infection as compared with the uninfected microbiota community. Finally, our analysis highlights the inter-individual variation of the proportion of Btg (in Btg-infected samples) and the Enterococcus spp. occurring in the microbiota community between larvae, which is an important aspect to consider and is often lost in group-level analyses.

Isolated E. mundtii from the conventional larvae gut microbiota inhibited Btg in vitro

The inhibition assays using the "agar-spot-on-lawn" technique ( 43) with E. mundtii against Bacillus thuringiensis subsp. galleriae 69-6 yielded inhibition zones around the spot of E. mundtii (Figure 4). This showed that the E. mundtii isolated from the conventional larvae gut had some antimicrobial activity against Btg in vitro.

Basal immune gene expression is stimulated by gut microbiota

The results of the qRT-PCR of several immune response genes revealed that strong differences were observed in the basal (uninfected control group) levels of expression of gallerimycin, gloverin, and IMPI between the axenic and conventional larvae (Figure 5). This was statistically significant for gallerimycin at 20 h (p = 0.0024) and 40 h (p ≤ 0.0001), as well as at 96 h for gloverin (p = The clearance of Btg in the gut of surviving larvae over 96 hours: the dissected gut from surviving larvae post infection by Btg at 0, 24-, 48-, 72 and 96 hours was plated on LBA to count the resulting colony-forming units (CFU). By 72 h post infection the conventional larvae (in blue) were able to clear Btg to a sublethal concentration and, furthermore, completely clear the infection by 96 h. Comparatively, the number of spores in the axenic larvae (in red) remained high, even at 72 h, resulting in 100% mortality, and, therefore, no clearance data for the axenic group by 96 h. Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 07 0.0048) and IMPI (p = 0.007). However, during Btg infection, there was a similar level of gene expression between the axenic and conventional larvae. In fact, there were no significant differences found during Btg infection at either 20 h or 40 h post exposure. Notably, no significant differences were observed in the relative levels of expression of lysozyme at either 20 h or 40 h between the axenic and conventional larvae, at both basal conditions as well as post-Btg infection.

The levels of gene expression in the gut post-topical infection of Mr were different from the orally infected Btg infection. At 20 h post infection, there was a significant difference in the expression of gallerimycin (p = 0.0003) between the axenic and conventional larvae, with the conventional larvae experiencing higher levels of expression (Figure 5). It is expected that the levels of gene expression in the gut would be lower following a topical infection than an oral infection; however, at 96 h only the conventional larvae experienced an increase in important AMP expression (gallerimycin, p ≤ 0.0001; gloverin, p = 0.0027). At basal (uninfected) conditions a significant difference was observed in gallerimycin (p = 0.0017), and almost significantly for gloverin (p = 0.0508) at 20 h post infection; however, no significant differences in the levels of gene expression were observed at 20 h for IMPI, and at 96 h, no genes were significantly more expressed in either the axenic or conventional larvae.

A global view of the fold change in gene expression from basal to infected state

During Btg infection, the axenic larvae had a greater fold change in gene expression from an uninfected to an infected state, which FIGURE 4 Zone of inhibition using the agar-spot-on-lawn technique between 24-h culture of Enterococcus mundtii ("spot") and a 24-hour culture of Bacillus thuringiensis subsp. galleriae 69-6 ("lawn"/indicator strain); the area from edge to edge of the inhibition zone (measurement indicated by white bar) is 19 mm ± 1 mm.

FIGURE 3

The relative abundances of the bacterial species in the gut of the conventional larvae at two time points for orally infected larvae with Bacillus thuringiensis subsp. galleriae 69-6 (Btg), and topically infected with Metarhizium robertsii (Mr), as well as the controls (larvae force-fed sterile water for the Btg group and submerged in 0.05% Triton X for the Mr group). The plots display the proportion of reads post rarefied (35,000), normalized (per 100), and filtered (0.1/100). Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 08 was significantly different from conventional larvae at 20 h post infection for gallerimycin (p = 0.0226) as well as at 40 h post infection for gallerimycin (p ≤ 0.0001), gloverin (p = 0.0183), and IMPI (0.0004). During the Mr infection, the fold change in gene expression was significantly greater only in the conventional larvae gut than in the axenic group for the expression of gallerimycin (p = 0.0009) and gloverin (p = 0.025) at 96 h post infection, and no fold change in the level of expression in these important immune response genes was found in the axenic group (Figure 6).

PO activity increased post Btg infection but decreased post Mr infection

We measured the PO activity in the hemolymph of orally and topically infected larvae reared axenically or conventionally to further investigate the role of the microbiota in different immune responses in its host (Figure 7). The larvae, independent of microbiota status, experienced an increase in PO activity post-Btg infection as compared with the uninfected controls. For the Btg infection, at 20 h both the axenic and conventional larvae experienced significant increases in PO activity (p = 0.005 and p ≤ 0.0001 for axenic and conventional larvae, respectively). The significant increase was maintained at 40 h for the axenic larvae (p ≤ 0.0001), but not for the conventional larvae. For the topical Mr infection, a slight increase in PO activity was observed at 20 h post infection (although this was not statistically significant), followed by a decline in activity at 96 h post infection for both the axenic and conventional larvae. No significance was found at 96 h for either the conventional or axenic larvae; however, there was considerable variation in the amount of PO activity, potentially corresponding to the variation in cadaver phenotypes post death by Mr (Supplementary Figure S9), in which some larvae experienced higher levels of melanization with a complete absence of melanization in others. The relative expression of several immune response genes in the gut of axenic (red) and conventional larvae (blue), expressed as a ratio over 18S House keeping gene (HKG), at different time points post infection, along with respective controls. For the oral Btg infection and uninfected controls, relative expression is assessed at 20 h and 40 h, as for the topical Mr infection and uninfected controls, relative expression is assessed at 20 h and 96 h. Significance was determined by one-way ANOVA on log2-transformed values; ns (not significant) = P > 0.05; * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001. Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 09 3.9 Total free radicals (ROS/RNS) were greater in the gut of uninfected axenic larvae than in uninfected conventional larvae

The level of total free radicals (ROS/RNS) was slightly higher in uninfected axenic larvae than in uninfected conventional larvae (Figures 8A,B). During infection by Btg, surviving axenic larvae experienced an increase in total ROS/RNS in the gut environment at 20 h post infection (4.2× increase from the uninfected state average), as well as at 40 h post infection (1.9× increase). Post infection (1.7× increase), although not to the same level as axenic larvae. No increase in ROS/RNS was observed at 40 h post infection for the conventional group, and ROS/RNS production was not significantly induced by the topical Mr infection, although a slight increase for both the axenic and conventional larvae was observed during infection compared with the controls.

Discussion

Understanding the relationship between the gut microbiota and immune response is important to improve strategies for maintaining insect health. We have provided data on this relationship between the microbiota and immune response in the gut following infection by pathogens with different routes of entry into the host. We have made a comparative analysis of the immune response and growth parameters between conventional larvae and those reared in the absence of the involvement of the gut microbiota. Broad-spectrum antibiotics are often used to generate germ-free insects to gain insights into the role of the gut microbiota in host tolerance to various foreign microbes. However, this approach can have indirect detrimental effects on the host, such as interfering with protein synthesis mechanisms (46). Furthermore, the use of antibiotics in generating an axenic host may not be suitable for studies that require targeted infection of the host with bacterial strains, as they may themselves be susceptible to antibiotics (47,48). Through the combination of UV sterilization of the egg surface coupled with rearing on a sterile diet, we achieved a successful and effective method of generating G. mellonella axenic larvae, as was verified using PCR and CFU counts. For certain insects, such as Aedes aegypti (Diptera: family) and Apis mellifera (Hymenoptera: family), the gut microbiota is required to complete its life cycle (49,50), and in the Lepidoptera, the microbiota has been shown to improve the growth rate of larvae on suboptimal diets (51). Thus, we examined the effects of harboring a gut microbiota, or not, on G. mellonella growth and development.

Our results indicate that the gut microbiota is not required for a successful completion of the life cycle in G. mellonella. The insect could successfully pupate and reach the adult life stage in the absence of a gut microbiota, albeit with more variation, particularly at the larval stage (Figures 1A,B,C). There was no difference in pupal weight, suggesting that the larvae pupate once they reach a critical weight, even if it delays their time to pupation (Figure 1D). Therefore, it appears that in G. mellonella, the resource allocation of harboring a microbiota and/or its role in regulating the immune response to maintain its homeostasis and tolerance to beneficial microbes has no visible effects on the biological parameters we have measured and does not seem to compromise other important physiological functions that would impact the insect life cycle. To investigate whether or not the gut microbiota plays a role in protecting G. mellonella against infection, we then used two pathogens to challenge axenic and conventional G. mellonella larvae via different infectious routes. The results show that when both the axenic and conventional larvae were force fed by a spore crystal preparation of Btg at a half-lethal dose for the conventional group, the percentage mortality was always greater for the axenic larvae, with between 90% to 100% mortality by 96 h. These results demonstrate that G. mellonella larvae with a gut microbiota are less susceptible to Btg infection than larvae with an absence of microbiota (Figure 1E). In contrast, for the Mr infection there is no significant difference in the mortality in the infected larvae when they were given a half-lethal dose, indicating that the presence of the gut microbiota does not aid in the protection from fungal pathogens entering topically (Figure 1F).

Dominance of Enterococcus and its role in maintaining gut microbiota composition

Our metagenomic analysis of the bacterial microbiota of our G. mellonella population revealed that our strain is associated with a relatively small number of bacterial taxa, dominated by a subset of Enterococcus species with a very high prevalence of E. mundtii, E. gallinarum, E. casseliflavus, and E. inessii (Figure 3). Any observed effects on the immune response were as a result of the presence of Frontiers in Insect Science frontiersin.org 10 this genus of bacteria. A previous study also found only Enterococcus species in the G. mellonella gut (52); however, more recently published studies have also identified, along with Enterococcus but in lower abundances, Enterobacter, Bacillus, Halomonas, Shewanella, Pseudomonas, Variovorax, Staphylococcus, Serratia, and Escherichia-Shigella present in the late-larval instar of G. mellonella (28,41,(53)(54)(55)(56). With a more diverse microbiota, shifts in the dominance between bacterial groups post-pathogen infection have been observed. In particular, studies led by Dubovskiy et al. (41) and [START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF] found that Enterobacter (a genus of Gramnegative bacteria) maintained a minor presence in the gut community of G. mellonella at basal conditions, but proliferated during infection by Btg to overwhelm and dominate the community. Similar shifts have been observed post-fungal infection with Beauveria bassiana (54). These observed shifts are in contrast to what we have found, as the microbiota community remained stable without drastic changes in response to oral Btg or topical Mr infection. Indeed, the alpha-and beta-diversity analyses (Supplementary Figures S5,S6) showed no significant differences in the microbiota communities from uninfected control samples to the infected samples. This is interesting as the development of infections is not only restricted to the pathogen. For example, an increase in mortality due to enteric bacteria of the microbiota playing a synergistic role with pathogenic bacteria and fungi during infections has been found, increasing the mortality rates in its host (16,47,57). However, we did not test for absolute abundance, which may have provided some more information on the status of the microbiota during these infections. We nevertheless found that when both axenic and conventional larvae were topically infected with a half-lethal dose of Mr, their mortality rates were similar, indicating that the microbiota did not play a role in assisting the fungal infection. We did, however, observe an improved fitness in the conventional larvae with the oral Btg infection. The improved fitness could be in part due to the presence of E. mundtii, which was identified by 16S sequencing as well as by isolating it from the conventional larvae gut. [START_REF] Tang | Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae[END_REF] previously discovered that E. mundtii accounted for 40% of the entire microbial gut community in Spodoptera littoralis (Lepidoptera: Noctuidae) larvae, and that it exhibited the ability to eliminate both other Enterococcus bacteria and other harmful invading bacteria that posed a significant threat to the larvae's survival (58). As E. mundtii was a dominant member in the microbiota community of our G. mellonella population, and we observed some variation in the relative abundances of the Enterococcus species at different times and larval cohorts, it would be interesting to further test the competition and community dynamics between the Enterococcus species, to try to uncover and understand their colonization strategies.

We were driven to assess if the E. mundtii isolated from the conventional larva gut exhibited inhibition to Btg, as E. mundtii is known to secrete bacteriocins that have previously been shown to have broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, including Bacillus thuringiensis (17,43). Grau et al. (43) found that when Tribolium castaneum (Coleoptera: Tenebrionidae) beetle larvae were inoculated with a A B FIGURE 7 The phenoloxidase (PO) activity in the hemolymph of surviving axenic and conventional larvae infected either (A) orally by Btg, or (B) topically by Mr. Significance was determined by ANOVA-Kruskal-Wallis followed by Dunn's multiple comparison test. Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 11 probiotic isolate of E. mundtii (or the supernatant), the larvae experienced improved survival to Btg but not to Gram-negative Pseudomonas entomophila. Furthermore, a recent study on Hyphantria cunea (Lepidoptera: Erebidae) found that germ-free larvae inoculated with E. mundtii reduced the mortality of larvae infected with Btg as well as a nucleopolyhedrovirus (NPV). However, germ-free larvae inoculated with Klebsiella oxytoca (a species of Gram-negative Enterobacter) were found to be significantly synergistic with the effects of both Btg and the NPV, resulting in increased mortality (17). Using the same zone of inhibition technique as Grau et al. (43), we indeed observed inhibition zones between the E. mundtii against Btg, confirming that this dominant microbiota member has antimicrobial activity against Btg in vitro (Figure 4). Shao et al. (59) found that a strain of E. mundtii, isolated from the gut of S. littoralis, produced a stable class IIa bacteriocin, mundticin KS, which strongly affects Grampositive pathogens while having no or little effect on other resident gut bacteria. It would therefore be interesting to further test the antimicrobial activity of the E. mundtii isolate on other commensal bacteria to understand if it plays a role in the establishment and development of the microbiota community.

Immunostimulatory role of Enterococcus species

We were able to identify a faster rate of clearance of the Btg spores from the gut of conventional larvae (Figure 2), which was likely due to colonization resistance factors of the commensal gut microbiota such as space utilization, the secretion of antimicrobial inhibitors, and immune "readiness", as found by the increased basal expression of immune-relevant genes (Figure 5). The conventional larvae had increased levels of expression of all three immune response genes tested (gallerimycin, gloverin, and IMPI), which could result in a faster response to the Btg infection. Krams et al. (60) also observed an upregulation of AMP genes (including gallerimycin and gloverin) in larvae with a higher number of enterococci symbionts than in antibiotic-treated larvae. Krams et al. (60) further hypothesized that the elevated expression of AMP genes may be a prophylactic response to pathogens, which, from observations in our study of the improved response to Btg infection in larvae harboring the enterococci symbionts, agrees with this proposed hypothesis. However, more research is required to understand the specificity of the prophylactic response, as the mortality rates were similar during the topical fungal infection in larvae with and without a gut microbiota, even though larvae harboring the microbiota also maintained an upregulation of intestinal AMP gene expression at basal conditions. Furthermore, no significant differences were found in the fold change of gene expression from the control to the infected state at 20 h posttopical infection. This may simply suggest that at an early time point of infection, the fungi, entering its host topically, may not yet strongly induce the immune response in the gut (Figure 6), and although the focus of this study was on the interaction of the microbiota community and localized gut immune responses during infection, the gene expression in the body fat may have been an important tissue to evaluate. We did, however, measure the PO activity in the hemolymph (Figure 7), which would likely be a faster indicator of a topical infection, and indeed a slight increase in PO activity in the hemolymph was observed in both axenic and conventional larvae at 20 h post infection, followed by a decrease in activity by 96 h. The initial increase may be a response to the presence of the germinating conidia on the cuticle (Supplementary Figure S10); furthermore, an increase in PO activity is commonly reported in the initial stages of mycoses in both Beauveria bassiana and Metarhizium species, followed by a decrease at acute stages (61)(62)(63)(64). Entomopathogenic fungi possess the ability to inhibit melanization and other immune defenses through the production of metabolites that interfere with anti-microbial processes (65,66). We also observed a large variation in the melanization of cadavers, post-topical Mr infection, in which some had completely melanized while others died without experiencing any melanization. This observation was seen in both conventional and axenic larvae. It is worth noting that at 96 h, the immune-stimulatory effect on gene expression by the microbiota in conventional larvae was diminished, as no significant differences in basal immune expression were detected between the conventional and axenic larvae. This absence of immune stimulation in conventional larvae at 96 h post Mr infection could be attributed to the age of the larvae, which were close to pupation. A decrease in midgut AMPs close to pupation has also previously been observed in prepupal G. mellonella larvae [START_REF] Suraporn | Effects of Probiotic Bacteria on the Growth Parameters of the Thai Silkworm, Bombyxmori[END_REF]. Therefore, the diminished immune stimulation by 96 h, and the purported lack of participation in the fungal infection by the microbiota (due to no significant changes in the relative abundance or significant differences in alpha-and beta-diversity metrics between the control and infected larvae), resulted in similar mortality rates between the axenic and conventional larvae. The total ROS/RNS in the gut of nine surviving larvae (three guts per sample × 3) during (A) Btg and (B) Mr infections at two time points corresponding to early and late infections. No significance was found using non-parametric one-way ANOVA. We also examined the inducibility of ROS/RNS in the gut environment during infections (Figure 8). The axenic larvae, more susceptible to the dose of Btg than the conventional larvae, experienced greater oxidative stress in the gut environment. An excessive production of ROS/RNS can contribute to cell structure damage, likely causing significant cell lysis and aiding pore formation in the gut, contributing to mortality [START_REF] Suraporn | Supplementation of Lactobacillus Casei Reduces the Mortality of Bombyx Mori Larvae Challenged by Nosema Bombycis[END_REF][START_REF] Singh | Effect of Use of Lactobacillus Plantarumas a Probiotics to Improve Cocoon Production of Mulberry Silkworm, Bombyxmori (L.)[END_REF]. In general, there were no significant changes to the level of gut ROS/ RNS between all Mr infected and uninfected larvae. The slightly higher levels of ROS/RNS in the gut of all uninfected axenic larvae could suggest that the Enterococcus-dominated microbiota may potentially produce some antioxidants and enzymes that help scavenge ROS in normal conditions, thereby maintaining the gut environment's oxidative balance, which may have also played a role in attenuating oxidative stress post-Btg infection in the conventional larvae. The antioxidant properties in Enterococcus species have been increasingly studied, as these bacteria have exhibited the potential to inhibit pathogenic microbes as well as reduce oxidative spoilage in foods and feed [START_REF] Saranya | Fortification of Mulberry Leaves with Indigenous Probiotic Bacteria on Larval Growth and Economic Traits of Silkworm (Bombyx Mori, L.)[END_REF][START_REF] Esaivani | Impact of Probiotic Saccharomyces Cerevisiae on the Enzymatic Profile and the Economic Parameters of Sylkworm Bombyx Mori[END_REF][START_REF] Alcosaba | Effect of Fungus, Trichoderma Harzianumas Probiotic on the Growth, Cocoon Parameters, Silk Characters and Resistance of Silkworms (Bombyx Mori) Challenged by Muscardine Disease-Causing Metarhizium[END_REF].

Implications and future directions

In addition, we recognize that the immune response is modulated by additional genes than those tested in this study, as well as molecules not traditionally considered "immune genes" but which are also important for the immune response (41,[START_REF] Rajakumari | Efficacy of Probiotic and Neutraceutical Feed Supplements against Flacherie Disease in Mulberry Silkworm, Bombyx Mori[END_REF]. Therefore, a wider view using a larger transcriptomic approach may provide a more comprehensive understanding of the immune system stimulation by the gut microbiota both before and during infections. The dynamics of the immune stimulation of the microbiota under different stresses and environmental conditions also deserve further attention, including how the microbiota could influence more systematic immune responses. Understanding the insect gut microbiota-pathogen interactions offers potential benefits for future insect control and insect health strategies through targeted manipulation or exploitation of these interactions. For example, by introducing beneficial microbes into insects or their environments, it may be possible to develop probiotics or prebiotics that can modulate the gut microbiota composition and function to enhance host resistance or tolerance to pathogens. In particular, we plan to look into the role and the effect of the isolated E. mundtii strain on the immune response of G. mellonella larvae by assessing their ability to restore the resistance to Btg after repopulating axenic larvae. By generating gnotobiotic G. mellonella larvae, we could determine if the immune response can be improved by a single bacterial isolate or if a synergistic effect between the multiple bacterial Enterococcus species is needed.

Conclusion

In summary, our study revealed that a restricted number of bacterial strains in the larval gut significantly enhanced their survival rate following Btg infection but had a neutral role in the pathogenesis of topically inoculated Mr, in the economically important laboratory infection model, Galleria mellonella. Moreover, our results demonstrated a substantial impact of these Enterococcus species on the basal level of expression of immunerelated genes, underscoring the crucial role of the gut microbiota in the immune response of G. mellonella. These findings suggest that the gut microbiota of G. mellonella can serve as a protective mechanism against pathogenic infections caused by the bacterial pathogen Btg. This protection may be attributed to various factors, including, as discussed, the modulation or enhancement of the host immune system's basal level of immune activation or "readiness." In addition, the gut microbiota, dominated by Enterococcus species, might contribute to this defense through the production of antimicrobial compounds or by competing with pathogens for vital resources such as nutrients or space. Collectively, these mechanisms highlight the important role of the gut microbiota in safeguarding G. mellonella against certain pathogenic threats. This work adds value to the readily discussed role of the microbiota, reinforcing the reality that its role depends largely on the species composition of the microbiota, the host rearing conditions, and the type of pathogen. Moreover, some studies have shown that insects have the ability to mount specific and enhanced immune responses following prior exposure to a pathogen [START_REF] Scalfaro | Galleria Mellonella as an in Vivo Model for Assessing the Protective Activity of Probiotics against Gastrointestinal Bacterial Pathogens[END_REF]. Whether or not some members of the resident insect gut microbial communities can also function as "immune priming agents" capable of inducing the expression of genes involved in the recognition and elimination of pathogens is not yet clearly established and remains to be explored. Understanding these relationships will provide insight for new strategies on how to maintain insect health when they meet diseases from multiple and diverse pathogens. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 153

Isolating E. mundtii from the conventional larva gut

To test the efficacy of one symbiotic strain on host resistance to pathogens, as compared to larvae harboring a complex microbiota and those that are sterile, we isolated Enterococcus mundtii from conventional larvae (Suppl. Fig. S3). To isolate the bacteria, final instar larvae were chilled on ice before being surface sterilized with 70% EtOH. Three guts were dissected, pooled and homogenized in sterile H2O before plating the contents on to Brain-Heart Infusion (BHI) agar media. The plates were incubated for 72 hours at 30°C, before being inspected for colonies with different size, shape and colour morphologies. The majority of the colonies growing were uniform in their morphologies, and from microscope evaluation it was suspected that the colonies were E. mundtii. A colony was re-streaked to obtain a pure colony. For molecular verification, the isolate was cultured in liquid BHi for 24 hours. Following this, DNA was extracted and sequenced using the V3-V4 region of the 16S rRNA gene sequence and positively identified as E. mundtii.

Introduction

Insects, as like other animals, maintain a diverse microbial community in their gut which can contribute to their overall health and abilities (Gupta & Nair, 2020;[START_REF] Muñoz-Benavent | Insects' Potential: Understanding the Functional Role of Their Gut Microbiome[END_REF]. However, disruption to this gut microbiota community by antibiotics, pathogens or toxins can alter host susceptibility to diseases by influencing the immune system functioning [START_REF] Belkaid | Role of the Microbiota in Immunity and Inflammation[END_REF]. Alternatively, probiotics, can potentially help restore, maintain and therefore confer a health benefit on the host (Hill et al., 2014). It is therefore of interest to pinpoint bacterial strains with potential applications, whether it's enhancing an animal's growth and survival or improving immune functions. Probiotics utilize various mechanisms, including generating antimicrobial compounds [START_REF] Plaza-Diaz | Mechanisms of Action of Probiotics[END_REF][START_REF] Daliri | Probiotic Effector Compounds: Current Knowledge and Future Perspectives[END_REF][START_REF] Fijan | Probiotics and Their Antimicrobial Effect[END_REF], suppressing virulence genes via quorum sensing6 inhibitors [START_REF] Salman | Anti-Quorum Sensing Activity of Probiotics: The Mechanism and Role in Food and Gut Health[END_REF], promoting epithelial barrier functions [START_REF] Plaza-Diaz | Mechanisms of Action of Probiotics[END_REF][START_REF] Liu | Surface Components and Metabolites of Probiotics for Regulation of Intestinal Epithelial Barrier[END_REF][START_REF] Gou | How Do Intestinal Probiotics Restore the Intestinal Barrier?[END_REF], and stimulating the intestinal immune cells along with the microbiota to modulate specific immune functions [START_REF] Liu | Modulation of Gut Microbiota and Immune System by Probiotics, Pre-Biotics, and Post-Biotics[END_REF][START_REF] Mazziotta | Probiotics Mechanism of Action on Immune Cells and Beneficial Effects on Human Health[END_REF].

Enterococcus mundtii (Lactobacillales), is a common gut symbiont in laboratory-reared insects, especially the Lepidoptera (Table 1) and has also been found to show probiotic properties [START_REF] Todorov | An Antibacterial and Antiviral Peptide Produced by Enterococcus Mundtii ST4V Isolated from Soya Beans[END_REF][START_REF] Botes | Adhesion of the Probiotic Strains Enterococcus Mundtii ST4SA and Lactobacillus Plantarum 423 to Caco-2 Cells under Conditions Simulating the Intestinal Tract, and in the Presence of Antibiotics and Anti-Inflammatory Medicaments[END_REF][START_REF] Todorov | Evaluation of Potential Probiotic Properties of Enterococcus Mundtii, Its Survival in Boza and in Situ Bacteriocin Production[END_REF][START_REF] Pieniz | Assessment of Beneficial Properties of Enterococcus Strains[END_REF]Grau et al., 2017;Li et al., 2021b). Using an E. mundtii strain isolated from conventionally reared larvae, we tested the ability to resist Btg infection, in G. mellonella larvae recolonized with only one Enterococcus species, as compared to conventional larvae that host principally four Enterococcus species, and an axenic group which are devoid of gut bacteria.

Erebidae

Lymantria dispar mundtii [START_REF] Broderick | Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods[END_REF]Bai et al., 2023) faecalis

Gelechiidae

Pectinophora gossypiella na (Chaitra et al. 2022)

Phthorimaea operculella mundtii (Sevim et al., 2016;[START_REF] Zheng | Midgut Microbiota Diversity of Potato Tuber Moth Associated with Potato Tissue Consumed[END_REF] casseliflavus Sitotroga cerealella na (Sevim et al., 2016) Noctuidae Anticarsia gemmatalis mundtii (Visôtto et al., 2009) gallinarum Busseola fusca casseliflavus (Snyman et al., 2016) Brithys crini na (Serrano et al. 2020) Helicoverpa armigera mundtii [START_REF] Tang | Complexity and variability of gut commensal microbiota in polyphagous lepidopteran larvae[END_REF]Xiang et al. 2006;Tian et al., 2023) faecum casseliflavus avium gallinarum Helicoverpa zea casseliflavus [START_REF] Jones | Host Plant and Population Source Drive Diversity of Microbial Gut Communities in Two Polyphagous Insects[END_REF]Deguenon 2021) Chapter 3

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Heliothis virescens na (Staudacher et al., 2016) Mythimna separata mundtii (He et al., 2013;Xu et al., 2023) faecum Spodoptera exigua na (Gao et al. 2019) Spodoptera frugiperda mundtii hirae (Jones et

Plutellidae

Plutella xylostella mundtii (Xia et al., 2013;Lin et al., 2014;Lin et al., 2015;Li et al., 2017) Pyralidae Dioryctria abietella na [START_REF] Wang | An Entomopathogenic Fungus Exploits Its Host Humoral Antibacterial Immunity to Minimize Bacterial Competition in the Hemolymph[END_REF] Galleria mellonella mundtii innesii casseliflavus gallinarum faecalis (Dubovskiy et al., 2016;Allonsius et al., 2019;Polenogova et al., 2019;Cassone et al., 2020;Lou et al., 2020;[START_REF] Barrionuevo | The Bacterial and Fungal Gut Microbiota of the Greater Wax Moth, Galleria Mellonella L. Consuming Polyethylene and Polystyrene[END_REF]Gohl et al., 2022;Gooch et al., 2021;Upfold et al., 2023 -unpublished)

Sphingidae

Hyles euphorbiae casseliflavus (Vilanova et al., 2016) Manduca sexta gallinarum (Van Der Hoeven et al., 2008;Brinkmann et al., 2008) casseliflavus Saccharolyticus na -species level identification is not applicable (not reported in the study)

Chapter 3

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Material and Method

Monitoring the stability of recolonized E. mundtii in the gut

To verify the ability of the E. mundtii to recolonize the gut environment during late larval instar stages, or possibly be cleared from the gut. The axenic larvae were force-fed 10µl of E. mundtii at 5 x 10 8 CFU/ml, a similar concentration to that evaluated by CFU counts from dissected guts of conventionally reared larvae. At 0, 24, 48, and 72 hours, the guts of three larvae were dissected at each time point and individually plated on BHiA media. They were then incubated at 30°C for 48 hours. Colony counts were subsequently performed. This procedure was repeated twice, resulting in a total of six larvae examined for each time point.

Survival assay

Once we observed that the E. mundtii strain could successfully recolonize the gut, we generated gnotobiotic larvae to compare their susceptibility to Btg against conventional and axenic-reared larvae. For this, fifth instar axenic and conventional larvae were starved for 24 hours. The E. mundtii was prepared by growing the bacteria for 24 hours in liquid BHi medium at 30°C without shaking. The fresh preparation of E. munditi was adjusted to approximately 1 x 10 8 CFU/ml in sterile H2O. Half of the axenic larvae were reinoculated with the isolate by force-feeding 10µl using a cannula hypodermic needle (30G x 25mm) and 1mL syringe (Terumo Corporation). These are termed the 'gnotobiotic' larvae or 'Ax + Em.' The remaining axenic and conventional larvae were force-fed 10µl sterile H2O as a control. After 24 hours, the larvae were force-fed approximately 5 x 10 5 CFU of Btg in 10ul, the dose was estimated based on previous mortality assays with the conventional larvae, and was estimated to be lethal to 30-Chapter 3

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40% of the population. Mortality was recorded every 24 hours for up to 96 hours post Btg infection.

Results

Isolated E. mundtii has inhibition towards Btg in vitro, and can be recolonized in the gut of axenically reared larvae

Furthermore, the axenic larvae were able to be recolonized by the E. mundtii and maintain a high concentration, at least until 72 hours without clearance (Figure 4). No mortality was observed by E. mundtii on the larvae.

Gnotobiotic larvae have slight improvement to Btg infection by 96 hours

Probability of survival plots between axenic and gnotobiotic larvae was similar during the early stages of Btg infection however a slight improvement between the two groups 111

Discussion

Generating gnotobiotic larvae can be a powerful tool to decipher individual symbiotic relationships between a bacterial symbiont and its host [START_REF] Wu | Axenic and Gnotobiotic Insect Technologies in Research on Host-Microbiota Interactions[END_REF]. We have previously found an improved resistance to an oral infection of Btg in conventionally reared larvae where the gut microbiota was dominated by a few Enterococcus species, and did not experience significant shifts during the infection. Enterococcus mundtii was found to be a major presence and as such was isolated for further investigation to evaluate its ability to resist Btg in vitro, and Btg infection in vivo. Our results show that the E. mundtii strain isolated from the conventional larvae gut secreted bacteriocins, which inhibit the growth of Btg. E. mundtii is known to secrete a class IIa bacteriocin, mundticin, which has also shown inhibitory activity against many Gram-positive bacteria, including Listeria spp., Lactobacillus spp., Weissella spp., Pediococcus spp., as well as other Enterococcus spp. (E. faecalis and E. hirae) [START_REF] Bennik | A Novel Bacteriocin with a YGNGV Motif from Vegetable-Associated Enterococcus Mundtii: Full Characterization and Interaction with Target Organisms[END_REF], as well as some Gram-negative bacteria, which is an unusual characteristic for a bacteriocin produced by Lactic acid bacteria (LAB) [START_REF] Schelegueda | Synergistic Antimicrobial Action and Potential Application for Fish Preservation of a Bacteriocin Produced by Enterococcus Mundtii Isolated from Odontesthes Platensis[END_REF]. E.

mundtii was able to be recolonized in last instar larvae without significant clearance.

Furthermore, the axenic larvae recolonized with E. mundtii 24-hours before being infected with Btg, showed a slight increase in survival to Btg infection at 96-hours compared to axenic larvae (30% survival versus 20%, respectively), although this difference is not statistically significant.

Similar assays have been conducted notably in Triboleum castaneum (Coleoptera:

Tenebrionidae) beetle larvae, Hyphantria cunea (Lepidoptera: Erebidae) and Spodoptera exigua (Lepidoptera: Noctuidae) moth larvae (Grau et al., 2017;[START_REF] Li | Bt GS57 Interaction With Gut Microbiota Accelerates Spodoptera Exigua Mortality[END_REF]Chen et al., 2023a). For T. castaneum larvae, re-inoculating larvae with E.

Chapter 3

112 mundtii protected them against Bt but not against gram-negative Pseudomonas entomphila. Similarly, in H. cuneae larvae, E. mundtii was found to be antagonistic to both a nucleopolyhedrovirus (NPV) and Bt infections, whereas larvae reinoculated with gram-negative Klebsiella oxytoca showed significant synergistic effects with both NPV and Bt infections, resulting in higher mortalities. As for S. exigua, interestingly, when aseptic larvae were reintroduced with Bacillus cereus, the susceptibility to Bt was significantly enhanced, as compared to a reintroduction of E. mundtii, which improved the resistance to Bt as compared to the aseptic larvae. An E. mundtii strain from Spodoptera littoralis (Lepidoptera: Noctuidae) was found to produce an antimicrobial peptide mundticin KS, which can strongly inhibit some potential pathogens by weakening their colonization capability, which further promotes the stability of the intestinal microbiota against bacterial infections (Shao et al., 2017). However, E.

mundtii has also been found to play a contrary role in Plutella xylostella (Lepidoptera:

Plutellidae) where a reintroduction of E. mundtii into aseptic larvae regained its sensitivity to Cry1Ac protoxin (Li et al., 2021). The ability of some strains to be protective while others more pathogenic has also been observed between different Serratia marcescens strains in mosquito larvae, where one strain was found to be protective during Plasmodium infection in the Anopheles sinensis mosquito but another strain had no impact on the Plasmodium parasite development [START_REF] Bai | A Gut Symbiotic Bacterium Serratia Marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses[END_REF].

Therefore, depending in insect species, microbiota composition, interaction types or environmental factors, certain intestinal bacteria can elevate the risk of death from infectious pathogens, while others may provide protective benefits. These interactions between introduced bacteria and their effects on the immune response needs further research to tease apart the specificity and conditioning of the type of bacteria on the Chapter 3 113 immune response. In our study we used a similar concentration to re-inoculate axenic larvae to that of what is observed in CFU counts of dissected conventional larvae guts, however, not all resulting colonies may be from E. mundtii. Enterococcus gallinarum was also found to be a major presence based on the 16S rRNA illumina sequencing of the conventional larvae microbiota. Further tests should prolong the recolonization duration and experiment with different doses of E. mundtii or by a combination of the different Enterococcus species found in the conventional larvae microbiota. There is not a lot of published information on the functional role of these Enterococcus species in G. mellonella, future research should also be conducted to try identify the potential role and benefits these species provide to their host or play in supporting host resistance to pathogen infections. Additionally, it would be valuable to test the bacteriocin separately, using its supernatant, to assess its standalone efficacy.

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CHAPTER 4

General Discussion

Thesis overview

The role of the gut microbiota in host resistance to pathogens has yielded varied results. However, numerous studies have highlighted the microbiota's ability to enhance the growth, functionality, and overall capabilities of its host [START_REF] Gichuhi | Influence of Inoculated Gut Bacteria on the Development of Bactrocera Dorsalis and on Its Susceptibility to the Entomopathogenic Fungus, Metarhizium Anisopliae[END_REF]Franks et al., 2021;Zhang et al., 2021a;Chen et al., 2022Chen et al., , 2023b)). Insects, in particular, encounter a broad spectrum of pathogens that have evolved efficient and distinct strategies for overwhelming and subverting insect defenses. This thesis explored the intricate partnership between the gut microbiota and immune response in G. mellonella larvae confronted by two pathogens, each employing distinct entry routes -oral and topical. G. mellonella has emerged as a go-to model organism due to its cellular and humoral immune system being functionally analogous to mammals.

Therefore, previous studies have characterized the insect's immune effectors and functions. Moreover, discernible shifts in this insect's gut microbiota upon pathogen invasion have been reported (Dubovskiy et al., 2016;Polenogova et al., 2019;[START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF]. This prompted us to utilize G. mellonella as our candidate, and by generating axenic larvae we could probe the immune response in the stark absence of microbiota. Drawing from prior research, it's well-established that G. mellonella's microbial community is predominantly composed of a select few bacterial genera, especially Enterococcus and Enterobacter. However, it's worth noting that this dominance can fluctuate based on factors like diet and differences between wild and laboratory-reared populations. It is also vital to reiterate the importance of the sterilization technique used in our experiments, particularly in light of the controversy to which previous studies have generated aseptic larvae using antibiotics (Broderick et al., 2006). Instead, we used UV sterilization to clear the eggs of surface contaminants, and employed gamma-irradiation to ensure the complete sterility of the Chapter 4

116 diet. The larvae were subsequently reared within autoclaved sterile glass containers and any manipulations of these larvae was strictly performed within a sterile biosafety cabinet, ensuring an uncontaminated environment at all times.

The effects of the host intestinal microbiota composition, or its absence, on the insect's growth parameters

We firstly assessed the impact of maintaining a gut microbiota on its host by measuring the growth and developmental rate between G. mellonella reared axenically and conventionally. Our findings demonstrated that axenic larvae, those without their typical gut microbiota, exhibited a more variable time to pupation. This suggests that the presence or absence of certain microbial communities within the gut can influence the developmental pace of G. mellonella larvae. Corroborating this notion, another study pinpointed that when Enterococcus spp. were removed from the gut of later instar larvae, there was a faster transition from the larval to pupal stage [START_REF] Kong | Population Dynamics of Intestinal Enterococcus Modulate Galleria Mellonella Metamorphosis[END_REF]. However, this study also argues that Enterococcus, in particular E. innesii, drives metamorphosis, due to the reduction of this genera and dominance of Enterobacter spp. in the pupal stage. We however observed axenic larvae pupating at a similar average time to the conventional when reared their whole life axenically, albeit with more variation. It is therefore more likely that the drivers for pupation are hormonal which can then alter the microbiota by suppressing certain immune responses during this process. Nevertheless, Collectively, these results highlight the intricate relationship between the gut microbiota and insect development, suggesting that specific gut bacteria can either accelerate or decelerate the developmental processes.

Importantly, the axenic larvae were fully capable of completing their life cycle, and we did not observe any significant consequences for its fitness which indicates that our Chapter 4 117 axenic larvae were physiologically comparable to conventional insects. Further studies would be invaluable in understanding the exact mechanisms and implications gut microbial symbionts have on insect growth and maturation, considering different nutritional properties in the diet, environmental stress factors such as temperature, humidity and CO2, as well as the fluctuating hormonal drivers over the insect's life cycle, and assessing the impacts of this hormonal fluctuation on microbial communities.

The interaction of the microbiota and intestinal immunity during infection by oral (Bt) and topical (Mr) infections

The connection between the microbiota and immune response was then investigated, by conducting a dual-analysis experiment. In this study, we simultaneously analyzed the composition and state of the gut microbiota during infections, as well as the immune-related gene expression of two antimicrobial peptides (Gallerimycin and Gloverin), the inducible metalloproteinase inhibitor (IMPI) and lysozyme, within the same group of larvae. A notable observation in the microbiota of our laboratory-reared G. mellonella population was the dominance of Firmicutes. This phylum primarily consisted of four Enterococcus species: E. casseliflavus, E. gallinarum, E. innessi, and E. mundtii. Remarkably, regardless of the mode of infection-oral or topical-the composition of this microbiota remained relatively stable and we did not observe shifts from the Gram-positive to Gram-negative bacteria as has previously been observed by other authors (Dubovskiy et al., 2016a;Polenogova et al., 2019;[START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF]. We observed that the Enterococcus species, dominant within the microbiota of the conventionally reared larvae, acted as potential stimulators of the gut's immunological response which could result in a faster response to the Btg infection.

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118 This is substantiated by the increased basal expression of the immune response genes (Gallerimycin and Gloverin) in conventionally reared larvae relative to their axenic counterparts, highlighting the role of this intestinal microbiota in the immune response of G. mellonella. This suggest that certain resident intestinal microbial communities could act as 'immune priming' agents, stimulating the host's immune response against certain pathogens. Indeed, the conventional larvae, even with their limited microbiota diversity, demonstrated higher resistance to an oral Btg infection compared to axenic larvae. The latter group was more vulnerable to the same infection dosage, calculated to be half-lethal for the conventional group. While no significant differences in mortality post topical infection by Mr were observed in axenic and conventional larvae. The protection observed against Btg in the conventional larvae can be achieved by several mechanisms (Fig. 15), in particular by activating or modulating the host immune system through an increase in the basal level of activation or 'readiness' of the host immune system. In addition, the intestinal microbiota, dominated by Enterococcus species, could contribute to this defence by producing antimicrobial compounds and/or competing with pathogens for resources and colonisation sites.

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119 Figure 15. The different activities of the Enterococcus dominated gut microbiota promoting colonization resistance in the gut of conventional larvae, and the subsequent result by the lack of these symbionts in the gut of the axenic larvae; A1) Enterococci stimulates the production of AMPs (Gallerimycin and Gloverin) in the gut epithelial cells, 'activating' the immune system to be prepared for future challenge, A2) mundticin is produced by E. mundtii which has antimicrobial compounds against Btg, helping to reduce the load of Btg vegetative cells resulting from the germination of spores, A3) carbohydrates and resources are already utilized and metabolized by Enterococcus spp. reducing the amount of available nutrients for Btg, A4) finally, once the surviving Btg have evaded the various obstacles introduced by the established symbionts, the host immune response will destroy surviving Btg, by increasing the expression of Gallerimycin (for example) 3x. In comparison, the absence of the established microbiota population, B1) will ease the adaptation of Btg into the environment, in particular, they will not be met with cell-wall degrading mundticin, B2)

therefore the Btg will utilize available nutrients that will be in less competition than in the conventional gut environment, allowing the Btg to grow, and eventually release cry-toxins which can B3) generate pores in the gut and release the vegetative cells into the hemocoel causing septicemia and death, lastly, B4) the host will also defend itself from the Btg by releasing AMPs such as Gallerimycin, but at a level that of 12x from basal conditions, as there is no pre-stimulation by the Enterococcus spp.

The host gut microbiota and Bt

An increased susceptibility to an oral infection of Bacillus thuringiensis kurstaki HD-1 (Foray 48B) has also been observed in antibiotic-treated Lymantria dispar larvae, at every instar and inoculation method (leaf disk or droplet assay) [START_REF] Frankenhuyzen | Interactions between Bacillus Thuringiensis Subsp. Kurstaki HD-1 and Midgut Bacteria in Larvae of Gypsy Moth and Spruce Budworm[END_REF]. However, the role of the microbiota in Bt-mediated killing has been evaluated in a number of hosts and Bt strains, with somewhat varying results. Raymond et al. (2009), found that aseptic Plutella xylostella larvae were more vulnerable to purified Bt Cry1Ac toxin, and spore/toxin combinations. Similarly, Johnston & Crickmore (2009) found that axenic Manduca sexta larvae were susceptible to both Bt spores and the Cry1Ac toxin. In contrast however, Broderick et al. (2006) found that sterile L. dispar larvae were less susceptible to Bacillus thuringiensis subspecies kurstaki (DiPel DF, Valent Biosciences, Libertyville, IL) infections, although the use of the antibiotic cocktail 7 to generate the aseptic larvae was implicit in the results of this study. Our results show that the microbiota is not necessary for the spore-crystal suspension of Bacillus thuringiensis ssp. galleriae 69-6 to exert its pathogenicity on G. mellonella larvae. Notably, we did not recover Enterobacter species from the gut microbiota of our G. mellonella population, furthermore, this species was also absent in the gut of the L. dispar larvae where the conventional individuals were more resistant to Bt HD 1 (Foray 48B) [START_REF] Frankenhuyzen | Interactions between Bacillus Thuringiensis Subsp. Kurstaki HD-1 and Midgut Bacteria in Larvae of Gypsy Moth and Spruce Budworm[END_REF]. Interestingly, [START_REF] Frankenhuyzen | Interactions between Bacillus Thuringiensis Subsp. Kurstaki HD-1 and Midgut Bacteria in Larvae of Gypsy Moth and Spruce Budworm[END_REF] reported that they used larvae from the same source as [START_REF] Broderick | Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods[END_REF]2006;2009) where Broderick did recover Enterobacter from the gut of conventional larvae.

Furthermore, when Broderick et al (2006) re-inoculated antibiotic treated larvae with the Enterobacter species previously isolated from the conventional larvae gut, Btmediated killing was recovered to a similar level to that of conventional larvae. This led Chapter 4

121 to their hypothesis that the microbiota (but they only specifically tested an Enterobacter sp.) is responsible (or contributes -directely or indirectly -to Bt virulence) in L. dispar larvae, a hypothesis that has since been contested. Even further, Raymond et al (2009) reported that the majority of the 16S sequences from the less susceptible P. xylostella larvae were mostly colonized by Enterococcus species and Enterobacter species were relatively rare. More recently, Chen et al (2023) found Enterobacter to be synergistic with Bt killing in Hyphantria cunea larvae, in comparison E. mundtii played antagonistic effects. However, it should not be overlooked that certain resident bacteria in the microbiota are also involved in Bt infections. In the absence of Bt the resident bacteria are not pathogenic but when Bt is present they somehow increase its virulence, by helping to promote/accelerate septicemia, for example (Broderick et al., 2006;[START_REF] Mason | From Commensal to Pathogen: Translocation of Enterococcus Faecalis from the Midgut to the Hemocoel of Manduca Sexta[END_REF]Chen et al., 2023a). Consequently, it is quite evident that further work needs to be conducted to understand the specificity of the immune response by the microbiota, as Gram-positive or Gram-negative bacteria are likely playing a role in stimulating, or 'priming' certain immune responses. A study starting to investigate this phenomena was conducted where G. mellonella larvae primed with a sublethal dose of Gram-positive Bt were more resistant to a second dose of Bt, but not to the Gramnegative Pseudomonas aeruginosa, yeast, Candida albicans, as well as the fungi Beauveria bassiana [START_REF] Taszłow | Humoral Immune Response of Galleria Mellonella after Repeated Infection with Bacillus Thuringiensis[END_REF]. Unfortunately, the study did not infect with a different Gram-positive bacterium, to test whether the response was Bt or Grampositive specific.

The shift in the community structure from Gram-positive to Gram-negative bacteria has been observed in G. mellonella larvae during infection by different pathogens and parasitoids (Dubovskiy et al., 2016;Polenogova et al., 2019;[START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF].

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In particular, the replacement of Enterococcus by Enterobacter has been observed post-Bt infection (Dubovskiy et al., 2016;[START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF] as well as postenvenomation 8 by Habrobracon hebetor (Hymenoptera: Braconidae) venom, and postinfection by Beauveria bassiana (Polenogova et al., 2019). It was proposed by [START_REF] Grizanova | Virulent and Necrotrophic Strategies of Bacillus Thuringiensis in Resistant and Susceptible Insects, Galleria Mellonella[END_REF] that the shift from Gram-positive to Gram-negative bacteria in the gut post Bt infection was a potential mechanism by the more Bt resistant larvae to clear the gut of gram-positive pathogens, however shifts from Gram-positive to Gramnegative bacteria were observed in larvae infected with fungi as well as post envenomation, and it is therefore clearly not a specific response to Bt or Bt resistance.

Furthermore, an analysis of the microbiota community post Bt infection was previously conducted on the same susceptible and resistant larvae (Dubovskiy et al., 2016), which reported that a common phenomenon in both lines during Bt infection was the disappearance of several genera (in particular Micrococcinae) and a huge shift in dominance from Enterococcus to Enterobacter, which contradicts Grizanova's later findings.

The host gut microbiota and Mr

The gut environment remained stable during topical Mr infection, coupled by the similar mortality rates between axenic and conventional larvae, it is not likely that this microbiota community contributed to mortality. In some instances, opposing or counteracting effects between gut microbes and susceptibility to fungal pathogens has been observed. For the German cockroach, Blattella germanica (L. Blattodea:

Ectobiidae), germ-free individuals were far more susceptible to both Metarhizium anisopliae and Beauveria bassiana cuticular infections than conventional ones.

Furthermore, gut homogenates and aqueous fecal extracts from conventional Chapter 4

123 individuals showed antifungal activity, whereas germ-free cockroaches did not (Zhang et al., 2018). However, for mosquito larvae, an infection by Beauveria bassiana induced dysbiosis in the gut and increased mortality rates [START_REF] Ramirez | Entomopathogenic Fungal Infection Leads to Temporospatial Modulation of the Mosquito Immune System[END_REF]. The reported occurrences of entomopathogenic fungi triggering dysbiosis is very high (Wei et al., 2017;[START_REF] Bai | A Gut Symbiotic Bacterium Serratia Marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses[END_REF][START_REF] Xu | Gut Microbiota in an Invasive Bark Beetle Infected by a Pathogenic Fungus Accelerates Beetle Mortality[END_REF]Zhang et al., 2021b). Some potential benefits to this phenomenon may be that by allowing opportunistic bacteria to spread into the hemocoel, the fungi can quickly kill the insect host. This might allow for rapid conidia release and infection of other nearby hosts, promoting a faster spread of the fungi. It may also be that entomopathogenic fungi have not evolved precise mechanisms to regulate the extent of dysbiosis they cause. They might be aiming to slightly suppress the host immune system to establish an infection, but sometimes this suppression goes too far, causing unintended consequences like dysbiosis.

Furthermore, even if opportunistic bacteria proliferate in the hemolymph, fungi might still be able to harness resources effectively. A study by [START_REF] Wang | An Entomopathogenic Fungus Exploits Its Host Humoral Antibacterial Immunity to Minimize Bacterial Competition in the Hemolymph[END_REF] proposed a novel strategy by Metarhizium rileyi whereby the fungi induced translocation of gut bacteria, but then activated and exploited its host's humoral antibacterial immunity to eliminate the opportunistic bacteria. Thereby this prevents competition for nutrients in the hemolymph. Nevertheless, the interaction of fungi, the microbiota and host immune responses clearly requires further investigation to unravel all the mechanisms at play.

As stated, the gut environment remained stable during topical Mr infection, with no notable alterations in the larvae's bacterial microbiota community composition and no major changes in ROS production. This indicates that topical Mr infections primarily elicit localized hemolymph responses. Kryukov et al., (2020) observed that following Mr infection by injection of the blastospores into G. mellonella larvae's hemocoel, there was a decrease in hemolymph PO activity at 24-and 48-hours post infection. However, Chapter 4

124 in our study, we first detected a rise in PO activity at 24-hours, which was subsequently suppressed by 96-hours. An increase in PO activity is commonly reported in the initial stages of mycoses to both Beauveria bassiana and Metarhizium species, followed by a decrease at acute stages [START_REF] Charnley | Fungal pathogens of insects: cuticle degrading enzymes and toxins[END_REF]Dubovskiy et al., 2013;Tyurin et al., 2016;Vivekanandhan et al., 2022). Metarhizium genomes possess more detoxification genes than other fungi, which potentially enhances their ability to interact with insect hosts [START_REF] Lovett | Stress Is the Rule Rather than the Exception for Metarhizium[END_REF][START_REF] Iwanicki | Genomic Signatures and Insights into Host Niche Adaptation of the Entomopathogenic Fungus Metarhizium Humberi[END_REF]. For successful colonization and resource utilization, Mr has to neutralize or degrade the host's toxic compounds.

Specifically, the detoxification genes in Mr, especially monooxygenases, are thought to be pivotal in rapidly removing insect polyphenolics, converting phenols to catechols [START_REF] Itoh | Monooxygenase Activity of Type 3 Copper Proteins[END_REF][START_REF] Gao | Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium Anisopliae and M. Acridum[END_REF]. Kryukov et al.'s (2020) infection method of injecting blastospores directly into the hemocoel might instigate a swifter immune suppression by the fungi. Our observed 24-hour increase could be due to the fungi's growth mechanisms: the germination of conidia on the cuticle and the appressorium's swelling, this process elicits toxins by the fungi which initiates cuticle degradation. This fungal action probably activates the immune response in the hemolymph, before the fungi proliferates in the environment and its metabolites inhibit the proPO system [START_REF] Pal | Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila Melanogaster[END_REF]. However, the mechanisms of PO inhibition during mycoses development and the precise fungal toxin and insect immune system interactions remain areas that necessitate further study.

The ability of isolated bacteria from the gut microbiota to re-establish in axenic late instar larvae and restore resistance to pathogens

The final section of the thesis explored the possibility of generating gnotobiotic larvae to further explore the interaction between Bt and E. mundtii, which was isolated from the conventional larvae gut. Zone of inhibition assays revealed that indeed this Chapter 4

125 dominant microbiota member inhibits the growth of Btg in vitro, and improved the resistance to Btg in vivo, as compared to the axenic larvae. Various isolates of E.

mundtii have demonstrated an ability to enhance resistance against Bt in several insects and inhibit Bt growth in vitro (Grau et al., 2017;[START_REF] Li | Bt GS57 Interaction With Gut Microbiota Accelerates Spodoptera Exigua Mortality[END_REF]Chen et al., 2023), however one study also found E. mundtii restoring sensitivity to Bt Cry1Ac toxin in an axenic P. xylostella population (Li et al., 2021b). This suggests not all E. mundtii strains are capable of protecting the host against pathogen infection. A similar case has been observed with different S. marcescens strains, where one strain was found to be protective during Plasmodium infection in the mosquito but another strain had no impact on parasite development [START_REF] Bai | A Gut Symbiotic Bacterium Serratia Marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses[END_REF].

Research in the greater context of insects reared for food and feed

The work presented in this thesis investigated an aspect of insect health that demands greater attention, the dynamic interplay between gut immunity and immune responses.

The balance and health of the gut microbiota can be a key factor influencing the insects' ability to thrive, reproduce, resist environmental stressors and overall immunity. Therefore, understanding the role of the microbiota during infection by diverse pathogens such as Bacillus thuringiensis and Metarhizium robertsii is crucial information needed to sustain mass-rearing operations. Thus, bridging the gap in our knowledge of the role of the gut immunity during infection by pathogens which will likely be infecting insects under mass rearing conditions becomes paramount for the sustainable development of insect-based food and feed production. The INSECT DOCTORS programme as a whole has worked to address these knowledge gaps, not only have we enhanced our understanding of insect physiology and pathology, but we also equip farmers with information and tools needed to optimize rearing conditions which can bolster insect health and mitigate the risk of pathogen emergence.

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126

Study Limitations

There are some aspects to the study which may be improved or investigated further to have a greater understanding of the subject at large and are described further.

The influence of the diet

Firstly, the diet plays a pivotal role in shaping and maintaining the gut microbiota [START_REF] Jones | Host Plant and Population Source Drive Diversity of Microbial Gut Communities in Two Polyphagous Insects[END_REF]Schmidt & Engel, 2021;[START_REF] Šigut | Fungi Are More Transient than Bacteria in Caterpillar Gut Microbiomes[END_REF]. For this study, larvae were fed a natural diet consisting of bee-collected pollen and beeswax. Pollen is highly nutritious, rich in proteins, essential amino acids, sugars, fatty acids, vitamins, macro and microelements (Komosinska-Vassev et al., 2015;[START_REF] Didaras | Antimicrobial Activity of Bee-Collected Pollen and Beebread: State of the Art and Future Perspectives[END_REF]. The beeswax complements this, offering a rich source of carbohydrates. However, despite our understanding of the nutritional composition of the diet, we lacked some information of its microbial content. Preliminary data had suggested that both pollen and beeswax are not highly diverse in bacterial species, but this assessment was based on a different batch of diet. Furthermore, given that different cohorts of larvae showed variable relative abundances of four key species, it underscores the potential influence of diet-specific microbes. Therefore, a detailed analysis, perhaps involving 16S rRNA and Illumina sequencing of the DNA extracted from the diet, would have enriched our understanding of the diet's contribution to the microbial landscape within the larvae. This could have shed more light on the observed fluctuations, particularly in the Enterococcus species, across different larvae cohorts.

The microbiota is not just bacteria

Secondly, many microbiome investigations face inherent biases that might skew the data towards certain microorganisms, primarily due to the techniques employed. A common limitation, which our study was not exempt from, is the prokaryote-centric nature of many microbiota datasets. This bias often arises from the use of bacteria-Chapter 4

127 specific primers which, while efficiently detecting bacterial species, can inadvertently sideline the eukaryotic constituents of the microbiome, such as fungi. In an attempt to include this fungal mycobiota community, we used ITS2a primers, which are known for their efficacy in detecting and distinguishing the fungal components of microbial communities. However, while we sought to uncover the diversity and abundance of fungi within our samples, the use of the ITS2a primers also amplified substantial quantities of plant material, likely originating from the pollen grain diet. This unintended amplification clouded our understanding of the true fungal composition, as it impeded further analysis to investigate the relative abundances or alpha and beta community metrices. We could however detect Metarhizium in all samples infected with Metarhizium robertsii which were notably absent from the uninfected samples. Going forward, there's a clear need to refine methodologies to investigate and include the fungal mycobiota in microbiome studies. Optimizing extraction and amplification protocols to reduce the unintended amplification of plant material would greatly enhance the resolution and specificity of fungal datasets. Additionally, considering the rapid advancements in sequencing technologies and bioinformatics tools, integrating a multi-primer or multi-technique approach may offer a more holistic and unbiased view of the microbiome. By addressing these limitations in subsequent studies, we can aspire to gain a more comprehensive and accurate picture of the microbial communities under investigation.

The influence of age

Thirdly, by focusing our evaluation primarily on the gut microbiome of older larvae, we risk oversimplifying the dynamic interplay of microbial communities throughout an insect's life. There's potential that we might be sidelining or downplaying critical interactions that are particularly pronounced during the younger life stages. Numerous

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128 studies have shown that the composition and role of the gut microbiota undergo significant shifts as insects' transition through their life stages. The implications of these shifts is that the microbiota's influence on an insect's immune response or capability to resist infections can vary based on its life stage. This immune 'readiness' determines how prepared an insect is to fend off diseases and adapt to environmental changes. Given that younger insects, particularly during their early life stages, are more susceptible to threats, it becomes even more crucial to understand how the microbiota shapes their immunity. Therefore, a comprehensive study, that investigates microbial influence throughout an insect's lifecycle, especially during its early and more vulnerable stages, could provide a holistic view of microbiota-host interactions and their consequences for insect health and survival.

Future Perspectives

Enhancing the potential of the innate immune system

The current research therefore highlights the need to understand how the microbiota, or bacteria in general, can "prime" or prepare the immune system in specific ways.

"Priming" in this context refers to the phenomenon wherein prior exposure to certain bacteria or microbiota components can enhance or modify the immune response upon subsequent encounters with pathogens [START_REF] Sheehan | Immune Priming: The Secret Weapon of the Insect World[END_REF]. This is akin to a "memory" function, albeit not in the same manner as adaptive immunity in vertebrates.

Gram-positive and Gram-negative bacteria differ in terms of the molecules they present to the host immune system. For instance, Gram-positive and Gram-negative bacteria possess distinct peptidoglycans on their cell walls, which can be potent immune stimulators but interact differently with the immune system. The different molecular signatures of these bacteria could, therefore, lead to distinct priming effects.

If certain microbiota components specifically prime the immune system, it could mean Chapter 4

129 that insects harboring a particular gut bacterial composition might be more resistant to specific pathogens and less so to others. In essence, as the literature on this topic continues to expand, there are increasing results showing specificity of the priming response, where the specific bacterial makeup-down to the Gram-positive vs. Gramnegative distinction-could play a pivotal role in determining the nature and magnitude of the immune response. There are also some interactions which are not always specific, for example an Enterococcus strain (HcM7) upregulated gloverin in H. cunea which protected the larvae from subsequent NPV infections [START_REF] Li | A Gut-Isolated Enterococcus Strain (HcM7) Triggers the Expression of Antimicrobial Peptides That Aid Resistance to Nucleopolyhedrovirus Infection of Hyphantria Cunea Larvae[END_REF], and

Tenebrio molitor (Coleoptera: Tenebrionidae) larvae were observed as having an increased resistance to several Gram-positive and Gram-negative bacterial infections when they were initially primed with a Gram-positive pathogen due to the persistence of an antimicrobial response, although interestingly the same level of immune priming could not be achieved with the Gram-negative bacterium. The specificity of this priming effect is intriguing and has profound implications for understanding insect immunity.

In our own research, we observed significant zones of inhibition against Btg, suggesting that the commensal E. mundtii strain, isolated from the conventional larvae gut microbiota, inhibit Btg growth via the secretion of bacteriocins. Yet, it's crucial to further investigate whether E. mundtii and its antibacterial compounds can serve as a safe and effective probiotic in insect mass rearing. This is especially relevant due its prevalence in the gut microbiota of numerous insect species, and given its potential to target various Gram-positive, Gram-negative bacteria as well as NPV. In general, and despite the promise, there's a notable gap in our understanding of the potential applications of probiotics (and prebiotics) for insects reared for food and feed.

Especially considering that mass rearing environments pose heightened risks of emerging pathogens and diseases due to factors like high densities, fluctuating Chapter 4 130 temperatures, and various abiotic and biotic stresses. Probiotics offer a potential solution to maintain healthy insect populations under these conditions. However, a thorough grasp of how probiotics interact with insect gut microbiota and their overall health impact is vital. Together with two other authors I have written a review to provide an overview of the role of probiotics in insects reared for food and feed, discussing in particular, how bacterial strains have been selected for and used to test its effectiveness as a probiotic. Additionally, we outline future perspectives on probiotic applications in mass-reared insects (Appendix A).

Concluding remarks

This thesis examined the interactions between the gut microbiota, immune response, and host susceptibility to various pathogens in the insect model, Galleria mellonella (wax moth) larvae. The central hypothesis was that symbiotic bacterial isolates in the insect gut may enhance the host's immune defense against pathogens. However, the effectiveness of this symbiotically-boosted immune response could vary based on the specific pathogen and its entry route into the host. Indeed, we observed larvae maintaining Enterococci symbionts were less susceptible to oral Btg infections than axenic counterparts, however no significant difference in mortality post topical fungal infection was observed. Notably, these Enterococi species exerted a marked influence on the basal gene expression linked to immunity, emphasizing the gut microbiota's pivotal role in G. mellonella's immune response. This research enriches our understanding of the microbiota, emphasizing that its function is intricately tied to the microbiota's composition, host environment, and the nature of the pathogen. Certain studies have established that insects can develop enhanced immune responses after previous pathogen exposure. Whether certain gut microbial communities in insects act Chapter 4

131 as "immune priming agents", stimulating genes vital for pathogen recognition and elimination, is still an open question warranting further exploration. Unraveling these intricacies could pave the way for innovative strategies to bolster insect health amidst multifarious pathogenic threats.

Introduction

Insect mass-rearing for food and feed has been identified as a valuable industry with current predictions expecting the market to grow by 47% from 2019 to 2026 [1]. This growth will see production volumes reaching an expected 730,000 tonnes by 2030 [2]. Over 1900 insect species have been reported in literature as being consumed worldwide [3]; however, only a few of these species are mass-reared in a more intensive manner. Much like traditional intensive livestock production, mass-rearing of insects faces similar challenges of high densities, a high rate of pathogen transmission, higher susceptibility to pathogens due to lack of oxygen, high temperatures, and nutrient deficiencies [4,5]. These challenges associated with farming insects need to be investigated with urgency in order to develop a successful industry, which has the potential to contribute to global food security.

A solution to mitigate some of these challenges has been the administration of antimicrobials, but this comes with the potential risk of emerging multiple antibiotic resistant 'superbugs' that threaten both animal and human health. The trend has therefore shifted to the use of probiotics which are defined as "live microorganisms that, when administered in adequate amounts confer a health benefit on the host" [6]. It is now apparent that every organism is associated with a microbial community ranging from parasitic to mutualistic, and this is true for all animals, from humans to invertebrates, insects included. In particular, the gut microbiota has been of focus to researchers in recent years due to its link to the health status of its host, with more and more studies finding that exploiting these microorganisms may improve animal productivity and maintain their health and wellbeing [7,8].

Interactions between insects and their microbiota play an important role in behaviour and evolution of many insect species [9,10]. Several microorganisms are able to manipulate host behaviour to increase their transmission. For example, Wolbachia which is able to modify the mating preference of its hosts when it acts as a symbiont, and the lack of microbiota or the presence of foreign gut bacteria can distort the feeding behaviour of insects by changing their sense of smell [9]. Reproduction, conversion, and growth performances have been related to specific microorganisms in mass-reared insects [11]. Insect-microorganism communication is bi-directional and social interactions in insects can impact the distribution of microorganisms within the population.

Insect diets have, in this context, an important role in providing nutrients both to the insects and the microorganisms. Within its composition, it is possible to highlight specific nutrients that act as prebiotics and that have been defined as "selectively fermented ingredients that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host wellbeing and health" [12].

Our goal is to provide an overview of the use of probiotics, and in brief, prebiotics, in the rapidly growing industry of insects for food and feed. As previously mentioned, there are over 1900 species of insects consumed worldwide [3]; however, along with some other relevant examples, this review will focus on the important species currently mass reared in this industry, which includes Acheta domesticus (L.) (Orthoptera: Grylloidea), Gryllus bimaculatus (De Geer) (Orthoptera: Gryllidae), Hermetia illucens (L.) (Diptera: Stratiomyidae), Musca domestica (L.) (Diptera: Muscidae), Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), as well as two other insects of economic importance that are mass-reared, Bombyx mori (L.) (Lepidoptera: Bombycidae) for their role in sericulture, and Galleria mellonella (L.) (Lepidoptera: Pyralidae), for their role as a non-mammalian model for the study of human pathogens.

This review will highlight the relationship between insects and their gut microbiome and discuss the mechanisms by which probiotics may exert their beneficial effects. It also reviews some of the methods that have been used to reduce the occurrence of disease in reared colonies and gives a summary of the probiotics that have been tested in the seven insect species mentioned. Lastly, we highlight some of the techniques used in isolation of probiotics and ways of testing microorganisms for probiotic potential as well as future perspectives on industrial applications.

Defining Probiotics and Prebiotics: The Bugs That Debug the Bugs

Every organism is associated with a microbial community that may promote its health. The identification of probiotics opened the possibility of exploring the health of insects when they are provided with beneficial microorganisms. In particular, the effect of the microorganisms is assessed for improving growth and reproductive performance and for decreasing the occurrence of diseases in stressful rearing conditions. The World Health Organization's internationally endorsed definition of probiotics is "live microorganisms that, when administered in adequate amounts confer a health benefit on the host" [6]. This definition, however, is still unclear in some circumstances, thus causing controversy and confusion. A distinction is made between microorganisms given to insects as a supplement and those that are commensal gut microbes that putatively confer health benefits to the insect [13]. The latter are often erroneously termed as probiotics, but this requires that they be isolated and characterized and their subsequent health-promoting effects validated [13]. Other terms that have been used synonymously are direct-fed microbials given in animal diets and live biotherapeutic products that are more pharmaceutical and take the form of drugs rather than food supplements, even though they are intended for the same use. Overall, the scope of probiotic intervention is expanding, leading to various ways in which the product reaches the market and different regulatory requirements, and this comes with various terms/definitions as a result.

The concept of prebiotics came to light when, in the early 1950s, scientists discovered that there was a special growth-promoting factor in human milk that aided the growth of the probiotic Bifidobacterium bifidus (Tissier) [14]. These components were later named by Gibson and Robertfroid [15] as prebiotics and defined as "nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health". To put it another way, these are the nutrients that feed the probiotic microorganisms enabling their proliferation in the gut [16]. These nutrients are mostly fibers, and they include inulin, oligofructose (produced from inulin), and fructooligosaccharides (FOS) synthetically produced from sucrose, as well as galactosecontaining and xylose-containing oligosaccharides, resistant starch (RS), pectin, and other fermentable fibers [17]. It was not long before probiotics and prebiotics were conveniently combined in one synergistic pack known as synbiotics [15,18]. For the insect mass rearing industry, this highlights the fundamental importance of different types of diet and diet quality for the success of the probiotic application.

The Crosstalk between the Insect and Their Intestinal Microbiota

The ubiquitous nature of gut bacteria and increasing knowledge of their numerous advantages to insect hosts has led to their application as probiotics in the insect mass rearing industry. The use of probiotics is based on the interaction between the host and their gut microbes; hence, understanding the nature of this crosstalk is fundamental to understanding the process. The insect gut microbiota and their collective genomes ('microbiome') have captured the interest of many researchers today, as an 'organ' in itself that plays a core role in influencing key insect traits [19]. The insect microbiome consists of a large diversity of microorganisms including bacteria (bacteriome), fungi (mycobiome), viruses (virome), and archaea (archaeome), but bacteria are the most abundant and the most studied [19].

Studies examining this spectrum of symbiotic relationships have pointed to the beneficial effect of a variety of bacteria and yeast species in different insects and thus their application as probiotics. For example, probiotic application of Klebsiella oxytoca and an Enterobacter strain increased larval growth of Ceratitis capitata (Wiedemann) (Diptera: Tephrtidae) used in Sterile Insect Technique (SIT) application [20] by increasing larval growth, especially due to bacterial synthesis of nutrients and protection against pathogens through the release of some antimicrobial compounds. A similar effect was also observed in transgenic Plutella xylostella (L.) (Lepidoptera: Plutellidae) where aseptically reared larvae that otherwise had low pupal weight and poor survival registered a substantial increase in pupal weight and male fitness when inoculated with Enterobacter cloacae (Jordan) [21]. However, to initiate any form of host-microbe interaction, a series of steps occur beginning with the microbiota acquisition followed by gut colonization or adhesion and progressing to an establishment in the gut and further transmission back to the environment or to new hosts [22]. It has been shown that insects can acquire their microbiota horizontally from the environment mainly through diet [23,24]. Dietary habits have been shown to affect both the composition and robustness of gut communities by regulating nutrient availability for the microbes [25]. Microbiota are also acquired by social interaction through trophallaxis and coprophagy transplantation as seen in termites and bees, and thirdly, through vertical transmission from parent to offspring via the egg surface, which is exposed to microbes from the ovaries of the mothers [26].

Adherence of the microorganisms to the gut lumen then follows, but the mechanisms vary across the different species, likely due to diversity in the physiology, morphology, and ecology of insects [27,28]. Gut colonization is affected by many different factors, including physicochemical gut conditions, i.e., pH, redox potential, and oxygen content. Gut compartmentalization can affect microbiota distribution presenting increased microbial density from anterior to posterior compartments. Moreover, the presence of enzymes and immunological compounds in the gut and the life history characteristics causes changes in community abundance [29,30]. Depending on the insect species, different bacterial communities have to develop strategies that allow them to survive and persist in the harsh conditions of the host such as the highly alkaline guts of lepidopteran species. To illustrate this, an RNA-sequencing study showed that the gut symbiont E. mundtii had upregulated pathways for tolerating high alkaline stress during its passage in the gut of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) [31,32].

After successful colonization, these microbes may participate in many different symbiotic, pathogenic, or vectoring activities within the host [7,33]. Focusing mainly on the symbiotic or mutualistic roles, microbes play a key role in metabolism by breaking down indigestible plant-derived polysaccharides through microbiota encoded carbohydratedegrading enzymes in the midgut and hindgut of caterpillars (Lepidoptera), termites (Dictyoptera: Isoptera), honey bees (Hymenoptera), beetles (Coleoptera), crickets (Orthoptera), and other herbivorous insects [34]. These enzymes include cellulases, hemicellulases, and pectinases that are generally absent in insects [35]. Remarkably, recent phylogenetic studies have shown that the genes encoding these enzymes in gut microbes have also been encoded in the genomes of some hosts such as the mustard leaf beetle, Phaedon cochleariae (Fabricius) (Coleoptera: Chrysomelidae), signifying horizontal gene transfer [36,37]. The breakdown of these plant cell wall materials culminates in the formation of short-chain fatty acids that impact both the microbe and host's nutrition and help in maintaining the integrity of the gut barrier [27,34]. In this way, gut microbes also compensate for the sometimes nutrient-poor diets of their hosts through nutrient provisioning, for instance, Buchnera aphidicola (Munson et al.) in pea aphids that provide essential amino acids that are lacking in the insect diet [38].

Through provisioning of nutrients and aiding digestion, gut symbionts then positively influence the growth and development of hosts, as shown in numerous studies. A study in Drosophila melanogaster (Meigen) (Diptera; Drosophilidae), for example, showed that aseptically reared insects had reduced growth and slower development as compared to conventionally reared insects. However, when inoculated with Acetobacter pomorum (Sokkollek), a gut commensal, the growth and development was restored to a similar rate as that of conventionally reared insects [19]. It was also observed that the addition of Leuconostoc spp. as a probiotic in the diet of the fruit fly Bactrocera tryoni (Froggatt) reduces the mean development time from egg to adult [39].

During herbivory, insects also encounter a variety of toxic plant defense chemicals, the detoxification of which is aided by their gut symbionts which enables their success as pests [40]. This type of detoxifying-symbiosis confers resistance not only to plant allelochemicals but also to insecticides [41]. This is achieved through diverse mechanisms, for example, enzymatic degradation of potential toxic phytochemical compounds as aglycones by gut symbionts of T. molitor [42,43]. Gut symbionts also play a protective role by increasing the host's resistance to pathogens. Several mechanisms come into play here, including the inhibition of infection, as seen in Tsetse fly Glossina morsitans Westwood (Diptera: Glossinidae) colonized with the commensal Kosakonia cowanii Zambiae which inhibits Serratia marcescens and Trypanosoma by raising the pH of the gut [26]. Gut symbionts also protect the host by nutrient and space competition with invading pathogens, thus edging them out as observed in mammals and also suspected to be the case in insect hosts [34]. The microbiota also enhances the epithelial barrier function to prevent systemic infection by pathogens, as seen in mosquitoes [44] and the stimulation of the host immune system or immune priming [34].

Overall, the host-microbiota relationship is intricate and intriguing but there are still some aspects that are yet to be explored and well understood for instance the influence of abiotic factors on the interaction and how microbiota impact insect population dynamics as well as behaviour manipulation, all of which could be advantageous in the advancement of probiotic use in the insect mass rearing industry.

Probiotics Applications as a Means of Decreasing Disease Occurrence in Mass Reared Insects Mass-reared insects are highly susceptible to diseases caused by organisms that belong to different Phyla within bacteria, viruses, fungi, protista, and nematoda [45]. Finding a method for preventing these diseases and increasing growth and reproductive performances in insects reared for food and feed purposes is thus a key goal of both industries and researchers [46]. Maciel-Vergara et al. [5] presented an overview of good production practices for reducing risks of pathogen occurrences such as daily adherence to good hygiene practices, differential breeding, mechanical pest control, and techniques such as heat shock/thermal therapy, breeding of tolerant strains, biological control, and RNA interference. In this context, probiotics have also been considered an option for decreasing the impact of diseases due to their ability to positively influence host performance and enhance immune responses against pathogens [5,47].

The most known probiotic bacteria are Lactic Acid Bacteria (LAB) that present a high immune system activity in humans [48]. In insects, antibacterial activity and immune regulatory effects have been widely recorded within the Lactobacillus genus in the microbiota of silkworm Bombyx mori and the honeybee Apis mellifera (L.) (Hymenoptera: Apidae) when infected with the pathogen Pseudomonas aeruginosa (Gessard) and Nosema spp., respectively [49,50]. Observations of Galleria mellonella have highlighted the antimicrobial effects of Lactobacillus reuteri and Lactobacillus rhamnosus on Staphylococcus aureus, Escherichia coli, Candida albicans and Pneumonia aeruginosa [51][52][53]. Probiotics strains have been tested against Nosema ceranae on honeybee alongside small-molecule RNA interference techniques and supplements for decreasing the bee spore load and viability and increasing its survival and performance [54]. At the genus level, Lactobacillus and Bifidobacteria had an impact on the pathogens, but when L. rhamnosus and sucrose were provided to honeybees to decrease the impact of N. ceranae, higher mortality rates and lower phenol oxidase production were recorded [54][55][56]. The positive effects of Lactobacillus spp. on hosts infected by fungi have been highlighted in studies focused on Drosophila melanogaster and Galleria mellonella, respectively, infected with diaporthe FY and C. albicans [8,57]. Other genera such as Enterococcus have been studied for decreasing the occurrence of bacterial diseases. E. mundtii showed positive effects on Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) immune responses against Bacillus thuringiensis infections [58]. As for viral diseases, the activation of insect-specific or generic immune responses by probiotics is still to be clarified [59]. Although endosymbionts do not fall under the definition of probiotics, it is worth mentioning that the main positive results are related to the presence of Wolbachia and Spiroplasma that increase resistance to viral diseases in T. molitor and G. mellonella [59,60]. It is interesting to note, however, that some bacteria species isolated from shrimps have been shown to have antiviral activity in a plaque assay and demonstrated positive effects on fitness performance in shrimps infected with white spot syndrome virus, which sheds light on some possible techniques that could be used in screening insect microbiota for the same [16,61].

Although the use of probiotics in disease management is very promising, it is still hampered by a scarcity of knowledge on insect-pathogen dynamics and the influence of the stressors on production performances and on insects' susceptibility to diseases. This calls for the need to maintain a holistic approach in the general management of mass rearing systems, taking into account environmental factors, diet, microbiota and genetic factors.

The Most Common Microorganisms Used as Probiotics in Insects

The most commonly tested probiotics for insects reared for food and feed belong to the genera Lactobacillus, Saccharomyces, Streptococcus, and Bacillus which, according to the World Gastroenterology Organization [48], are among the seven 'core' microorganisms most often used as probiotics. Enterococcus is a largely underrepresented group, especially given that it is a common symbiont of the insect gut and especially in Lepidoptera. This may be in part due to the fact that Enterococcus species have both pathogenic and probiotic strains [62,63]. Nevertheless, an example of its potential use as a probiotic was described by Grau et al. [58] when they isolated an E. mundtii strain from the feces of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae), which showed antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, and increased survival in Tribolium castaneum beetles after infection with Bacillus thuringiensis.

Aside from summarizing probiotics tested for insects reared for food and feed, Table 1 highlights the lack of research into probiotics for crickets. However, the primary disease observed in reared A. domesticus populations is caused by a densovirus (AdDNV). An effective approach to reduce the impacts of the virus on A. domesticus populations may not be through the use of probiotics but rather through RNA interference technologies. This approach was used by La Fauce and Owens [64] on A. domesticus to reduce PmergDNV titres and subsequent mortality from the virus, by feeding the insect with dsRNA specific to the capsid protein by mixing it into their food. Similarly, there has been little work into probiotics for Acheta domesticus (L.) (Orthoptera: Gryllidae), another cricket species mass reared in particular for pet food and human consumption. However, more towards the direction of prebiotics, a recent study found the incorporation of Jew weeds, Comellina sinensis (L.) Kuntze, into the diet of the cricket usually just fed chicken feed, resulted in an increase in body weight and improved microbial quality [65].

Table 1. A summary of probiotics tested on insects mass-reared for food and feed with the objective of improving insect performance or fitness against natural pathogens in the mass rearing environment. The table does not include data where bacteria/yeast have been provided to the insect as a probiotic to test its efficacy against a specific human pathogen in vivo, nor does it include insects reared for sterile insect technique programmes.

Insect Species

Probiotics Effects on Performance and Yield Ref.

Silkworm Bacteria

Bombyx mori Bifidobacteria

Bifidobacterium bifidum

Found to be an immunomodulating agent (increase in the activity of protease, amylase and invertase); increased raw silk production with fewer cocoons [66]

Lactobacilli

Lactobacillus acidophilus

Stimulated growth factors leading to an increase in the silk yield and to an improvement of the silk harvest [START_REF] Suraporn | Effects of Probiotic Bacteria on the Growth Parameters of the Thai Silkworm, Bombyxmori[END_REF] L. casei Improved larval weight, cocooning ratio, pupation ratio, and economic characters (cocoon weight and size) when larvae were infected with microsporidium Nosema bombycis [START_REF] Suraporn | Supplementation of Lactobacillus Casei Reduces the Mortality of Bombyx Mori Larvae Challenged by Nosema Bombycis[END_REF] L. plantarum Helped to increase body weight, cocoon, shell, and pupation rate [START_REF] Singh | Effect of Use of Lactobacillus Plantarumas a Probiotics to Improve Cocoon Production of Mulberry Silkworm, Bombyxmori (L.)[END_REF] Table 1. Cont.

Insect Species Probiotics

Effects on Performance and Yield Ref.

Staphylococci

Staphylococcus gallinarum strain SWGB 7 & S. arlettae strain SWGB 16

Increased larval growth and cocoon characters (filament length and weight, finer denier) [START_REF] Saranya | Fortification of Mulberry Leaves with Indigenous Probiotic Bacteria on Larval Growth and Economic Traits of Silkworm (Bombyx Mori, L.)[END_REF] Yeast

Saccharomyces cerevisiae

Immunomodulating agent; increased raw silk production with fewer cocoons; increased protein content [66,[START_REF] Esaivani | Impact of Probiotic Saccharomyces Cerevisiae on the Enzymatic Profile and the Economic Parameters of Sylkworm Bombyx Mori[END_REF]

Fungi

Trichoderma harzianumas

Improved food digestion leading to increased growth and resistance to mortality by Metarhizium anisopliae and Beauveria bassiana [START_REF] Alcosaba | Effect of Fungus, Trichoderma Harzianumas Probiotic on the Growth, Cocoon Parameters, Silk Characters and Resistance of Silkworms (Bombyx Mori) Challenged by Muscardine Disease-Causing Metarhizium[END_REF] Commercial products

Lact-Act a

Larvae reared on leaves sprayed with Lact-Act had increased survival when exposed to bacterial pathogens (Bacillus thuringiensis var. sotto. and Staphylococcus aureus) [START_REF] Rajakumari | Efficacy of Probiotic and Neutraceutical Feed Supplements against Flacherie Disease in Mulberry Silkworm, Bombyx Mori[END_REF] Insect species Probiotics Effects on performance and yield Ref.

Greater wax moth Bacteria

Galleria mellonella Clostridiaceae

Clostridium butyricum Miyairi 588

Induced immune response and increased survival rates against Salmonella enterica serovar Typhimurium, enteropathogenic Escherichia coli or Listeria monocytogenes.

[74]

Lactobacilli

Lactobacillus acidophilus ATCC 4356

Increased survival from Candida albicans infection [START_REF] Vilela | Lactobacillus Acidophilus ATCC 4356 Inhibits Biofilm Formation by C. Albicansand Attenuates the Experimental Candidiasis in Galleria Mellonella[END_REF] L. kunkeei b Reduces infection of Pseudomonas aeruginosa through biofilm formation and affecting their stability [START_REF] Berríos | Inhibitory Effect of Biofilm-Forming Lactobacillus Kunkeei Strains against Virulent Pseudomonas Aeruginosa in Vitro and in Honeycomb Moth (Galleria Mellonella) Infection Model[END_REF] L. rhamnosus ATCC 7469 Promoted greater protection in larvae infected with Staphylococcus aureus or Escherichia coli.

[52]

L. rhamnosus ATCC 9595 Reduces infection of Pseudomonas aeruginosa through biofilm formation and affecting their stability [51] L. rhamnosus GG Induced immune response and increased survival rates against Salmonella enterica serovar Typhimurium, enteropathogenic Escherichia coli or Listeria monocytogenes.

[74]

Yellow mealworm Bacteria

Tenebrio molitor Bacilli

Bacillus subtilis

Enhanced growth and nutritional fortification [START_REF] Rizou | Amelioration of Growth, Nutritional Value, and Microbial Load of Tenebrio Molitor (Coleoptera: Tenebrionidae) through Probiotic Supplemented Feed[END_REF] B. toyonensis Enhanced growth and increased dry matter weight of produced feed [START_REF] Rizou | Amelioration of Growth, Nutritional Value, and Microbial Load of Tenebrio Molitor (Coleoptera: Tenebrionidae) through Probiotic Supplemented Feed[END_REF] Table 1. Cont.

Insect Species Probiotics

Effects on Performance and Yield Ref.

Enterococcaceae

Enterococcus faecalis

Increased larval weight gain and overall size and shorter time to pupation, also increased the crude protein content [START_REF] Rizou | Amelioration of Growth, Nutritional Value, and Microbial Load of Tenebrio Molitor (Coleoptera: Tenebrionidae) through Probiotic Supplemented Feed[END_REF]

Lactobacilli

Pediococcus pentosaceus (Isolated from the gut of Tenebrio larvae)

Reduces mortality in larvae and accelerates the rate of development. The strain has antimicrobial activity towards a number of pathogenic bacteria including several Bacillus thuringiensis, Serratia, and Pseudomonas spp.

[78]

Insect species Probiotics Effects on performance and yield Ref.

Black soldier fly Bacteria

Hermetia illucens Actinomycetia

Arthrobacter AK19

Enhanced growth rate at early life stages culminating in larger larvae than control [START_REF] Kooienga | Effects of Bacterial Supplementation on Black Soldier Fly Growth and Development at Benchtop and Industrial Scale[END_REF] Bacilli Bacillus subtilis S15 S16 S19; B. subtilis natto D1

Increased larval weight and total development time compared to control larvae [START_REF] Yu | Inoculating Poultry Manure With Companion Bacteria Influences Growth and Development of Black Soldier Fly (Diptera: Stratiomyidae) Larvae[END_REF]

Bifidobacteria

Bifidobacterium breve

Larvae had lower weights and appeared weak/slow/discolored compared to control [START_REF] Kooienga | Effects of Bacterial Supplementation on Black Soldier Fly Growth and Development at Benchtop and Industrial Scale[END_REF]

Nocardiaceae

Rhodococcus rhodochrous

Increased conversion rate, which could result in larger larvae with less feed. Larvae had increased proteins content related to energy production and storage. Larvae without the probiotic which had higher content of proteins related to stress responses.

[81]

Commercial product

Actisaf ® Sc47 c Increased bioconversion rate, lipid and protein yield in processed larvae [START_REF] Richard | Live Yeast Probiotic Can Boost Growth Performances of Yellow Mealworm and Black Soldier Fly Larvae[END_REF] House fly Bacteria

Musca domestica Enterobacteriaceae

Enterobacter hormaechei

Increased body length and weight, pupal weight, and shortened growth cycle, which is a considerable advantage that can contribute to cost savings and boost production in large-scale feeding facilities.

[83]

a -Probiotic powder containing Lactobacillus sporogens, Bacillus thuringiensis, yeast hydrolysate, a-amylase, vita. min and mineral mix; b -Strain was isolated from honeybee guts and tested against gram-pathogen Pseudomonas aeruginosa; c -Yeast-Saccharomyces cerevisiae CNCM I-4407.

Isolating Potential Probiotic Strains and Their Characterization

There is no standard method for identifying probiotic strains; however, potential probiotic candidates are usually identified in the core microbiota of the insect [START_REF] Yan | Probiotics and Immune Health[END_REF][START_REF] Cai | Intestinal Probiotics Restore the Ecological Fitness Decline of Bactrocera Dorsalis by Irradiation[END_REF][START_REF] Dierking | Receptors Mediating Host-Microbiota Communication in the Metaorganism: The Invertebrate Perspective[END_REF], primarily through a metagenomic approach, as illustrated in Figure 1 The microbiota is readily characterized via DNA extraction followed by 16S rRNA gene sequencing. The 16S rRNA gene region is used for sequencing, as it is a short (approximately 300-500

Insects 2022, 13, 376 9 of 18 bases), conserved gene specific to bacterial genus, and even some species [START_REF] Clarridge | Impact of 16S RRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases[END_REF]. However, as discussed in a recent review by Winand et al. [88], next-generation sequencing such as Illumina and Nanopore Technologies offers a reliable identification of bacterial genera but can have reduced accuracy in the identification of bacterial species, necessitating the combination of omics with classical microbiological techniques to get down to species and strain level. Once the core microbiota is classified, further culturing steps can be utilized to target specific bacterial isolates. Yeruva et al. [89] utilized this approach when they assessed the midgut of B. mori to identify potential probiotics. Through this approach, Enterococcus, Lactobacillus, and Bacillus species were found to be dominant in the microbiota, and upon further evaluation, these species are well-known producers of coenzymes, antimicrobial substances and extracellular enzymes [START_REF] Yeruva | Identification of Potential Probiotics in the Midgut of Mulberry Silkworm, Bombyx Mori Through Metagenomic Approach[END_REF].

conserved gene specific to bacterial genus, and even some species [START_REF] Clarridge | Impact of 16S RRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases[END_REF]. However, as discussed in a recent review by Winand et al. [88], next-generation sequencing such as Illumina and Nanopore Technologies offers a reliable identification of bacterial genera but can have reduced accuracy in the identification of bacterial species, necessitating the combination of omics with classical microbiological techniques to get down to species and strain level. Once the core microbiota is classified, further culturing steps can be utilized to target specific bacterial isolates. Yeruva et al. [89] utilized this approach when they assessed the midgut of B. mori to identify potential probiotics. Through this approach, Enterococcus, Lactobacillus, and Bacillus species were found to be dominant in the microbiota, and upon further evaluation, these species are well-known producers of coenzymes, antimicrobial substances and extracellular enzymes [START_REF] Yeruva | Identification of Potential Probiotics in the Midgut of Mulberry Silkworm, Bombyx Mori Through Metagenomic Approach[END_REF].

The analysis of health-promoting properties of probiotic bacteria and yeast should be conducted using in vitro and in vivo approaches before providing the strain on a large scale [START_REF]Food and Agriculture Organization of the United Nations Probiotics in Food, Health Nutritional Proprieties and Guidelines for Evaluation[END_REF]. Papadimitriou et al. [91] and Byakika et al. [92] provided an overview on the assays that can be performed on bacterial strains for observing if the safety and biological and chemical characteristics of the strain fulfill the characteristics of being classified as probiotics. The same procedures have to be performed on yeast strains [START_REF] Kumura | Screening of Dairy Yeast Strains for Probiotic Applications[END_REF]. The analysis of health-promoting properties of probiotic bacteria and yeast should be conducted using in vitro and in vivo approaches before providing the strain on a large scale [START_REF]Food and Agriculture Organization of the United Nations Probiotics in Food, Health Nutritional Proprieties and Guidelines for Evaluation[END_REF]. Papadimitriou et al. [91] and Byakika et al. [92] provided an overview on the assays that can be performed on bacterial strains for observing if the safety and biological and chemical characteristics of the strain fulfill the characteristics of being classified as probiotics. The same procedures have to be performed on yeast strains [START_REF] Kumura | Screening of Dairy Yeast Strains for Probiotic Applications[END_REF].

Safety Assays

All intrinsic characteristics relevant to the strains have to be fully evaluated before considering the strain safe for probiotic purposes. Safety assays on the production of bio-genic amines by the decarboxylation of amino acids by substrate specific decarboxylases of potential probiotic bacteria should be conducted; for example, the production of histamine, which can persist in the food chain and lead to severe allergies through consumption of edible insects [START_REF] Papadimitriou | Discovering Probiotic Microorganisms: In Vitro, in Vivo, Genetic and Omics Approaches[END_REF][START_REF] Chomchai | Histamine Poisoning from Insect Consumption: An Outbreak Investigation from Thailand[END_REF].

Moreover, the determination of the minimal inhibition concentrations (MIC) related to antimicrobials in probiotic strains reduces the risk of the addition of antimicrobial-resistant genes in the insects' gut environment and avoidance of the horizontal transmission of these genes to other microorganisms. The official protocols created by the European Food Safety Agency [START_REF]European Food Safety Authority Guidance on the Assessment of Bacterial Susceptibility to Antimicrobials of Human and Veterinary Importance[END_REF] have to be performed following international standard recognized methods cultivating the strain on seven antibiotics and the cut-off values, taking into considerations strain, growing conditions, and dilution variability (CLSI; www.clsi.org accessed on 2 February 2022; ISO; www.iso.org accessed on 15 January 2022). Other safety considerations include the production of virulent genes and toxin production, which could be deleterious to reared insects and consumers of insects.

Analysis of Antimicrobial Potential

The analysis of the antimicrobial potential is performed to assess the strain's ability to produce antimicrobial compounds (AMCs) active against selected pathogens and can be performed by running in vitro assays. The agar spot method is efficient for recording the probiotic's zone of pathogens' inhibition on selected media in selected growth conditions [53,[START_REF] Lecocq | Probiotic Properties of an Indigenous Pediococcus Pentosaceus Strain on Tenebrio Molitor Larval Growth and Survival[END_REF][START_REF]European Food Safety Authority Guidance on the Assessment of Bacterial Susceptibility to Antimicrobials of Human and Veterinary Importance[END_REF]. The technique is used for quantifying the effect of antimicrobial agents such as bacteriocins and organic acids on selected pathogens. As agar gradient and culturing conditions can distort the effective concentration/production of the molecules, other methods can be applied. The paper-disk diffusion assay [START_REF] Tagg | Assay System for Bacteriocins[END_REF] and the well diffusion assay work on the same principle. For reducing the effect of the concentration of agar, liquid-medium techniques have been proposed by recording nisin production in Leuconostoc mesenteroides (De Moss et al.) [START_REF] Cabo | A Method for Bacteriocin Quantification[END_REF][START_REF] Juturu | Microbial Production of Bacteriocins: Latest Research Development and Applications[END_REF]. Microbiota interactions and environmental conditions such as pH and temperature can influence the production of antimicrobial compounds. For instance, in vitro studies highlighted optimal conditions of pH 6.2 and a temperature of 37 • C for some Lactobacillus spp. for producing bacteriocins [START_REF] Yang | Influence of Culture Media, PH and Temperature on Growth and Bacteriocin Production of Bacteriocinogenic Lactic Acid Bacteria[END_REF]. It is also important to take into consideration the consistent differences that are present within and between insect orders in terms of gut structure and gut environment, which also affect the production of AMCs [19].

As probiotics can decrease pathogen inference by competition, co-aggregation abilities can be recorded by in vitro assays based on absorbance measurements and fluorescence and radiolabelling detection [100]. Other in vitro methods used for observing the microbiota interactions and competition for space acquisition are focused on autoaggregation and on the probiotic's effects on the pathogen's capacity to produce biofilms. The methods are based on spectroscopy measurements. The autoaggregation of Lactobacillus spp. varies from 10 to 23% influencing competition processes against pathogens, therefore decreasing the ability of microorganisms such as L. monocytogenes to produce biofilm and to infect the host [START_REF] Byakika | A Review of Criteria and Methods for Evaluating the Probiotic Potential of Microorganisms[END_REF]101,102]. Hydrophobicity properties related to gut adhesion and colonization can be measured by performing spectrometry on isolated strains of the potential probiotic culture in several organic solvents [START_REF] Byakika | A Review of Criteria and Methods for Evaluating the Probiotic Potential of Microorganisms[END_REF]102,103]. Another important parameter that can influence gut colonization by adhesion is the production of exopolysaccharides (EPS). For in vitro analysis of the production of these molecules, an extraction followed by concentration estimation by phenol-sulfuric acid method is efficient [102]. The expression of these parameters increases the probiotic strain capabilities to compete with pathogens strains in gut space colonization by reducing the chance of incurring infection.

Assessing Immune Modulation

The influence of a potential probiotic strain on the immune response can be determined by monitoring the expression of important immune response genes, or by monitoring the expression of genes encoding immunologically important molecules by quantitative RT-qPCR [START_REF] Yan | Probiotics and Immune Health[END_REF]. The innate immune system comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT) [104]. The most commonly investigated groups of genes for probiotic studies are antimicrobial peptides (AMP) and pattern-recognition receptors of the Toll and Imd pathways due to the receptors mediating host-microbiota communication [START_REF] Dierking | Receptors Mediating Host-Microbiota Communication in the Metaorganism: The Invertebrate Perspective[END_REF]. Coupled with an assessment of immune-relevant gene expression, it is also common practice to collect hemolymph samples for measuring other immunomodulatory parameters. Ordinarily, these are assays involving measuring the level of phenoloxidase, total protein concentration, and total hemocyte counts and differential haemocytes circulating in the hemolymph after the administration of a probiotic [START_REF] Franks | The Effect of Rhodococcus Rhodochrous Supplementation on Black Soldier Fly (Diptera: Stratiomyidae) Development, Nutrition, and Waste Conversion[END_REF]105].

Ecological Fitness Assay

According to Peacock [106] fitness can be most usefully understood as all aspects resulting in survival, not only the properties of reproductive success. Furthermore, Rosenberg and Bouchard [107] clarify ecological fitness as interactions between organisms and environments. With this definition in mind, it is clear that the experimental design is critical when evaluating the effect of probiotics on overall fitness and health, as the results can be influenced by several factors: 1. method of delivery; 2. biological traits measured; 3. effect on different life stages; 4. pre-existing microbiota; and 5. diet [108]. Additionally, due to the diversity of life-history strategies and environments in which insects inhabit, there can be species-specific influences within these factors, further establishing a criterion for assessing probiotics in insects complicated. Nevertheless, indicators of biological fitness are often measured by the overall longevity, mortality, fertility, and fecundity of the insect. For those insect species that are holometabolous, the weight of pupae and adult emergence rate can also be monitored, as can, in some cases, the flight capacity [102].

Within these measurements, it can be important to distinguish the additive role of the probiotic on having an effect due to the interaction with the insect or simply as a source of nutrients. Some researchers have attempted to separate these responses by providing both dead and live probiotics. When assessing the effect of Klebsiella oxytoca and Enterobacter sp. AA26 as probiotics in larval and adult C. capitata mass-reared for SIT, Kyritsis et al. [109] provided both dead (inactivated via heat treatment) and live bacteria. In doing this, they were able to differentiate the responses as an effect of the live bacteria, or not. A reduction in the developmental time of the immature stages was found for flies fed both dead and live K. oxytica-enriched diets, but a positive effect on flight ability was only demonstrated in individuals provided the live bacteria. Similarly, Gavriel et al. [110] only found an increase in mating competitiveness in medfly adults provided live bacteria, with no beneficial effect on males fed dead bacteria. In general, this highlights the need to better understand the role of a supplement as either providing an additional source of nutrients, or as establishing in the gut and interacting with the host.

In summary, there is no 'best practice' for assessing the efficacy of probiotics in insects. However, assays involving the assessment of the effect of the probiotic on biological parameters have become a standard practice. An often-overlooked element in categorizing probiotic potential is the evaluation of the microbiota community composition after the administration of the probiotic as some strains are able to reset unbalanced microbiota phenotypes by modulating the host defense systems, as well as drive microbiome functional shifts. These shifts, if sufficient, could achieve a positive effect or trigger a decline in nutrients, energy, and metabolic activity of the insect and reduce overall growth and reproduction performance [START_REF] Cai | Intestinal Probiotics Restore the Ecological Fitness Decline of Bactrocera Dorsalis by Irradiation[END_REF]111].

Improving Mass Reared Insect Fitness by Probiotic Provision

The gut microbial composition has the potential to shape its host growth trajectory in a stressful environment. The manipulation of the gut microbial composition by providing selected probiotic feed additives in the rearing systems could be a way to improve insect fitness, reduce the effects of external factors such as stress, and reduce or altogether prevent the use of chemical growth promoters [112]. Studies on aseptically reared insects have mainly focused on the role of specific strains on nutrient absorption in poor nutrient conditions. A study on the popular insect model Drosophila melanogaster highlighted the importance of the beneficial metabolic dialogue between Lactobacillus plantarum and Acetobacter pomorum. The provision of these bacteria determines the boosting of the host juvenile growth despite the malnutrition, by each providing essential metabolites such as lactate, essential amino acids, and anabolic metabolites that foster growth [113].

Even if the selection of specific strains for improving fitness performances is still ongoing for several mass-reared insect species, some results have already been obtained on B. mori's body weight, cocoon, shell, and pupation rate with the addition of Lactobacillus species in the diet [START_REF] Yeruva | Identification of Potential Probiotics in the Midgut of Mulberry Silkworm, Bombyx Mori Through Metagenomic Approach[END_REF]. Saccharomyces cerevisiae, Staphylococcus gallinarum and Staphylococcus arlettae provided on mulberry leaves resulted in better performance in B. mori [START_REF] Saranya | Fortification of Mulberry Leaves with Indigenous Probiotic Bacteria on Larval Growth and Economic Traits of Silkworm (Bombyx Mori, L.)[END_REF][START_REF] Esaivani | Impact of Probiotic Saccharomyces Cerevisiae on the Enzymatic Profile and the Economic Parameters of Sylkworm Bombyx Mori[END_REF]. Positive connections between the provision of the strain Pediococcus pentosaceus (Figure 2) to T. molitor larvae and fitness performances have been proven allowing its definition as probiotic [START_REF] Lecocq | Probiotic Properties of an Indigenous Pediococcus Pentosaceus Strain on Tenebrio Molitor Larval Growth and Survival[END_REF]. The definition of a protocol for providing the strain on an industrial scale is ongoing. Initial studies on T. molitor have demonstrated that the provision of a mixed culture of probiotic bacteria can affect growth and weight gain positively [114].

The primary interests of providing probiotics to Hermetia illucens are mainly focused on waste conversion and their positive effect on larval growth. Arthrobacter AK19 and Rhodococcus rhodochrous 21198 increased the protein digestion and absorption by 20-30% with no impact on the microbial community. On the other hand, the provision of Bifidobacterium breve, caused an increase of 50% of larval final weight, 20% lower waste conversion, and the suppression of microbial community diversity at a benchtop and industrial scale [START_REF] Kooienga | Effects of Bacterial Supplementation on Black Soldier Fly Growth and Development at Benchtop and Industrial Scale[END_REF].

The nutritional content of the insects is affected by the manipulation of the microbial composition. The dry matter and crude protein percentage showed higher values in T. molitor larvae and H. illucens's fatty acids compositions and presented a shift to polyunsaturated fatty acids [START_REF] Kooienga | Effects of Bacterial Supplementation on Black Soldier Fly Growth and Development at Benchtop and Industrial Scale[END_REF]114]. The selection of targeted microorganisms plays a key role in shaping the microbial community and obtaining positive effects on fitness parameters.

Concluding Remarks and Future Perspectives

The occurrence of diseases in mass-reared insects and the need of reducing th of antibiotics in all the production systems to cope with the rising frequency of antib resistance, are demanding new solutions for preserving animals' health and impro their fitness performances. The identification and the selection of host-specific prob

Concluding Remarks and Future Perspectives

The occurrence of diseases in mass-reared insects and the need of reducing the use of antibiotics in all the production systems to cope with the rising frequency of antibiotic resistance, are demanding new solutions for preserving animals' health and improving their fitness performances. The identification and the selection of host-specific probiotics could represent a sustainable solution for stabilizing insect production and ensuring food and feed safety.

Once the probiotic strain is selected, the supplements' formulation and the role of prebiotics in synbiotic interactions could have an important role in stabilizing the commercial product and in assuring the probiotic gut colonization and persistence after the provision to the insects.

Insect-microbiota relationships can affect behaviour and fitness performances in several stressful situations. Interest in the probiotic provision of insect species for food and feed purposes has already led to the enhancement of fitness performances and immune responses. Therefore, the chance to shape the microbial community favouring probiotic microorganisms by providing prebiotics in the diet increase the opportunities to promote the health status of the insects and to decrease the occurrence of diseases in mass reared conditions. A lot of questions related to synbiotic-host relationship are still open and multivariate statistical models are needed for studying the effects of diet, environmental factors, and microbiota on these interactions. Further studies could be focused on the manipulation of mass-reared insect microbiota for breeding individuals with a selected starting gut microbiota that could allow better growth and reproductive performance, decreasing the occurrence of diseases.

Valorization of thesis

Change of environment:

The PhD was conducted between the MICALIS institute of INRAE (Université Paris-Saclay) for 2 years, and the Section for Organismal Biology at the University of Copenhagen for 1 year.

Internship:

Ynsect (NL and FR) Spent one week at the Ermelo (NL) R&D facility, assessing the health of the Alphitobius diaperinus (Buffalo beetle) population by evaluating the abundance of gregarines in their rearing facility. Along with two other early stage researchers we were able to collect gregarines and further aim to sequence and generate primers from these samples, to make it easier to assess the quantity of gregarines in the facility in the future by using qPCR techniques. I also Spent three days at the R&D facility in Evry (FR), understanding how the health and safety aspect of the rearing is assessed and discussing ways to improve this. Abstrakt : Patogener kan i høj grad påvirke insekternes fitness, hvilket ofte resulterer i død og yderligere spredning af patogenet. Dette er isaer problematisk i et masseopdraet, hvor der er et stort antal insekter, der lever taet sammen. Sådanne miljøer er udsatte og sårbare ved forekomsten af patogener da der er grobund for hurtig spredning af sygdomme. Samtidig har insekter fordelagtige symbionter i deres tarme, symbionter der kan facilitere vigtige funktioner i deres vaert, såsom naeringsstofoptagelse eller resistens over for patogener. Dette isaer når tarmmikrobiotaen, som mikrobielt samfund, er i balance. Der kan ske aendringer tarmens mikrobiota ved infektion med patogener, og det kan enten bidrage til -eller mindske vaertens modstandsdygtighed over for patogener. På grund af dette potentiale er der behov for mere forskning der stiller skarpt på det tredelte samspil mellem tarmmikrobiotaen, patogenet og vaertens respons. I dette projekt blev larver af Galleria mellonella (voksmøl) brugt som modelvaert til at undersøge mikrobiotaens rolle i forhold til sundhed og modstandsdygtighed over for kolonisering af entomopatogener der inficerer deres vaert enten oralt eller topikalt. Bacillus thuringiensis ssp. galleriae og Metarhizium robertsii (Mr) blev brugt til at fremkalde immunresponser via henholdsvis en oral og en topisk infektionsvej. Vores strategi var at måle forskellige fitness parametre på Galleria mellonella-larver opdraettet uden eller med en tarmmikrobiota (steril -"akseniske" vs. konventionelle larver) og at undersøge vaertsresponsen efter infektion. Vi antog, at symbiotiske bakterieisolater i insekttarmen kan forbedre vaertens immunrespons og hjaelpe i kampen mod patogeninfektion. Effektiviteten af dette symbiotisk forstaerkede immunrespons kan dog variere afhaengigt af det specifikke patogen, der er involveret, samt dets vej ind i vaertsorganismen. Vi observerede, at larver uden en tarmmikrobiota udviste større variation i deres udviklingstid, men at den overordnede fitness og evne til at fuldføre deres livscyklus ikke haemmes. Vi eksponerede derefter 250-300 mg larver for LD50 doser af Btg og Mr og fandt, at akseniske larver var mere modtagelige for en oral infektion af Btg, med 90-100% mortalitet i den akseniske gruppe mod 50 % mortalitet i den konventionelle gruppe. Der var dog ingen forskel i mortaliteten af de akseniske og konventionelle larver efter de blev udsat for topisk infektion med Mr. Endvidere karakteriseredes tarmmikrobiotaen via 16S rRNAsekventering og ekspressionsniveauet af immunrelateret gener blev analyseret via qRT-PCR for samme gruppe larver under infektion med Btg og Mr. Heraf kunne vi se at mikrobiotaen i vores konventionelt population var domineret af fire Enterococcus-arter, og at disse arter potentielt stimulerede immunresponset i tarmen, da vi observerede en øget basal ekspression af to AMP'er; Gallerimycin og Gloverin, i de konventionelle larver sammenlignet med de akseniske. Desuden udviste Enterococcus mundtii, isoleret fra tarmen hos konventionelle larver, haemmende aktivitet over for Btg in vitro, og da akseniske larver blev re-inokuleret med dette isolat, blev der observeret en forbedret resistens over for Btg 96 timer efter infektion. Disse resultater fremhaever den immunmodulerende rolle, som den Enterococcus-dominerede tarmmikrobiota spiller, en mikrobiotasammensaetning, der i stigende grad rapporteres hos laboratoriepopulationer af Lepidoptera, og dens indflydelse på vaertens reaktion på orale og topiske infektioner.

Figure 1 .Figure 3 . 12 . 14 .

 131214 Figure 1. The European Joint Doctoral Programme, INSECT DOCTORS. ____________________ Figure 2. The tripartite interactions between the host, its resident gut microbiota, and invading pathogens _____________________________________________________________________________ Figure 3. The different life stages of the holometabolous insect, G. mellonella ______________ Figure 4. Galleria mellonella as a model organism for different infection assays ____________ Figure 5. Light and fluorescence microscopic images of hemocytes isolated from Galleria mellonella hemolymph. _________________________________________________________________ Figure 6. Summary of the proPO activation in insects where foreign bodies are recognized based on their PAMPs by the hosts PRRs.._______________________________________________ Figure 7. Free spores and crystals of strain Bacillus thuringiensis subsp. galleriae 69-6 _____ Figure 8. Metarhizium robertsii characteristics ___________________________________________ Figure 9. The different infection routes by oral (Bacillus thuringiensis) and topical (Metarhizium robertsii) pathogens _______________________________________________________ Figure 10. The multitude of interactions between two organisms __________________________ Figure 11. A. The anatomy of G. mellonella displaying the underside of the larva, showing the foregut, midgut and hindgut regions of the digestive tract _________________________________ Figure 12. Methods of microbiota-mediated colonization resistance against foreign microbes. _____________________________________________________________________________ Figure 13. The sterilization procedure for producing axenic larvae. ________________________ Figure 14. External morphological similarities between axenic and conventional Galleria mellonella larvae ______________________________________________________________________

Figure 1 .

 1 Figure 1. The European Joint Doctoral Programme, INSECT DOCTORS. The PhD research projects (numbered 1 -15) are divided into three work packages, each with its own objectives under the umbrella of understanding insect health. The first work package focuses on pathogen-host interactions and determines abiotic and biotic triggers for disease outbreaks. The second work package looks at covert infections and pathogen detection while the third work package is focused on increasing insect resistance against pathogens, by looking at the interactions between pathogens and the insect microbiome and how nutritional adaptations can increase insect fitness. The figure to the right places each project into a Venn diagram describing which type of insect pathogen will be evaluated as part of the work.

Figure 3 .

 3 Figure 3. The different life stages of the holometabolous insect, G. mellonella starting with 1. the creamy white egg clusters, 2-6, the larval instars, 7, the silken pupal sac which protects from injury 8, the pupae, and 9. the adult moth. Image taken from (Jorjão et al., 2018) originally published in Virulence.

Figure 5 .

 5 Figure 5. Light (top) and fluorescence (bottom) microscopic images of hemocytes isolated from Galleria mellonella hemolymph. Hemocyte descriptions are well described by (Wu et al., 2016a; Mohamed & Amro, 2022) A. Prohemocytes are round or oval with a high N/C ratio B. Plasmatocytes are usually ovoid or spindle-shaped cells with a high N/C ratio, C. Granular cells usually spherical or ovoid with a low nucleus/cytoplasm (N/C) ratio, D. Oenocytes, usually the largest hemocyte, with a low N/C ratio, and originate from an immature prohemocyte E. Spherulocytes, comprise less than 10% of the total circulating hemocytes, have a low N/C ratio. It is important to note that hemocyte populations can vary considerably in morphology (especially for plasmatocytes) depending on different stages in differentiation or functional specialization (Jiang et al., 2010). Figure is taken from Arteaga Blanco et al., (2017), originally published in Cell and Tissue Research.

Figure 6 .

 6 Figure 6. Summary of the proPO activation in insects where foreign bodies are recognized based on their PAMPs by the hosts PRRs. The serine protease cascade is then activated by proPO which triggers active PO activating serine proteases (PAPs). The activated PAPs will further convert the zymogen, inactive proPO into active and functional PO. This results in the oxidization of phenols into melanin which are involved in the immune response in the insect. Figure from Marieshwari et al., (2023), originally published in the Journal of Comparative Physiology.

Figure 7 .

 7 Figure 7. Free spores and crystals of strain Bacillus thuringiensis subsp. galleriae 69-6 A) Free spores and crystals of strain Bacillus thuringiensis subsp. 69-6 grown in HCT + 0.5% glucose media for 96h, the spore and crystal are similar in size, scale = 10μm B) The elongated vegetative cell with the yellow arrow pointing to the spore, and the green arrow pointing to the crystal, C) Observation of spores and crystals of B. thuringiensis strain H29.3 by electron microscopy. Figure C taken from Palma et al., (2014), originally published in Toxins

Figure 8 .

 8 Figure 8. Elongated conidia of Metarhizium robertsii KVL 00-89 (scale = 10μm)Grown on SDA/4 medium for three weeks. The strain was collected from Danish soils and is part of the Section for Organismal Biology library of isolated entomopathogenic fungi. Galleria mellonella was used as a bait insect to collect the isolate, by the method described by[START_REF] Meyling | Occurrence and Distribution of Soil Borne Entomopathogenic Fungi within a Single Organic Agroecosystem[END_REF].

Figure 9 .

 9 Figure 9. The different infection routes by oral (Bacillus thuringiensis) and topical (Metarhizium robertsii)A. 1. The Bt spore-crystals are ingested during the intake of food which triggers peristalsis (the abrupt halting of feeding), 2. the toxins can then break down the peritrophic matrix (pm) and further destroy epithelial cells, 3. leading to pore formations in the gut where the spore and crystals can enter the hemocoel. 4. In the hemocoel the spores germinate into a vegetative state, where more spores and crystals are produced, overloading the hemocoel eventually causing death to its host. Upon death, 5. the insect cuticle melanizes and softens, allowing the spores to be released back into the environment. B. As for the topical infection by M. robertsii, 1. the conidia adheres to the surface of the cuticle and starts to secrete cuticle degrading enzymes before it 2. forms a penetration peg to enter the hemocoel and 3. form blastospores which are released into the circulating hemocoel allowing the fungi to spread throughout the host body until it 4. overwhelms the host tissues and can 5. sporulate and penetrate back out of the host by the production of hyphal bodies which will further prepare conidia to infect the next host.

Figure 11 .

 11 Figure 11. A. The anatomy of G. mellonella displaying the underside of the larva, showing the foregut, midgut and hindgut regions of the digestive tract B. Dissected digestive tract, stained with methylene blue. C. A schematic drawing of the digestive tract, where SV is the stomadeal valve which separates the foregut and midgut, as well as PV which is the proctodeal valve (PV) which separates the midgut and hindgut. 1 -mouth, 2esophagus, 3 -crop, 4 -glandular intestine, 5 -transition zone, 6 -cuticular intestine, 7 -rectum 8 -anus. Figure taken from Sänger et al., (2022), originally published in PLOS Pathogens

4 1 51

 1 Transient -Lasting only for a short time Chapter can also facilitate or hamper the colonization of other microbes, via colonization resistance mechanisms (Figure 12).

Figure 12 .

 12 Figure 12. Methods of microbiota-mediated colonization resistance against foreign microbes.

Figure 13 .

 13 Figure 13. The sterilization procedure for producing axenic larvae.The process begins within a sterile laminar flow hood, ensuring contamination-free handling. 24 -48 h old egg sheets are retrieved from the adult cage, and inspected for obstructions. Obstructions such as some paper covering the eggs, or eggs laid in clutches blocking one another could hinder the subsequent UV sterilization's effectiveness. These sheets, along with essential tools, undergo a thorough UV sterilization with a 10-minute exposure on either side of the sheet. The previously gamma-irradiated sterile diet of pollen and wax is then poured into the autoclaved sterilized jar. Sterilized egg sheets are then placed atop this diet, followed by a three-tier sealing process involving a meshed metal lid, a layer medical gauze, a layer of cotton wool, and a final layer of medical gauze which is taped together and sealed. The jar is then placed in an incubator maintained at 27°C with a 12/12 photoperiod cycle, allowing larvae to grow to an approximate weight of 250-300mg. Finally, their axenic status is verified via both culture-dependent and independent means, by monitoring the microbial growth of dissected gut homogenates on agar plates, or by molecular analysis targeting the v3-v4 region of the 16S gene from bacterial DNA extracted from dissected guts. This comprehensive workflow ensures the production of axenic larvae with validated sterility.

Figure 14 .

 14 Figure 14. External morphological similarities between axenic and conventional Galleria mellonella larvaeBoth axenic and conventional larvae have similar coloration, size, and can successfully pupate and reach the adult life stage.

1

 1 FIGURE 1Various life history traits measured between the axenic and conventional larvae (n = 75 per group), including (A) the number of days from hatching until pupation, (B) the length of the adult life span, (C) the total life span, and (D) the pupal weight. The percentage mortality for the axenic and conventional larvae was calculated for (E) Btg infected larvae by orally force-feeding the LD50 (2 × 106 in 10 µL of a spore crystal mix) and recorded over 96 hours (three replicates, with n = 60); (F) for Mr infected larvae by topically submerging larvae into conidial suspension (1 x 107 conidia in 3mL TritonX 0.05%) and recorded over 192 hours (three replicates, with n = 90). No mortality was recorded for the controls. The different phenotypes of the cadavers post infection by Btg and Mr are also displayed. Btg-infected larvae are completely melanized upon death, whereas the Mr-infected larvae have fungal hyphae growing out of the cadaver at 72 h post death. ns, not significant.

FIGURE 2

 2 FIGURE 2

FIGURE 5

 5 FIGURE 5

FIGURE 6

 6 FIGURE 6 Heatmap of the log2-fold change gene expression from basal to infected conditions in the axenic and conventional larvae by either Btg or Mr at two time points post infection. The significance was determined for the difference in the fold change in expression of each gene and the time point between the conventional and axenic larvae by one-way ANOVA on log2-transformed values. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001. T1 = 20 h post infection for both Btg and Mr, and T2 = 40 h or 96 h post infection for Btg and Mr, respectively

  FIGURE 8

66 .

 66 Ramirez JL, Dunlap CA, Muturi EJ, Barletta ABF, Rooney AP. Entomopathogenic fungal infection leads to temporospatial modulation of the mosquito immune system. PloS Negl Trop Dis (2018) 12(4):e00064335. doi: 10.1371/ journal.pntd.0006433 67. Kong HG, Son J-S, Chung J-H, Lee S, Kim J-S, Ryu C-M. Population dynamics of intestinal Enterococcus modulate Galleria mellonella metamorphosis. Microbiol Spectr (2023) 11(4):e02780-225. doi: 10.1128/spectrum.02780-22 68. de Maagd RA, Bravo A, Berry C, Crickmore N, Ernest Schnepf H. Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic b a c t e r i a ' . A n n u R e v G e n e t( 2 0 0 3 ) 3 7 ( 1 ) : 4 0 9 -3 3 5 . d o i : 1 0 . 1 1 4 6/ annurev.genet.37.110801.143042 69. Dubovskiy IM, Martemyanov VV, Vorontsova YL, Rantala MJ, Gryzanova EV, Glupov VV. Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae)'. Comp Biochem Physiol Part C: Toxicol Pharmacol (2008) 148(1):1-55. doi: 10.1016/j.cbpc.2008.02.003 70. Pieniz S, Andreazza R, Okeke BC, Camargo FAO, Brandelli A. Assessment of beneficial properties of Enterococcus strains. J Food Process Preserv (2014) 38(2):665-755. doi: 10.1111/jfpp.12016 71. Graham K, Rea R, Simpson P, Stack H. Enterococcus faecalis milk fermentates display antioxidant properties and inhibitory activity towards key enzymes linked to hypertension and hyperglycaemia. J Funct Foods (2019) 58(7):292-3005. doi: 10.1016/ j.jff.2019.04.052 72. Sharma V, Ghosh M. Characterization of immunomodulatory, anticancer and antioxidant properties of an extracellular polymer produced by Enterococcus sp. in vegetable waste medium. Environ Sustain (2021) 4(2):419-285. doi: 10.1007/s42398-021-00188-4 73. Unckless RL, Rottschaefer SM, Lazzaro BP. The complex contributions of genetics and nutrition to immunity in Drosophila melanogaster. PloS Genet (2015) 11(3):e10050305. doi: 10.1371/journal.pgen.1005030 74. Cooper D, Eleftherianos I. Memory and specificity in the insect immune system: current perspectives and future challenges. Front Immunol (2017) 8:539(5). doi: 10.3389/fimmu.2017.00539 Upfold et al.

Figure 4 .

 4 Figure 4. The mean concentration and standard error of E. mundtii in the gut of larvae

  the E. mundtii recolonized gnotobiotic larvae by 96 hours, albeit this was not significant (x 2 = 2.84; df = 1; p = 0.0921) (Figure4).

Figure 4 .

 4 Figure 4. Comparison of the probability of survival between larvae recolonized

Figure 1 .

 1 Figure 1. General workflow and screening techniques to characterize strains probiotic potential prior to commercialization. Phases 1 and 2 display the identification of candidate strains and isolation methods; phase 3 offers different in vitro and in vivo techniques for characterizing the probiotic potential. Fundamental in vitro safety assays are also listed. Phase 4 highlights two major factors that also need evaluation prior to the probiotic being used commercially.

Insects 2022 ,

 2022 13, x FOR PEER REVIEW 13

Figure 2 .

 2 Figure 2. Confocal microscopy observation of Pediococcus pentosaceus KVBL 19-01. Probiotic ac of the strain has been recorded on T. molitor by Lecocq et al. [78].

Figure 2 .

 2 Figure 2. Confocal microscopy observation of Pediococcus pentosaceus KVBL 19-01. Probiotic activity of the strain has been recorded on T. molitor by Lecocq et al. [78].

  RSA/ online) -presentation -KU PhD day (DK) -presentation -Insect Doctors Network meeting (FR) -presentation -Insects to Feed the World (Canada) -presentation (Award for outstanding presentation) 2022-2023 -SOBI seminar (DK) -presentation -Insect Doctors Network meeting (FR) -presentation -Royal Entomological Society (UK) -presentation

  Open Science (Université Paris-Saclay) -Scientific Integrity in Research Professions (Université Paris-Saclay) -Responsible Code of Research 1 (Copenhagen University) -Concepts and terms in insect pathology and their placement in the general context of diseases and health Copenhagen University) -Laboratory methods (diagnostics, bio-assays) in insect pathology across organisms (INRAE) -The value and pitfalls of metagenomics in pathogen detection and discovery (CNRS) -Mixtures and Combined Stressors: Use of multi-stressor theory for invertebrate functionbased end points (UKCEH -Transdisciplinary training on topics concerning insect as food and feed (University of Tours) -'Insect Doctors' and Stakeholder Symposium May 25 th 2023 (University of Tours) -ABIES Journal Club -French as a foreign language (INRAE) immunitaire locale appropriée et limite et module la réponse du système immunitaire inné. L'étude a plus particulièrement porté sur les changements qui se produisent dans la composition du microbiote intestinal suite à une infection par deux agents pathogènes qui infectent naturellement G. mellonella, à savoir : Bacillus thuringiensis et Metarhizium robertsii, et leurs conséquences sur la réponse immunitaire intestinale de l'hôte. En utilisant ce modèle et ces deux pathogènes, nous avons pu analyser quantitativement ou qualitativement l'influence du pathogène et du microbiote sur le degré de sensibilité de l'hôte et sur les réponses immunologiques mesurables induites par les changements dans la composition ou la diversité du microbiote. Nous avons émis l'hypothèse que les isolats bactériens symbiotiques résidant dans l'intestin de l'insecte pourraient renforcer la réponse immunitaire de l'hôte, aidant à combattre l'infection par les pathogènes. Cependant, l'efficacité de cette réponse immunitaire renforcée symbiotiquement peut varier en fonction du pathogène spécifique impliqué ainsi que de sa voie d'entrée dans l'organisme hôte. Pour induire la réponse immunitaire via la voie d'infection orale, nous avons utilisé une souche de la bactérie entomopathogène Bacillus thuringiensis ssp. galleriae 69-6 (Btg). Bacillus thuringiensis est l'une des bactéries les plus étudiées en pathologie des insectes en raison de son utilisation comme biopesticide. C'est une bactérie Gram-positive, formant des spores. Les utilisations expérimentales de Bt comme biopesticide pour protéger les plantes contre les larves de lépidoptères ont commencé à la fin des années 1920, avec des produits à base Bt commercialisés et disponibles en France dans les années 1930. L'action insecticide de Bt contre les larves d'insectes est due à la présence de corps d'inclusion cristallins parasporaux composés de protéines insecticides spécifiques appelées delta-endotoxines ou protéines Cry. Lorsqu'elles sont ingérées, les toxines sont d'abord solubilisées dans l'intestin moyen, puis activées par les protéases de l'intestin moyen ; elles peuvent ensuite se lier à des récepteurs spécifiques présents à la surface des cellules épithéliales de l'intestin moyen. Les toxines Cry activées et liées peuvent alors former des pores et provoquer la lyse de ces cellules épithéliales de l'intestin moyen. La destruction de l'épithélium est généralement suivie par une septicémie lorsque les bactéries franchissent la barrière intestinale et prolifèrent dans l'hémocèle, entraînant la mort de l'insecte. Pour induire la réponse immunitaire via la voie d'infection topique, nous avons utilisé une souche une infection par Bt, ainsi qu'après une envenimation par le venin d'Habrobracon hebetor (Hymenoptera : Braconidae), ou encore après une infection par Beauveria bassiana. En parallèle, l'analyse par qRT-PCR de l'expression de plusieurs gènes de la réponse immunitaire a montré que, dans le groupe contrôle (non infecté), les niveaux d'expression de base des gènes codant pour gallérimycine, la glovérine et l'IMPI étaient significativement plus élevés dans le groupe de larves conventionnelles que dans le groupe de larves axéniques. Ce résultat suggère que les espèces d'Enterococcus, dominantes au sein du microbiote des larves conventionnelle, contribuent à maintenir une expression basale plus élevée des gènes codant pour ces peptides antimicrobiens (AMPs) par rapport à leurs homologues axéniques. Cependant, lors d'une infection par Btg, le niveau final d'expression de ces gènes devient comparable dans les larves axéniques et conventionnelles, indiquant que Btg induit une reaction immunitaire qui se traduit par une forte augmentation du niveau d'expression de ces genes, en presence, ou non, du microbiote. Afin d'étudier davantage l'effet de la présence du microbiote sur la réponse immunitaire, nous avons également testé deux autres effecteurs de l'immunité innée, à savoir l'activité de la phénoloxidase (PO) dans l'hémolymphe et les niveaux de ROS/RNS dans l'intestin. Nos résultats indiquent que les larves axéniques infectées par Btg subissent un stress oxydatif plus important dans l'environnement intestinal, comme l'indique l'augmentation du niveau des ROS/RNS dans l'intestin de ces larves par rapport à celui mesuré dans le groupe conventionnel. Cette augmentation du niveau des ROS/RNS dans l'intestin des larves axéniques par rapport à l'intestin conventionnel reflète probablement un effet pathogénique plus important de Btg sur les larves axéniques, qui se traduit par une perturbation de l'homéostasie ROS/RNS. L'augmentation des niveaux de ces molécules réactives dans l'intestin des larves pourrait à son tour provoquer des dommages au niveau des cellules intestinales de l'hôte. Nous avons également pu observer qu'en absence d'infection (conditions basales) les larves axéniques maintenaient toujours des niveaux légèrement plus élevés de ROS/RNS dans l'intestin que les larves conventionnelles. Nous faisons l'hypothèse que microbiote dominé par des Enterococcus pourrait potentiellement produire certains antioxydants et enzymes qui aident à éliminer les ROS dans des conditions normales, basales, maintenant ainsi leur équilibre. Les propriétés antioxydantes des espèces d'Enterococcus sont de plus en plus étudiées car ces bactéries ont montré, qu'au-delà de leur capacité à inhiber certains microbes recolonisé des larves de G. mellonella axéniques de dernier stade larvaire avec cette souche afin d'obtenir des larves gnotobiotiques n'hébergeant que cette une seule espèce d'Enterococcus. Nous avons ensuite vérifié qu'E. mundtii était capable de s'implanter de manière stable dans l'intestin des larves de G. mellonella recolonisées. Le niveau de sensibilité des larves gnotobiotiques mono-colonisées par E. mundtii à une infection par Btg à ensuite été comparé à celui des larves conventionnelles qui hébergent les quatre espèces d'Enterococcus, et à celui des larves axéniques. Nos résultats semblent indiquer que les larves mono-recolonisées présentent une survie légèrement accrue à Btg à 96 heures par rapport aux larves axéniques (30% de survie versus 20%) bien que cette différence ne soit pas statistiquement significative. Des essais similaires ont été menés notamment sur des larves de Triboleum castaneum (Coleoptera : Tenebrionidae) et les larves de Hyphantria cunea (Lepidoptera : Erebidae). Pour les larves de T. castaneum, la réinoculation des larves avec E. mundtii les a protégées contre Bt mais pas contre Pseudomonas entomphila une bactérie à Gram négatif. De même, chez les larves de H. cuneae, E. mundtii s'est avéré être un antagoniste du virus de la nucléopolyédrose (NPV) et de Bt ; par contre, la réinoculation de larves d'H. cuneae, avec Klebsiella oxytoca, une bactérie à Gram négatif, a produit des effets inverses, à savoir un effet synergique significatif avec les infections par le NPV et par Bt, entraînant des mortalités plus élevées. Les résultats présentés dans cette thèse apportent un éclairage sur le rôle d'un microbiote intestinal dominé par un petit nombre d'espèces d'entérocoques et dont la composition est très peu affectée/modifiée au cours d'une infection par Btg ou Mr. Cette étude a cependant révélé que, chez une lignée de G. mellonella, possédant un nombre restreint de souches bactériennes Gram-positives présentes dans l'intestin larvaire, la présence de ce microbiote relativement restreint augmentait considérablement le taux de survie des larves à une infection par Btg, mais ne protégait pas vis-à-vis d'une infection par Mr inoculé par voie topique. Ces résultats suggèrent, que chez G. mellonella, un microbiote intestinal simple dominé par Enterococcus peut servir de mécanisme de protection contre les infections pathogènes causées par Bt chez les larves de G. mellonella, et que le microbiote ne contribue pas, ou n'exerce aucun effet synergique, qui pourrait contribuer à augmenter la virulence de Bt comme cela a été décrit chez d'autres espèces. De plus, notre étude a démontré que la présence de ces espèces d'Enterococcus avait un impact substantiel sur l'expression basale des gènes liés à l'immunité, soulignant le rôle de ce microbiote intestinal dans la réponse immunitaire de G. mellonella. Cette protection peut se manifester par plusieurs mécanismes, et notamment via l'activation ou la modulation du système immunitaire de l'hôte au travers de l'augmentation du niveau basal d'activation ou de "préparation" du système immunitaire de l'hôte. De plus, le microbiote intestinal, dominé par les espèces d'Enterococcus, pourrait contribuer à cette défense par la production de composés antimicrobiens et/ou la compétition avec les pathogènes pour les ressources et les sites de colonization. Il est cependant important de souligner que les interactions hôte-microbiote sont complexes, et que l'extrapolation de nos résultats au-delà du modèle d'infection utilisé pourrait conduire à interprétations érronées. Collectivement, nos résultats soulignent le rôle crucial que joue le microbiote intestinal de G. mellonella dans sa protection contre certains pathogènes, notamment Bacillus thuringiensis, et que la composition des espèces bactériennes présentes dans le microbiote influe sur la résistance de l'hôte aux infections. Ils permettent de conforter d'autres données de la littérature indiquant que la composition du microbiote, mais aussi les conditions d'élevage (densités élevées), des températures fluctuantes, la nourriture ou divers stress abiotiques, ainsi que le type de pathogène considéré, peuvent grandement influer sur le niveau de protection que les bactéries symbiotiques de l'hôte peuvent conférer à leurs hôtes invertébrés. Comprendre plus finement ces relations fournira des informations qui nous permettront de développer et proposer de nouvelles stratégies pour préserver la santé des insectes, lorsqu'ils sont confrontés à des maladies provenant de divers pathogènes. Enfin, nos observations suggèrent également que certaines communautés microbiennes intestinales résidentes pourraient agir comme des agents de "priming immunitaire" en stimulant la réponse immunitaire de l'hôte contre certains pathogènes. Certaines études ont déjà montré que les insectes ont la capacité de monter des réponses immunitaires spécifiques et renforcées suite à une exposition préalable à un pathogène, cependant chez G. mellonella cette question reste à explorer. Titel : En analyse af samspillet mellem tarmens mikrobiota og vaertens tarmimmunitet hos Galleria mellonella under infektion med Bacillus thuringiensis og Metarhizium robertsii Søgeord : Bacillus thuringiensis, Metarhizium robertsii, genekspression, tarmmikrobiota, trepartsinteraktioner, aksenisk.

Table 1 . The most studied AMPs and immune-response genes in Galleria mellonella

 1 . ...............

Table 2 . The dominant bacteria and fungi found in late larval instar G. mellonella

 2

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

  An E. mundtii strain isolated from the conventional larva gut, and a dominant presence in the microbiota, showed inhibition activity against Btg in vitro. The results indicate the microbiota dominated by limited Enterococcus species, stimulated basal immune response genes, and the presence of E. mundtii (a bacterium known to secrete bacteriocins which can inhibit the growth of Bt) resulted in the larvae hosting these symbionts being less susceptible to the Btg infection than the axenic larvae.

	The gut microbiota, dominated
	by Enterococcus species, did not exhibit shifts in the relative abundance, and the
	alpha-and beta-diversity metrices indicate the microbiota communities were not
	significantly different from basal (uninfected/control) conditions to that of infected

individuals. The gut microbiota, particularly dominated by four Enterococcus species, E. casseliflavus, E. gallinarum, E. innessi and E. mundtii significantly influenced the Chapter 3 71 basal expression of immune-related genes, in particular the expression of gallerimycin and gloverin in G. mellonella.

  1,2 * , Agnès Rejasse 1 , Christina Nielsen-Leroux 1 , Annette Bruun Jensen 2 and Vincent Sanchis-Borja 1

1 Universite ´Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France, 2 Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark

  Conventional larvae experienced a faster rate of Btg clearance from the gut

	Upfold et al.		10.3389/finsc.2023.1260333
	3.3
	Frontiers in Insect Science	06	frontiersin.org

12 Supplementary Table S2 .

 SupplementaryS2 Immune response genes targeting the gut tissue, used in this study

	Supplementary
	Primer name	Description	Immune	Forward	Reverse	Reference
			pathway /
			process
	18S rRNA	HKG	-	CACATCCAAGGAAGGCAG	AGTGTACTCATTCCGATTACGA	Polenogova et al
						2019, Lange et al
						2018
	EF1: Elongation	HKG	-	AACCTCCTTACAGTGAATCC	ATGTTATCTCCGTGCCAG	Polenogova 2019
	factor 1-alphs
	(Ef-1a)
	Gallerimycin	AMP	toll	GAAGTCTACAGAATCACACGA	ATCGAAGACATTGACATCCA	Dubovskiy 2016,
						Kryukov 2020,
						Pereira 2018, Tsai
						2016, Polenogova
						2019
	Gloverin-like	AMP	toll	AGATGCACGGTCCTACAG	GATCGTAGGTGCCTTGTG	Dubovskiy 2016,
	protein					Kryukov 2020,
						Pereira 2018, Tsai
						2016, Polenogova
						2019
	IMPI	Inducible	Immune	TAGTAAGCAGTAGCATAGTCC	GCCATCTTCACAGTAGCA	Polenogova 2019,
		metalloproteinase	response			Lange et al 2018
		inhibitor
	Lysozyme		Immune	GGACTGGTCCGAGCACTTAG	CGCATTTAGAGGCAACCGTG	Lange et al 2018
			response
						99

Table S4 .

 S4 The result of the NCBI BLAST search of the resulting ASVs from the conventional larvae gut microbiota, generated by Illumina miseq and DADA2

	bp length	NCBI BLAST	query cover	per. Ident.
	427	Enterococcus mundtii	100	100
	427	Enterococcus mundtii	100	99,77
	428	Enterococcus gallinarum	100	100
	428	Enterococcus gallinarum	100	100
	428	Enterococcus gallinarum	100	99,77
	428	Enterococcus gallinarum	100	99,77
	428	Enterococcus gallinarum	100	99,53
	428	Enterococcus casseliflavus	100	100
	428	Enterococcus innesii	100	99,77
	428	Enterococcus innesii	100	99,77

101 Supplementary

Table S5 .

 S5 The results of the PERMANOVA to test the signifance of the Beta community between samples, after subsetting and discarding Bacillus thuringiensis reads. No significance is observed in the beta community between infected samples, infected vs their controls, as well as between control samples at different time points.

	Group comparisons	F. model	R 2	P. value
	Infected t1 vs Infected t2
	Btg 20 h vs Btg 40 h	1.296	0.134	0.9
	Mr Topical 20 h vs Mr Topical 96 h	3.8	0.388	0.088
	Infected vs control
	Btg 20 h vs Control Btg 20 h	2.578	0.392	0.200
	Btg 40 h vs Control Btg 40 h	1.393	0.218	0.1
	Mr Topical 20 h vs Control Mr 20 h	0.469	0.072	0.634
	Mr Topical 96 h vs Control Mr 96 h	0.757	0.112	0.446
	Control t1 vs Control t2
	Control Btg 20 h vs Control Btg 40 h	0.315	0.073	0.983
	Control Mr 20 h vs Control Mr 96 h	4.595	0.434	0.109
	t1 = time point one (i.e. 20h) ; t2 = time point two (i.e. 40h or 96h)

Table 1 .

 1 Published papers between 2002 -2023 reporting Enterococcus species as symbionts in Lepidoptera, those also harboring Enterococcus mundtii are underlined. Members of the Enterococcus genus likely thrive in the guts of Lepidopterans due to their capability of cellulose digestion and detoxification of plant defense chemicals, which are often present in the plants caterpillars consume[START_REF] Mithöfer | Plant Defense Against Herbivores: Chemical Aspects[END_REF].

	Family -species	Enterococcus spp	Refs
	Bombycidae
			(Pandiarajan & Krishan 2018,
	Bombyx mori	mundtii	Yeruva et al., 2020; Zhang et al. 2021)
	Cossidae
	Camadia redtenbacheri	na	(Hernández-Flores et al., 2015)
	Crambidae
	Chilo suppressalis	na	(Chen et al. 2022)
	Cydalima perspectalis	casseliflavus	(Dantur et al., 2015)
	Diatraea saccharalis	na

(Dantur et al., 2015)

bacteria-like zeta toxins proteins -inhibits cell wall biosynthesis and it may act as a bactericide in nature

ISEA -Institute of Systematics and Ecology of Animals

CEMUBIO -Centro Multoplicador de Biocontroladores Nativos, National Institute of Agricultural Technology (INTA).

Lactobacillus was present in small quantities but increased along with Pseudomonas in polystyrene and polyethylene treated larvae

Notably, Firmicutes was the largest gut microbiota member but at early stages of development made up <1% of microbiota. (Dubovskiy et al., 2016) (Allonsius et al., 2019) (Cassone et al., 2020) (Lou et al., 2020) (Ruiz Barrionuevo et al., 2022) (Gohl et al., 2022) 1 ISEA (Russia)

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg, Denmark Published in: Frontiers in Insect Science | Insect Health and Pathology

(Schuhmann et al., 2003);

(Wu et al., 2016);

(Dubovskiy et al., 2016);

(Wu et al., 2022);

[START_REF] Taszłow | Humoral Immune Response of Galleria Mellonella after Repeated Infection with Bacillus Thuringiensis[END_REF];

(Axén et al., 1997);

(Lundström et al., 2002);

(Hwang & Kim, 2011);

(Moreno-Habel et al., 2012);

(Xu et al., 2012);

(Wojda, 2017) 100

Quorum sensing -cell-to-cell communication mechanisms occurring between inter-and intra-bacterial species which is regulated by signaling molecules called autoinducers

Antibiotic cocktail (0.5 mg/ml each of gentamicin, streptomycin, penicillin, and rifampicin; Sigma-Aldrich)(Broderick et al., 2006)

Insects 2022, 13, 376. https://doi.org/10.3390/insects13040376 https://www.mdpi.com/journal/insects

Acknowledgements

To my supervisors

Acknowledgments

We would like to thank Christophe Buisson for his assistance with insect rearing and guidance for gut dissections; Alicia Nevers (Micalis, INRAE) for her guidance on qRT-PCR analysis; and Olivier Rue from MaIAGE/BioinfOmics, MIGALE Bioinformatics Facility of INRAE for his assistance with the initial 16S rRNA analysis.

Acknowledgments: We appreciate the Insect Doctors consortium for their ongoing support and the reviewers for their insightful suggestions and comments on an earlier version of this manuscript.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This project has received funding from the European Union Horizon 2020 research and innovation program, under the Marie Skłodowska-Curie grant agreement 859850 to the Insect Doctors project.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Data Availability Statement: Not applicable.

PART 1

The immunostimulatory role of an Enterococcus dominated gut microbiota in host protection against bacterial and fungal pathogens, in Galleria mellonella larvae Jennifer Upfold 1,2* , Agnès Rejasse 1 , Christina Nielsen-Leroux 1 , Annette Bruun-Jensen ,2 , Vincent Sanchis-Borja 1 1 Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France

Ethics statement

Ethical review and approval was not required for the study on animals in accordance with the local legislation and institutional requirements.

Author contributions

JU: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Visualization; Writingoriginal draft; and Writingreview and editing. AR: Conceptualization; Data curation; Methodology; and Writingreview and editing. CN-L: Conceptualization; Funding acquisition; Methodology; Project administration; Resources; Supervision; Visualization; and Writingreview and editing. AJ: Conceptualization; Funding acquisition; Methodology; Project administration; Resources; Upfold et al. 10.3389/finsc.2023.1260333 Frontiers in Insect Science frontiersin.org 13 Supervision; Visualization; and Writingreview and editing. VS-B: Conceptualization; Funding acquisition; Methodology; Project administration; Resources; Supervision; Validation; Visualization; and Writingreview and editing.

Conflict of interest

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.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/finsc.2023.1260333/ full#supplementary-material

Supplementary Material

Jennifer Upfold 1,2* , Agnès Rejasse 1 , Christina Nielsen-Leroux 1 , Annette Bruun-Jensen 2 , Vincent Sanchis-Borja

Microbiota: Bioinformatics

The following modifications were made to the default functions proposed by the DADA2 workflow package. After inspecting the quality profiles of the forward and reverse reads, trimming was performed at 250 and 200 reads for forward and reverse reads, respectively (where the quality score started to drop below 30). To learn the error rates, the number of samples, bases and reads was increased with the arguments 'nbases = 1e + 09' and randomize = TRUE.' The dereplication, sample inference and merging steps were then performed as proposed by the workflow. Lastly, chimeric sequences were removed. Taxonomic affiliations were performed to genera level, and species level where possible, using the SILVA reference database, version 132. To further identify to species level, a BLAST search of the resulting ASVs against the NCBI rRNA/ITS database https://www.ncbi.nlm.nih.gov/ was conducted. Further details of the BLAST search are recorded in supplementary Table S4.

Statistical analyses

Analyses were performed using the R software, using the 'phyloseq ' (v.1.26.1) and 'microbiome' packages (Lahti et al. 2012;[START_REF] Mcmurdie | Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data[END_REF]. Sequence taxonomy, sample metadata and ASVs were combined into a phyloseq object for further analysis. Samples were firstly plotted to identify any outliers in the sequencing depth, followed by rarefying the data using the "ggrare" function by using the "rarefy_even_depth" function and setting "set.seed(400)" with a sample size of 35000 (Suppl. Fig. S11). The relative abundance of the taxa was calculated at the genus (Suppl. Fig. S6) and species level (Fig. 3), as a percentage of total read count. Alpha diversity (α-diversity) community differences within the samples was estimated using the Simpson's index which considers both the number of species present as well as the relative abundances of each species (suppl. Fig. S5). To visualize the Beta (β-diversity) community differences between the samples, a PCoA using jaccard distances was used. Jaccard distances was used as it only analyses the presence/ absence whereas braycurtis distances accounts for the presence and absence of bacteria, as well as their relative abundance within a sample (Paddock et al., 2021). As the relative abundances are influence by introducing Btg into the community, jaccard distance is used. To further investigate the beta community on the microbiota community, all ASVs assigned to Bacillus thuringiensis were subsetted and discarded using the "subset_taxa" function on Phyloseq. Now the impact of Btg on the rest of the community can be assessed without the influence of the introduced Btg affecting the results. As there is low diversity in the community, just the presence of the introduced Btg separates the infected samples into their own group (Fig. S8A and8B). However, in order to analyze the beta community in the rest of the community, these Btg reads need to be subsetted and removed, and then the PCoA was reconstructed. A PERMANOVA test, based on 999 permutations and significance at <0.05 was carried out using the 'vegan ' (v.2.6.4) package with the adonis function (Suppl. Table S5).

Supplementary Figures:

Axenic verification:

Verification of the axenic status of the larvae is very necessary prior to further experiments. The verification is done using three techniques (i) plating larval homogenates or the dissected gut on BHiA medium; (ii) using PCR to detect amplification of the 16S rRNA gene; (iii) illumina sequencing. These three methods used in conjunction are important as the culture method is straightforward but is not always able to detect all microbes, such as those that are anaerobic or cannot be cultured, and PCR tests will detect all microbes however nucleic acid contamination can cause false positives and amplification thresholds do not completely exclude false negatives ( showing the spectrum of non-melanized to melanic cadaver morphs. All cadavers are mummified and stiff, unlike the softened cadavers from Btg infected larvae. Larvae were further placed on dampened filter paper to monitor hyphal growth. This difference in melanization during the fungal infection may be reflected in the results of the PO activity whereby the vmax values are highly variable in the 96h larvae. S10. Infected larvae (left) with melanized spots indicating the cuticle degradation by the Metarhizium robertsii, as compared to a healthy, uninfected larvae (right).

Supplementary Figure

10 Supplementary Figure S11. The rarefaction curves for all samples using a sample size of 35000 and 'rngseed = 400' to ensure all ASVs were detected. S3. Immune response genes investigated in the study

Gene Literature

Gallerimycin Is reportedly active against filamentous fungi but not against bacteria or yeast (1) however, its expression is strongly induced by a bacterial infection in G. mellonella, as observed by infection of Photorhabdus luminescens (Gram-negative bacterium) (2) as well as infections by Bacillus thuringiensis (3,4,5).

Gloverin

Gloverins from different lepidopteran species shows broad-spectrum activity against E. coli, Gram-positive bacteria, fungi and even some viruses (3,4,6,7,8,9,10) IMPI The insect metalloproteinase inhibitor, an enzyme that can degrade pathogen metalloproteinases helping to prevent or limit pathogen colonization in the host (11) Lysozyme Lysozymes, along with their digestive function in the gut, possess defense properties. It is one of the first lines of the humoral defense which creates a hostile environment for intruding microbes (11).

Conflicts of Interest:

The authors declare no conflict of interest.

Résumé substantiel en Français

Les insectes constituent l'un des groupes d'animaux les plus anciens et les plus diversifiés sur Terre. Ils assument une multitude de rôles différents et fournissent des services écosystémiques essentiels, tels que la pollinisation ou la décomposition des déchets organiques ; ils peuvent être aussi utilisés pour contrôler les populations de nuisibles. Les insectes sont également une source de protéines de haute qualité, ce qui, au cours de ces dernières années, a favorisé le développement d'une industrie