Varroa destructor exacerbates the negative effect of cold contributing to honey bee mortality.

Several concurrent stress factors can impact honey bee health and colony stability. Although a satisfactory knowledge of the effect of almost every single factor is now available, a mechanistic understanding of the many possible interactions between stressors is still largely lacking. Here we studied, both at the individual and colony level, how honey bees are affected by concurrent exposure to cold and parasitic infection. We found that the parasitic mite Varroa destructor, further than increasing the natural mortality of bees, can induce an anorexia that reduces their capacity to thermoregulate and thus react to sub-optimal temperatures. This, in turn, could affect the collective response of the bee colony to cold temperatures aggravating the effect already observed at the individual level. These results highlight the important role that biotic factors can have by shaping the response to abiotic factors and the strategic need to consider the potential interactions between stressors at all levels of the biological organization to better understand their impact.

Age-related response to mite parasitization and viral infection in the honey bee suggests a trade-off between growth and immunity.

Host age at parasites' exposure is often neglected in studies on host-parasite interactions despite the important implications for epidemiology. Here we compared the impact of the parasitic mite Varroa destructor, and the associated pathogenic virus DWV on different life stages of their host, the western honey bee Apis mellifera. The pre-imaginal stages of the honey bee proved to be more susceptible to mite parasitization and viral infection than adults. The higher viral load in mite-infested bees and DWV genotype do not appear to be the drivers of the observed difference which, instead, seems to be related to the immune-competence of the host. These results support the existence of a trade-off between immunity and growth, making the pupa, which is involved in the highly energy-demanding process of metamorphosis, more susceptible to parasites and pathogens. This may have important implications for the evolution of the parasite's virulence and in turn for honey bee health. Our results highlight the important role of host's age and life stage at exposure in epidemiological modelling. Furthermore, our study could unravel new aspects of the complex honey bee-Varroa relationship to be addressed for a sustainable management of this parasite.

A deeper understanding of system interactions can explain contradictory field results on pesticide impact on honey bees.

While there is widespread concern regarding the impact of pesticides on honey bees, well-replicated field experiments, to date, have failed to provide clear insights on pesticide effects. Here, we adopt a systems biology approach to gain insights into the web of interactions amongst the factors influencing honey bee health. We put the focus on the properties of the system that depend upon its architecture and not on the strength, often unknown, of each single interaction. Then we test in vivo, on caged honey bees, the predictions derived from this modelling analysis. We show that the impact of toxic compounds on honey bee health can be shaped by the concurrent stressors affecting bees. We demonstrate that the immune-suppressive capacity of the widespread pathogen of bees, deformed wing virus, can introduce a critical positive feed-back loop in the system causing bistability, i.e., two stable equilibria. Therefore, honey bees under similar initial conditions can experience different consequences when exposed to the same stressor, including prolonged survival or premature death. The latter can generate an increased vulnerability of the hive to dwindling and collapse. Our conclusions reconcile contrasting field-testing outcomes and have important implications for the application of field studies to complex systems.

Epidemiology of a major honey bee pathogen, deformed wing virus: potential worldwide replacement of genotype A by genotype B.

The western honey bee (Apis mellifera) is of major economic and ecological importance, with elevated rates of colony losses in temperate regions over the last two decades thought to be largely caused by the exotic ectoparasitic mite Varroa destructor and deformed wing virus (DWV), which the mite transmits. DWV currently exists as two main genotypes: the formerly widespread DWV-A and the more recently described and rapidly expanding DWV-B. It is an excellent system to understand viral evolution and the replacement of one viral variant by another. Here we synthesise published results on the distribution and prevalence of DWV-A and -B over the period 2008-2021 and present novel data for Germany, Italy and the UK to suggest that (i) DWV-B has rapidly expanded worldwide since its first description in 2004 and (ii) that it is potentially replacing DWV-A. Both genotypes are also found in wild bee species. Based on a simple mathematical model, we suggest that interference between viral genotypes when co-infecting the same host is key to understanding their epidemiology. We finally discuss the consequences of genotype replacement for beekeeping and for wild pollinator species.

The Beneficial Effect of Pollen on Varroa Infested Bees Depends on Its Influence on Behavioral Maturation Genes.

Honey bees collect nectar and pollen to fulfill their nutritional demands. In particular, pollen can influence longevity, the development of hypopharyngeal glands, and immune-competence of bees. Pollen can also mitigate the deleterious effects caused by the parasitic mite Varroa destructor and related deformed wing virus (DWV) infections. It has been shown that V. destructor accelerates the physiological and behavioral maturation of honey bees by influencing the interaction between two core physiological factors, Vitellogenin and juvenile hormone. In this study, we test the hypothesis that the beneficial effects of pollen on Varroa-infested bees are related to the hormonal control underpinning behavioral maturation. By analyzing the expression of genes associated to behavioral maturation in pollen-fed mite-infested bees, we show that treatment with pollen increases the lifespan of mite-infested bees by reversing the faster maturation induced by the parasite at the gene expression level. As expected, from the different immune-competence of nurse and forager bees, the lifespan extension triggered by pollen is also correlated with a positive influence of antimicrobial peptide gene expression and DWV load, further reinforcing the beneficial effect of pollen. This study lay the groundwork for future analyses of the underlying evolutionary processes and applications to improve bee health.

Honeybees use propolis as a natural pesticide against their major ectoparasite.

Honeybees use propolis collected from plants for coating the inner walls of their nest. This substance is also used as a natural antibiotic against microbial pathogens, similarly to many other animals exploiting natural products for self-medication. We carried out chemical analyses and laboratory bioassays to test if honeybees use propolis for social medication against their major ectoparasite: Varroa destructor. We found that propolis is applied to brood cells where it can affect the reproducing parasites, with a positive effect on honeybees and a potential impact on Varroa population. We conclude that propolis can be regarded as a natural pesticide used by the honeybee to limit a dangerous parasite. These findings significantly enlarge our understanding of behavioural immunity in animals and may have important implications for the management of the most important threat to honeybees worldwide.

Behavioural Evidence and Chemical Identification of a Female Sex Pheromone in Anagrus atomus (Hymenoptera: Mymaridae).

Anagrus atomus (L.) is an egg parasitoid involved in the biological control of Empoasca vitis (Göthe) in vineyards. Sex pheromones play a crucial role in mate finding for several parasitoid species and could be used for monitoring under field conditions. We carried out laboratory and field studies aimed at assessing the existence and identity of a possible A. atomus sex pheromone. We found that males were significantly attracted by virgin females independent of age. Males were not attracted to individuals of the same sex, but they were attracted by a crude extract from an unmated female and its polar fraction. Eugenol (4-allyl-2-methoxyphenol) was identified as the attractive substance and proved to be attractive not only in the olfactometer but also in another laboratory bioassay and under field conditions. Attraction of males, but not females, confirms that this is not an aggregation pheromone. This is the first sex-pheromone component identified in Mymaridae, however more compounds could be involved in the mating behaviour of A. atomus. The utility of a sex pheromone in A. atomus is discussed in the context of fitness returns.

Neonicotinoid Clothianidin reduces honey bee immune response and contributes to Varroa mite proliferation.

The neonicotinoid Clothianidin has a negative impact on NF-κB signaling and on immune responses controlled by this transcription factor, which can boost the proliferation of honey bee parasites and pathogens. This effect has been well documented for the replication of deformed wing virus (DWV) induced by Clothianidin in honey bees bearing an asymptomatic infection. Here, we conduct infestation experiments of treated bees to show that the immune-suppression exerted by Clothianidin is associated with an enhanced fertility of the parasitic mite Varroa destructor, as a possible consequence of a higher feeding efficiency. A conceptual model is proposed to describe the synergistic interactions among different stress agents acting on honey bees.

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model.

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses.

Elucidating the mechanisms underlying the beneficial health effects of dietary pollen on honey bees (Apis mellifera) infested by Varroa mite ectoparasites.

Parasites and pathogens of the honey bee (Apis mellifera) are key factors underlying colony losses, which are threatening the beekeeping industry and agriculture as a whole. To control the spread and development of pathogen infections within the colony, honey bees use plant resins with antibiotic activity, but little is known about the properties of other substances, that are mainly used as a foodstuff, for controlling possible diseases both at the individual and colony level. In this study, we tested the hypothesis that pollen is beneficial for honey bees challenged with the parasitic mite Varroa destructor associated to the Deformed Wing Virus. First, we studied the effects of pollen on the survival of infested bees, under laboratory and field conditions, and observed that a pollen rich diet can compensate the deleterious effects of mite parasitization. Subsequently, we characterized the pollen compounds responsible for the observed positive effects. Finally, based on the results of a transcriptomic analysis of parasitized bees fed with pollen or not, we developed a comprehensive framework for interpreting the observed effects of pollen on honey bee health, which incorporates the possible effects on cuticle integrity, energetic metabolism and immune response.

Transcriptional signatures of parasitization and markers of colony decline in Varroa-infested honey bees (Apis mellifera).

Extensive annual losses of honey bee colonies (Apis mellifera L.) reported in the northern hemisphere represent a global problem for agriculture and biodiversity. The parasitic mite Varroa destructor, in association with deformed wing virus (DWV), plays a key role in this phenomenon, but the underlying mechanisms are still unclear. To elucidate these mechanisms, we analyzed the gene expression profile of uninfested and mite infested bees, under laboratory and field conditions, highlighting the effects of parasitization on the bee's transcriptome under a variety of conditions and scenarios. Parasitization was significantly correlated with higher viral loads. Honey bees exposed to mite infestation exhibited an altered expression of genes related to stress response, immunity, nervous system function, metabolism and behavioural maturation. Additionally, mite infested young bees showed a gene expression profile resembling that of forager bees. To identify potential molecular markers of colony decline, the expression of genes that were commonly regulated across the experiments were subsequently assessed in colonies experiencing increasing mite infestation levels. These studies suggest that PGRP-2, hymenoptaecin, a glucan recognition protein, UNC93 and a p450 cytocrome maybe suitable general biomarkers of Varroa-induced colony decline. Furthermore, the reliability of vitellogenin, a yolk protein previously identified as a good marker of colony survival, was confirmed here.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens.

BACKGROUND: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. RESULTS: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. CONCLUSIONS: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

A mutualistic symbiosis between a parasitic mite and a pathogenic virus undermines honey bee immunity and health.

Honey bee colony losses are triggered by interacting stress factors consistently associated with high loads of parasites and/or pathogens. A wealth of biotic and abiotic stressors are involved in the induction of this complex multifactorial syndrome, with the parasitic mite Varroa destructor and the associated deformed wing virus (DWV) apparently playing key roles. The mechanistic basis underpinning this association and the evolutionary implications remain largely obscure. Here we narrow this research gap by demonstrating that DWV, vectored by the Varroa mite, adversely affects humoral and cellular immune responses by interfering with NF-κB signaling. This immunosuppressive effect of the viral pathogen enhances reproduction of the parasitic mite. Our experimental data uncover an unrecognized mutualistic symbiosis between Varroa and DWV, which perpetuates a loop of reciprocal stimulation with escalating negative effects on honey bee immunity and health. These results largely account for the remarkable importance of this mite-virus interaction in the induction of honey bee colony losses. The discovery of this mutualistic association and the elucidation of the underlying regulatory mechanisms sets the stage for a more insightful analysis of how synergistic stress factors contribute to colony collapse, and for the development of new strategies to alleviate this problem.

Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

How does the mite Varroa destructor kill the honeybee Apis mellifera? Alteration of cuticular hydrcarbons and water loss in infested honeybees.

Several factors threaten the health of honeybees; among them the parasitic mite Varroa destructor and the Deformed Wing Virus play a major role. Recently, the dangerous interplay between the mite and the virus was studied in detail and the transition, triggered by mite feeding, from a benign covert infection to a devastating viral outbreak, characterized by an intense viral replication, associated with some characteristic symptoms, was described. In order to gain insight into the events preceding that crucial transition we carried out standardized lab experiments aiming at studying the effects of parasitization in asymptomatic bees to establish a relationship between such effects and bee mortality. It appears that parasitization alters the capacity of the honeybee to regulate water exchange; this, in turn, has severe effects on bee survival. These results are discussed in light of possible novel strategies aiming at mitigating the impact of the parasite on honeybee health.

Synergistic parasite-pathogen interactions mediated by host immunity can drive the collapse of honeybee colonies.

The health of the honeybee and, indirectly, global crop production are threatened by several biotic and abiotic factors, which play a poorly defined role in the induction of widespread colony losses. Recent descriptive studies suggest that colony losses are often related to the interaction between pathogens and other stress factors, including parasites. Through an integrated analysis of the population and molecular changes associated with the collapse of honeybee colonies infested by the parasitic mite Varroa destructor, we show that this parasite can de-stabilise the within-host dynamics of Deformed wing virus (DWV), transforming a cryptic and vertically transmitted virus into a rapidly replicating killer, which attains lethal levels late in the season. The de-stabilisation of DWV infection is associated with an immunosuppression syndrome, characterized by a strong down-regulation of the transcription factor NF-κB. The centrality of NF-κB in host responses to a range of environmental challenges suggests that this transcription factor can act as a common currency underlying colony collapse that may be triggered by different causes. Our results offer an integrated account for the multifactorial origin of honeybee losses and a new framework for assessing, and possibly mitigating, the impact of environmental challenges on honeybee health.

Octanoic acid confers to royal jelly varroa-repellent properties.

The mite Varroa destructor Anderson & Trueman is a parasite of the honeybee Apis mellifera L. and represents a major threat for apiculture in the Western world. Reproduction takes place only inside bee brood cells that are invaded just before sealing; drone cells are preferred over worker cells, whereas queen cells are not normally invaded. Lower incidence of mites in queen cells is at least partly due to the deterrent activity of royal jelly. In this study, the repellent properties of royal jelly were investigated using a lab bioassay. Chemical analysis showed that octanoic acid is a major volatile component of royal jelly; by contrast, the concentration is much lower in drone and worker larval food. Bioassays, carried out under lab conditions, demonstrated that octanoic acid is repellent to the mite. Field studies in bee colonies confirmed that the compound may interfere with the process of cell invasion by the mite.