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.

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

Previous surveys have shown that adult honeybee (Apis mellifera) workers harbor a characteristic gut microbiota that may play a significant role in bee health. For three major phylotypes within this microbiota, we have characterized distributions and abundances across the life cycle and among gut organs. These distinctive phylotypes, called Beta, Firm-5, and Gamma-1 (BFG), were assayed using quantitative PCR, fluorescent in situ hybridization (FISH) microscopy, and the experimental manipulation of inoculation routes within developing bees. Adult workers (9 to 30 days posteclosion) contained a large BFG microbiota with a characteristic distribution among gut organs. The crop and midgut were nearly devoid of these phylotypes, while the ileum and rectum together contained more than 95% of the total BFG microbiota. The ileum contained a stratified community in which the Beta and Gamma-1 phylotypes dominated, filling the longitudinal folds of this organ. Deep sequencing of 16S rRNA genes showed clear differences among communities in midgut, ileum, and rectum. In contrast with older workers, larvae and newly emerged workers contain few or no bacteria, and their major food source, bee bread, lacks most characteristic phylotypes. In experiments aimed at determining the route of inoculation, newly emerged workers (NEWs) sometimes acquired the typical phylotypes through contact with older workers, contact with the hive, and emergence from the brood cell; however, transmission was patchy in these assays. Our results outline a colonization pattern for the characteristic phylotypes through A. mellifera ontogeny. We propose the names “Candidatus Snodgrassella alvi” and “Candidatus Gilliamella apicola” for the Beta and Gamma-1 phylotypes, respectively.

Establishment of Characteristic Gut Bacteria during Development of the Honeybee Worker

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.

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

Studies of newly emerged Apis mellifera worker bees have demonstrated that their guts are colonized by a consistent core microbiota within several days of eclosure. We conducted experiments aimed at illuminating the transmission routes and spatiotemporal colonization dynamics of this microbiota. Experimental groups of newly emerged workers were maintained in cup cages and exposed to different potential transmission sources. Colonization patterns were evaluated using quantitative real-time PCR (qPCR) to assess community sizes and using deep sequencing of 16S rRNA gene amplicons to assess community composition. In addition, we monitored the establishment of the ileum and rectum communities within workers sampled over time from natural hive conditions. The study verified that workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. Typical communities, resembling those of workers within hives, were established in the presence of nurse workers or nurse worker fecal material, and atypical communities of noncore or highly skewed compositions were established when workers were exposed only to oral trophallaxis or hive components (comb, honey, bee bread). The core species of Gram-negative bacteria, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara, were dependent on the presence of nurses or hindgut material, whereas some Gram-positive species were more often transferred through exposure to hive components. These results indicate aspects of the colony life cycle and behavior that are key to the propagation of the characteristic honey bee gut microbiota.

Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera.

Gut microbiomes play crucial roles in animal health, and shifts in the gut microbial community structure can have detrimental impacts on hosts. Studies with vertebrate models and human subjects suggest that antibiotic treatments greatly perturb the native gut community, thereby facilitating proliferation of pathogens. In fact, persistent infections following antibiotic treatment are a major medical issue. In apiculture, antibiotics are frequently used to prevent bacterial infections of larval bees, but the impact of antibiotic-induced dysbiosis (microbial imbalance) on bee health and susceptibility to disease has not been fully elucidated. Here, we evaluated the effects of antibiotic exposure on the size and composition of honeybee gut communities. We monitored the survivorship of bees following antibiotic treatment in order to determine if dysbiosis of the gut microbiome impacts honeybee health, and we performed experiments to determine whether antibiotic exposure increases susceptibility to infection by opportunistic pathogens. Our results show that antibiotic treatment can have persistent effects on both the size and composition of the honeybee gut microbiome. Antibiotic exposure resulted in decreased survivorship, both in the hive and in laboratory experiments in which bees were exposed to opportunistic bacterial pathogens. Together, these results suggest that dysbiosis resulting from antibiotic exposure affects bee health, in part due to increased susceptibility to ubiquitous opportunistic pathogens. Not only do our results highlight the importance of the gut microbiome in honeybee health, but they also provide insights into how antibiotic treatment affects microbial communities and host health.

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Antibiotic exposure perturbs the gut microbiota and elevates mortality in honeybees.

(2009). Bee cups: single-use cages for honey bee experiments. Journal of Apicultural Research: Vol. 48, No. 4, pp. 300-302.

Virginia Williams

Papers

Bee cups:single-use cages for honey bee experiments