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.

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.

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.

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.

Ecology of Varroa destructor, the Major Ectoparasite of the Western Honey Bee, Apis mellifera.

Varroa destructor is the most important ectoparasite of Apis mellifera. This review addresses the interactions between the varroa mite, its environment, and the honey bee host, mediated by an impressive number of cues and signals, including semiochemicals regulating crucial steps of the mite's life cycle. Although mechanical stimuli, temperature, and humidity play an important role, chemical communication is the most important channel. Kairomones are used at all stages of the mite's life cycle, and the exploitation of bees' brood pheromones is particularly significant given these compounds function as primer and releaser signals that regulate the social organization of the honey bee colony. V. destructor is a major problem for apiculture, and the search for novel control methods is an essential task for researchers. A detailed study of the ecological interactions of V. destructor is a prerequisite for creating strategies to sustainably manage the parasite.

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.

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.

Assessing lethal and sublethal effects of pesticides on honey bees in a multifactorial context.

The registration of novel pesticides that are subsequently banned because of their unexpected negative effects on non-target species can have a huge environmental impact. Therefore, the pre-emptive evaluation of the potential effects of new compounds is essential. To this aim both lethal and sublethal effects should be assessed in a realistic scenario including the other stressors that can interact with pesticides. However, laboratory studies addressing such interactive effects are rare, while standardized laboratory-based protocols focus on lethal effects and not on sub-lethal effects. We propose to assess both lethal and sublethal effects in a multifactorial context including the other stressors affecting the non-target species. We tested this approach by studying the impact on honey bees of the insecticide sulfoxaflor in combination with a common parasite, a sub-optimal temperature and food deprivation. We studied the survival and the transcriptome of honey bees, to assess both the lethal and the potential sublethal effects of the insecticide, respectively. With this method we show that a field realistic concentration of sulfoxaflor in food does not affect the survival of honey bees; however, the significant impact on some key genes indicates that sublethal effects are possible in a realistically complex scenario. Moreover, our results demonstrate the feasibility and reliability of a novel approach to hazard assessment considering the interactive effects of pesticides. We anticipate our approach to be a starting point for a paradigm shift in toxicology: from an unifactorial, mortality-centered assessment to a multifactorial, comprehensive approach. This is something of the utmost importance to preserve pollination, thus contributing to the sustainability of our food production system.

Pesticide immunotoxicity on insects - Are agroecosystems at risk?

Recent years have witnessed heightened scrutiny of the non-target sublethal effects of pesticides on behavioural and physiological traits of insects. Traditionally, attention has focused on investigating pesticides' primary modes of action, often overlooking the potential secondary mechanisms. This review brings forth the nuanced impacts of sublethal pesticide exposure on the immune system of target and non-target insect species. Pesticides, such as for example neonicotinoids, suppress immune response, while others, like certain organophosphates and some insect growth regulators (IGRs), appear to bolster immunocompetence under certain circumstances. Beyond their individual impacts, the synergic effects of pesticide mixtures on insect immunity are garnering increasing interest. This review thus summarizes recent advances in the immunomodulatory effects of pesticides, detailing both mechanisms and consequences of such interactions. The implications of these effects for ecosystem preservation and viability of beneficial organisms, such as pollinators and natural enemies of pests, are discussed. The review also considers further research directions on pesticide secondary modes of action and explores potential implications for integrated pest management (IPM) programs, as several model organisms studied are crop pest species. While current data provide an expansive overview of how insect innate immunity is modulated, concrete endpoints remain elusive requiring further research into pesticide secondary modes of actions.

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.

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.

Emerging threats and opportunities to managed bee species in European agricultural systems: a horizon scan.

Managed bee species provide essential pollination services that contribute to food security worldwide. However, managed bees face a diverse array of threats and anticipating these, and potential opportunities to reduce risks, is essential for the sustainable management of pollination services. We conducted a horizon scanning exercise with 20 experts from across Europe to identify emerging threats and opportunities for managed bees in European agricultural systems. An initial 63 issues were identified, and this was shortlisted to 21 issues through the horizon scanning process. These ranged from local landscape-level management to geopolitical issues on a continental and global scale across seven broad themes-Pesticides & pollutants, Technology, Management practices, Predators & parasites, Environmental stressors, Crop modification, and Political & trade influences. While we conducted this horizon scan within a European context, the opportunities and threats identified will likely be relevant to other regions. A renewed research and policy focus, especially on the highest-ranking issues, is required to maximise the value of these opportunities and mitigate threats to maintain sustainable and healthy managed bee pollinators within agricultural systems.

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.

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.

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.

Repellent effect of sweet basil compounds on Ixodes ricinus ticks.

Diseases transmitted by ticks are causing increasing concern in Europe and all around the world. Repellents are an effective measure for reducing the risk of tick bite; products based on natural compounds represent an interesting alternative to common synthetic repellents. In this study the repellency of sweet basil (Ocimum basilicum L.) was tested against the tick Ixodes ricinus L., by using a laboratory bioassay. A bioassay-assisted fractionation allowed the identification of a compound involved in the biological activity. Eugenol appeared to be as repellent as DEET at two tested doses. Linalool, which was identified in the active fraction too, failed to give any response. Repellency of eugenol was proved also in the presence of human skin odour using a convenient and practical bioassay.

Honey Bee Antiviral Immune Barriers as Affected by Multiple Stress Factors: A Novel Paradigm to Interpret Colony Health Decline and Collapse.

Any attempt to outline a logical framework in which to interpret the honey bee health decline and its contribution to elevated colony losses should recognize the importance of the multifactorial nature of the responsible syndrome and provide a functional model as a basis for defining and testing working hypotheses. We propose that covert infections by deformed wing virus (DWV) represent a sword of Damocles permanently threatening the survival of honey bee colonies and suggest that any factor affecting the honey bee’s antiviral defenses can turn this pathogen into a killer. Here we discuss the available experimental evidence in the framework of a model based on honey bee immune competence as affected by multiple stress factors that is proposed as a conceptual tool for analyzing bee mortality and its underlying mechanisms.