Varroa destructor from the Laboratory to the Field: Control, Biocontrol and IPM Perspectives-A Review.

Varroa destructor is a real challenger for beekeepers and scientists: fragile out of the hive, tenacious inside a bee colony. From all the research done on the topic, we have learned that a better understanding of this organism in its relationship with the bee but also for itself is necessary. Its biology relies mostly on semiochemicals for reproduction, nutrition, or orientation. Many treatments have been developed over the years based on hard or soft acaricides or even on biocontrol techniques. To date, no real sustainable solution exists to reduce the pressure of the mite without creating resistances or harming honeybees. Consequently, the development of alternative disruptive tools against the parasitic life cycle remains open. It requires the combination of both laboratory and field results through a holistic approach based on health biomarkers. Here, we advocate for a more integrative vision of V. destructor research, where in vitro and field studies are more systematically compared and compiled. Therefore, after a brief state-of-the-art about the mite's life cycle, we discuss what has been done and what can be done from the laboratory to the field against V. destructor through an integrative approach.

How to stand the heat? Post-stress nutrition and developmental stage determine insect response to a heat wave.

Organisms are increasingly confronted with intense and long-lasting heat waves. In insects, the effects of heat waves on individual performance can vary in magnitude both within (e.g. from one larval instar to another) and between life stages. However, the reasons underlying these stage-dependent effects are not fully understood. There are several lines of evidence suggesting that individual ability to withstand a heat stress depends on mechanisms based on nutrition and supporting energetically physiological stress responses. Hence, we tested the hypothesis that the efficiency of these food-based buffering mechanisms may vary between different larval instars of a phytophagous insect. Using larvae of the moth Lobesia botrana, we examined the importance of post-stress food quality in insect response to a non-lethal heat wave at two distinct larval instars. Three major conclusions were drawn from this work. First, heat waves induced an overall decline in larval performance (delayed development, depressed immunity). Second, food quality primarily mediated the insect's ability to respond to the heat stress: the reduction in performance following heat wave application was mostly restricted to individuals with access to low-quality food after the heat stress. Third, larval instars differed in their susceptibility to this combination of thermal and food stressors, but conclusions about the instar being the most vulnerable differed in a trait-specific manner. In a global warming context, this study may shed additional light on the combination of direct and indirect (through alteration of plant nutritional value) effects of rising temperatures on the ecology and the evolution of phytophagous insects.

Immune challenge reduces gut microbial diversity and triggers fertility-dependent gene expression changes in a social insect.

BACKGROUND: The gut microbiome can influence life history traits associated with host fitness such as fecundity and longevity. In most organisms, these two life history traits are traded-off, while they are positively linked in social insects. In ants, highly fecund queens can live for decades, while their non-reproducing workers exhibit much shorter lifespans. Yet, when fertility is induced in workers by death or removal of the queen, worker lifespan can increase. It is unclear how this positive link between fecundity and longevity is achieved and what role the gut microbiome and the immune system play in this. To gain insights into the molecular regulation of lifespan in social insects, we investigated fat body gene expression and gut microbiome composition in workers of the ant Temnothorax rugatulus in response to an experimental induction of fertility and an immune challenge. RESULTS: Fertile workers upregulated several molecular repair mechanisms, which could explain their extended lifespan. The immune challenge altered the expression of several thousand genes in the fat body, including many immune genes, and, interestingly, this transcriptomic response depended on worker fertility. For example, only fertile, immune-challenged workers upregulated genes involved in the synthesis of alpha-ketoglutarate, an immune system regulator, which extends the lifespan in Caenorhabditis elegans by down-regulating the TOR pathway and reducing oxidant production. Additionally, we observed a dramatic loss in bacterial diversity in the guts of the ants within a day of the immune challenge. Yet, bacterial density did not change, so that the gut microbiomes of many immune challenged workers consisted of only a single or a few bacterial strains. Moreover, the expression of immune genes was linked to the gut microbiome composition, suggesting that the ant host can regulate the microbiome in its gut. CONCLUSIONS: Immune system flare-ups can have negative consequence on gut microbiome diversity, pointing to a previously underrated cost of immunity. Moreover, our results provide important insights into shifts in the molecular regulation of fertility and longevity associated with insect sociality.

Offspring reverse transcriptome responses to maternal deprivation when reared with pathogens in an insect with facultative family life.

Offspring of species with facultative family life are able to live with and without parents (i.e. to adjust to extreme changes in their social environment). While these adjustments are well understood on a phenotypic level, their genetic underpinnings remain surprisingly understudied. Investigating gene expression changes in response to parental absence may elucidate the genetic constraints driving evolutionary transitions between solitary and family life. Here, we manipulated maternal presence to observe gene expression changes in the fat body of juvenile European earwigs, an insect with facultative family life. Because parents typically protect offspring against pathogens, expression changes were recorded in pathogen-free and pathogen-exposed environments. We found that manipulating maternal presence changed the expression of 154 genes, including several metabolism and growth-related genes, and that this change depended on pathogen presence. Specifically, localization and cell transporter genes were downregulated in maternal absence without pathogens but upregulated with pathogens. At least one immunity gene (pathogenesis-related protein 5) was affected by pathogen exposure regardless of maternal presence. Overall, our findings explicate how offspring adjust to parental deprivation on a molecular level and reveal that such adjustments heavily depend on pathogens in the environment. This emphasizes the central role of pathogens in family life evolution.

Age, sex, mating status, but not social isolation interact to shape basal immunity in a group-living insect.

Immunity is a crucial but costly trait. Individuals should therefore adjust their investment into immunity to their condition and infection risks, which are often determined by their age, sex, mating status and social environment. However, whether and how these four key factors can interact to shape basal immunity remains poorly understood. Here, we tested the simultaneous effects of these factors on hemocyte concentration and phenoloxidase activity in adults of the European earwig. We found that hemocyte concentration increased with age, and that this increase was stronger in males. We also found an age-dependent increase in phenoloxidase activity in males and virgin females, but not in mated females. However, the two immune parameters were independent of social isolation. Overall, our results reveal that a complex interplay between age, sex and mating status determines basal immunity and stress the importance of interactions in our understanding of immune investment.

Condition-Dependent Trade-Off Between Weapon Size and Immunity in Males of the European Earwig.

Investigating the expression of trade-offs between key life-history functions is central to our understanding of how these functions evolved and are maintained. However, detecting trade-offs can be challenging due to variation in resource availability, which masks trade-offs at the population level. Here, we investigated in the European earwig Forficula auricularia whether (1) weapon size trades off with three key immune parameters - hemocyte concentration, phenoloxidase and prophenoloxidase activity - and whether (2) expression and strength of these trade-offs depend on male body condition (body size) and/or change after an immune challenge. Our results partially confirmed condition dependent trade-offs between weapon size and immunity in male earwigs. Specifically, we found that after an immune challenge, weapon size trades off with hemocyte concentrations in low-condition, but not in good-condition males. Contrastingly, weapon size was independent of pre-challenge hemocyte concentration. We also found no trade-off between weapon size and phenoloxidase activity, independent of body condition and immune challenge. Overall, our study reveals that trade-offs with sexual traits may weaken or disappear in good-condition individuals. Given the importance of weapon size for male reproductive success, our results highlight how low-condition individuals may employ alternative life-history investment strategies to cope with resource limitation.

No evidence of an immune adjustment in response to a parasitoid threat in Lobesia botrana larvae.

Immune function is a key determinant of an organism's fitness, and natural insect populations are highly variable for this trait, mainly due to environmental heterogeneity and pathogen diversity. We previously reported a positive correlation between infection prevalence by parasitoids and host immunity in natural populations of the vineyard pest Lobesia botrana. Here, we tested whether this correlation reflects a plastic adjustment of host immunity in response to the local presence of parasites. To this end, we measured immunity of non-parasitized L. botrana larvae exposed, respectively, to one of the two most common species of parasitoids in vineyards, over 6days. Larvae were able to sense the parasitoid through visual, chemical, or mechanical cues, but contact larvae-parasitoid were excluded. Contrary to our hypothesis, we found that L. botrana larvae did not increase their immune defenses in the presence of parasitoids, despite their ability to sense a potential threat. Our results therefore suggest that the positive correlation between infection prevalence by parasitoids and L. botrana immunity among natural populations may result from micro-evolutionary changes resulting from long-term local selection pressures imposed by parasitoids in wild populations rather than plastic adjustments of immunity.

Age, pathogen exposure, but not maternal care shape offspring immunity in an insect with facultative family life.

BACKGROUND: To optimize their resistance against pathogen infection, individuals are expected to find the right balance between investing into the immune system and other life history traits. In vertebrates, several factors were shown to critically affect the direction of this balance, such as the developmental stage of an individual, its current risk of infection and/or its access to external help such as parental care. However, the independent and/or interactive effects of these factors on immunity remain poorly studied in insects. RESULTS: Here, we manipulated maternal presence and pathogen exposure in families of the European earwig Forficula auricularia to measure whether and how the survival rate and investment into two key immune parameters changed during offspring development. The pathogen was the entomopathogenic fungus Metarhizium brunneum and the immune parameters were hemocyte concentration and phenol/pro-phenoloxidase enzyme activity (total-PO). Our results surprisingly showed that maternal presence had no effect on offspring immunity, but reduced offspring survival. Pathogen exposure also lowered the survival of offspring during their early development. The concentration of hemocytes and the total-PO activity increased during development, to be eventually higher in adult females compared to adult males. Finally, pathogen exposure overall increased the concentration of hemocytes-but not the total-PO activity-in adults, while it had no effect on these measures in offspring. CONCLUSIONS: Our results show that, independent of their infection risk and developmental stage, maternal presence does not shape immune defense in young earwigs. This reveals that pathogen pressure is not a universal evolutionary driver of the emergence and maintenance of post-hatching maternal care in insects.

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes.

Food-mediated modulation of immunity in a phytophagous insect: An effect of nutrition rather than parasitic contamination.

Inherent to the cost of immunity, the immune system itself can exhibit tradeoffs between its arms. Phytophagous insects face a wide range of microbial and eukaryotic parasites, each activating different immune pathways that could compromise the activity of the others. Feeding larvae are primarily exposed to microbes, which growth is controlled by antibiotic secondary metabolites produced by the host plant. The resulting variation in abundance of microbes on plants is expected to differentially stimulate the insect antimicrobial immune defenses. Under the above tradeoff hypothesis, stimulation of the insect antimicrobial defenses is expected to compromise immune activity against eukaryote parasites. In the European grape berry moth, Eupoecilia ambiguella, immune effectors directed towards microbes are negatively correlated to those directed towards eukaryotic parasites among host plants. Here, we hypothesize this relationship is caused by a variable control of the microbial community among host plants by their antibiotic metabolites. To test this hypothesis, we first quantified antimicrobial activity in berries of several grape varieties. We then measured immune defenses of E. ambiguella larvae raised on artificial diets in which we mimicked levels of antimicrobial activity of grape berries using tetracycline to control the abundance of growing microbes. Another group of larvae was raised on artificial diets made of berry extracts only to control for the effect of nutrition. We found that controlling microbe abundance with tetracycline in diets did not explain variation in the immune function whereas the presence of berry extracts did. This suggests that variation in immune defenses of E. ambiguella among grape varieties is caused by nutritional difference among host plants rather than microbe abundance. Further study of the effects of berry compounds on larval immune parameters will be needed to explain the observed tradeoff among immune system components.

Immune benefits from alternative host plants could maintain polyphagy in a phytophagous insect.

The tritrophic interactions hypothesis, integrating bottom-up (plant-herbivore) and top-down (herbivore-natural enemies) effects, predicts that specialist herbivores should outcompete generalists. However, some phytophagous insects have generalist diets, suggesting that maintenance of a diverse diet may confer certain fitness advantages that outweigh diet specialization. In field conditions, the European grapevine moth, Lobesia botrana, feeds on diverse locally rare alternative host plants (AHPs) although grapevines are a highly abundant and predictable food source. The laboratory studies presented here show that survival, growth, and constitutive levels of immune defences (concentration of haemocytes and phenoloxidase activity) of L. botrana larvae were significantly enhanced when they were fed AHPs rather than grape. These results indicated a strong positive effect of AHPs on life history traits and immune defences of L. botrana. Such positive effects of AHPs should be advantageous to the moth under heavy selective pressure by natural enemies and, as a consequence, favour the maintenance of a broad diet preference in this species. We therefore believe that our results account for the role of immunity in the maintenance of polyphagy in phytophagous insects.

Defense strategies used by two sympatric vineyard moth pests.

Natural enemies including parasitoids are the major biological cause of mortality among phytophagous insects. In response to parasitism, these insects have evolved a set of defenses to protect themselves, including behavioral, morphological, physiological and immunological barriers. According to life history theory, resources are partitioned to various functions including defense, implying trade-offs among defense mechanisms. In this study we characterized the relative investment in behavioral, physical and immunological defense systems in two sympatric species of Tortricidae (Eupoecilia ambiguella, Lobesia botrana) which are important grapevine moth pests. We also estimated the parasitism by parasitoids in natural populations of both species, to infer the relative success of the investment strategies used by each moth. We demonstrated that larvae invest differently in defense systems according to the species. Relative to L. botrana, E. ambiguella larvae invested more into morphological defenses and less into behavioral defenses, and exhibited lower basal levels of immune defense but strongly responded to immune challenge. L. botrana larvae in a natural population were more heavily parasitized by various parasitoid species than E. ambiguella, suggesting that the efficacy of defense strategies against parasitoids is not equal among species. These results have implications for understanding of regulation in communities, and in the development of biological control strategies for these two grapevine pests.

Geographical variation in parasitism shapes larval immune function in a phytophagous insect.

Two of the central goals of immunoecology are to understand natural variation in the immune system among populations and to identify those selection pressures that shape immune traits. Maintenance of the immune system can be costly, and both food quality and parasitism selection pressure are factors potentially driving immunocompetence. In tritrophic interactions involving phytophagous insects, host plants, and natural enemies, the immunocompetence of phytophagous insects is constrained by selective forces from both the host plants and the natural enemies. Here, we assessed the roles of host plants and natural enemies as selective pressures on immune variation among natural populations of Lobesia botrana. Our results showed marked geographical variation in immune defenses and parasitism among different natural populations. Larval immune functions were dependent of the host plant quality and were positively correlated to parasitism, suggesting that parasitoids select for greater investment into immunity in moth. Furthermore, investment in immune defense was negatively correlated with body size, suggesting that it is metabolically expensive. The findings emphasize the roles of host plants and parasitoids as selective forces shaping host immune functions in natural conditions. We argue that kinds of study are central to understanding natural variations in immune functions, and the selective forces beyond.

Lobesia botrana larvae develop faster in the presence of parasitoids.

To combat parasitism hosts often rely on their immune system, which is the last line of defense. However, the immune system may not always be effective, and other non-immunological defenses might be favored to reduce the cost of parasite infection. Here we report that larvae of the moth Lobesia botrana can rapidly accelerate their development and reach maturity earlier in response to cues perceived at a distance from parasitoids. Such a phenotypically plastic life history shift, induced by the perception of deadly enemies in the environment, is likely to be an adaptive defensive strategy to prevent parasitoid attack, and has important implications in host-parasite dynamics.