Bee Hosts Research Articles

Co-evolution and Gene Transfers Drive Speciation Patterns in Host-Associated Bacteria.

Microbial communities that maintain symbiotic relationships with animals evolve by adapting to the specific environmental niche provided by their host, yet understanding their patterns of speciation remains challenging. Whether bacterial speciation occurs primarily through allopatric or sympatric processes remains an open question. In addition, patterns of DNA transfers, which are pervasive in bacteria, are more constrained in a closed host-gut system. Eusocial bees have co-evolved with their specialized microbiota for over 85 million years, constituting a simple and valuable system to study the complex dynamics of host-associated microbial interactions. Here we studied the patterns of speciation and evolution of seven specialized gut bacteria from three clades of eusocial bee species: western honey bees, eastern honey bees and bumblebees. We conducted genomic analyses to infer species delineation relative to the patterns of homologous recombination (HR), and horizontal gene transfer (HGT). The studied bacteria presented various modes of evolution and speciation relative to their hosts, but some trends were consistent across all of them. We observed a clear interruption of homologous recombination between bacteria inhabiting different bee hosts, which is consistent with a mechanism of allopatric speciation, but we also identified interruptions of homologous recombination within hosts, suggesting recent or ongoing sympatric speciation. In contrast to HR, we observed that HGT events were not constrained by species borders. Overall, our findings show that in host-associated bacterial populations, patterns of HR and HGT have different impacts on speciation patterns, which are driven by both allopatric and sympatric speciation processes.

The effect of lambda-cyhalothrin nanocapsules on the gut microbial communities and immune response of the bee elucidates the potential environmental impact of emerging nanopesticides

Emerging nanopesticides are gradually gaining widespread application in agriculture due to their excellent properties, but their potential risks to pollinating insects are not fully understood. In this study, lambda-cyhalothrin nanocapsules (LC-NCs) were constructed by electrostatic self-assembly method with iron mineralization optimization, and their effects on bee gut microbial communities and host immune-related factors were investigated. Microbiome sequencing revealed that LC-NCs increase the diversity of gut microbial communities and reduce the complexity of network features, disrupting the overall structure of the microbial communities. In addition, LC-NCs also had systemic effects on the immune response of bees, including increased activity of SOD and CAT enzymes and expression of their genes, as well as downregulation of Defensin1. Furthermore, we noticed that the immune system of the host was activated simultaneously with a rise in the abundance of beneficial bacteria in the gut. Our research emphasizes the importance of both the host and gut microbiota of holobiont in revealing the potential risks of LC-NCs to environmental indicators of honey bees, and provides references for exploring the interactions between host-microbiota systems under exogenous stress. At the same time, we hope that more research can focus on the potential impacts of nanopesticides on the ecological environment.

Host specificity and cophylogeny in the “animal-gut bacteria-phage” tripartite system

Cophylogeny has been identified between gut bacteria and their animal host and is highly relevant to host health, but little research has extended to gut bacteriophages. Here we use bee model to investigate host specificity and cophylogeny in the “animal-gut bacteria-phage” tripartite system. Through metagenomic sequencing upon different bee species, the gut phageome revealed a more variable composition than the gut bacteriome. Nevertheless, the bacteriome and the phageome showed a significant association of their dissimilarity matrices, indicating a reciprocal interaction between the two kinds of communities. Most of the gut phages were host generalist at the viral cluster level but host specialist at the viral OTU level. While the dominant gut bacteria Gilliamella and Snodgrassella exhibited matched phylogeny with bee hosts, most of their phages showed a diminished level of cophylogeny. The evolutionary rates of the bee, the gut bacteria and the gut phages showed a remarkably increasing trend, including synonymous and non-synonymous substitution and gene content variation. For all of the three codiversified tripartite members, however, their genes under positive selection and genes involving gain/loss during evolution simultaneously enriched the functions into metabolism of nutrients, therefore highlighting the tripartite coevolution that results in an enhanced ecological fitness for the whole holobiont.

Lactobacillus Firm-5-derived succinate prevents honeybees from having diabetes-like symptoms

The gut microbiome plays an important role in honeybee hormonal regulation and growth, but the underlying mechanisms are poorly understood. Here, we showed that the depletion of gut bacteria resulted in reduced expression of insulin-like peptide gene (ilp) in the head, accompanied by metabolic syndromes resembling those of Type 1 diabetes in humans: hyperglycemia, impaired lipid storage, and decreased metabolism. These symptoms were alleviated by gut bacterial inoculation. Gut metabolite profiling revealed that succinate, produced by Lactobacillus Firm-5, played deterministic roles in activating ilp gene expression and in regulating metabolism in honeybees. Notably, we demonstrated that succinate modulates host ilp gene expression through stimulating gut gluconeogenesis, a mechanism resembling that of humans. This study presents evidence for the role of gut metabolite in modulating host metabolism and contributes to the understanding of the interactions between gut microbiome and bee hosts.

Heteromorphic deutonymph sex of the solitary bee parasite, Chaetodactylus krombeini (Baker) (Astigmata: Chaetodactylidae)

ABSTRACT Chaetodactylus krombeini (Acariformes: Astigmata: Chaetodactylidae) mites are kleptoparasites of solitary bees in the genus Osmia and depend on bee hosts for harbourage, nutrition, and dispersal, with infestations increasing morbidity and larval mortality. The life history traits of C. krombeini are unclear, complicating the control efforts of mites in managed bee nests. Mites in the family Chaetodactylidae have a facultative life stage, the heteromorphic deutonymph, used for either dispersal or dormancy when food resources are sparse. The heteromorphic deutonymph life stage is hypothesized to consist only of females, requiring arrhenotokous parthenogenesis to reproduce. This study examined this hypothesis by determining the sex of the adults that developed from each deutonymph morph. Phoretic deutonymphs were isolated after overwintering either with or without a bee host; inert deutonymphs were isolated from field collections from various locations. Mites were individually observed for 14 days, and development was recorded. Mites that reached adulthood were sexed. After 14 days, 32–38% of heteromorphic deutonymphs that reached adulthood were male. These results disprove the previous hypothesis that heteromorphic deutonymphs are exclusively female. The survival and reproductive strategies of C. krombeini are now in question, specifically the role of true arrhenotoky in founding new mite populations.

Deformed wing virus genotypes A and B do not elicit immunologically different responses in naïve honey bee hosts.

Iflavirus aladeformis (Picornavirales: Iflaviridae), commonly known as deformed wing virus(DWV), in association with Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae), is a leading factor associated with honey bee (Apis mellifera L. [Hymenoptera: Apidae]) deaths. The virus and mite have a near global distribution, making it difficult to separate the effect of one from the other. The prevalence of two main DWV genotypes (DWV-A and DWV-B) has changed over time, leading to the possibility that the two strains elicit a different immune response by the host. Here, we use a honey bee population naïve to both the mite and the virus to investigate if honey bees show a different immunological response to DWV genotypes. We examined the expression of 19 immune genes by reverse transcription quantitative PCR (RT-qPCR) and analysed small RNA after experimental injection with DWV-A and DWV-B. We found no evidence that DWV-A and DWV-B elicit different immune responses in honey bees. RNA interference genes were up-regulated during DWV infection, and small interfering RNA (siRNA) responses were proportional to viral loads yet did not inhibit DWV accumulation. The siRNA response towards DWV was weaker than the response to another honey bee pathogen, Triatovirus nigereginacellulae (Picornavirales: Dicistroviridae; black queen cell virus), suggesting that DWV is comparatively better at evading host antiviral defences. There was no evidence for the production of virus-derived Piwi-interacting RNAs (piRNAs) in response to DWV. In contrast to previous studies, and in the absence of V. destructor, we found no evidence that DWV has an immunosuppressive effect. Overall, our results advance our understanding of the immunological effect that DWV in isolation elicits in honey bees.

Land-use change alters specialist bee diet and drives body size declines

ContextLand-use change can cause decreases in plant abundance and richness and the replacement of wild plants with domesticated plants. Changes in plant community composition disrupt mutualistic plant-pollinator interactions with ecological consequences for plants and pollinators, and especially for specialists that rely on certain plants.ObjectivesWe assessed the effects of land-use change and subsequent shifts in plant communities on resource collection and body size for a bee pollinator, Habropoda laboriosa, a purported specialist of blueberries and related Ericaceae plants.MethodsWe collected Habropoda laboriosa across a gradient of land use in north-central Florida including agricultural, natural, and urban habitats. We assessed landcover and floral community composition at each site and related these to bee body size (intertegular distance) and resource use (proportion host plant pollen and pollen diversity collected).ResultsHost plant pollen collection and bee body size generally responded similarly to the landscape, both increasing with habitats containing host plants (blueberry farms and natural habitat) but decreasing with urban development. However, host pollen collection and bee body size responded in opposite ways to overall cropland in the landscape, with cropland negatively affecting body size despite positively affecting host pollen collection, indicating other factors associated with cropland might drive declines in bee body size.ConclusionsLand-use change can adversely affect bee diet and body size, and changes in diet likely contribute to changes in body size over time. Specialists are particularly at risk for negative impacts of land-use change due to their inability to shift plant hosts.

Identification of the mutual gliding locus as a factor for gut colonization in non-native bee hosts using the ARTP mutagenesis

BackgroundThe gut microbiota and their hosts profoundly affect each other’s physiology and evolution. Identifying host-selected traits is crucial to understanding the processes that govern the evolving interactions between animals and symbiotic microbes. Current experimental approaches mainly focus on the model bacteria, like hypermutating Escherichia coli or the evolutionary changes of wild stains by host transmissions. A method called atmospheric and room temperature plasma (ARTP) may overcome the bottleneck of low spontaneous mutation rates while maintaining mild conditions for the gut bacteria.ResultsWe established an experimental symbiotic system with gnotobiotic bee models to unravel the molecular mechanisms promoting host colonization. By in vivo serial passage, we tracked the genetic changes of ARTP-treated Snodgrassella strains from Bombus terrestris in the non-native honeybee host. We observed that passaged isolates showing genetic changes in the mutual gliding locus have a competitive advantage in the non-native host. Specifically, alleles in the orphan mglB, the GTPase activating protein, promoted colonization potentially by altering the type IV pili-dependent motility of the cells. Finally, competition assays confirmed that the mutations out-competed the ancestral strain in the non-native honeybee gut but not in the native host.ConclusionsUsing the ARTP mutagenesis to generate a mutation library of gut symbionts, we explored the potential genetic mechanisms for improved gut colonization in non-native hosts. Our findings demonstrate the implication of the cell mutual-gliding motility in host association and provide an experimental system for future study on host-microbe interactions.ArN8Xjm3N6Dubzus4DCSQmVideo

The Braconid Syntretomorpha szaboi Papp Is Posing a Great Threat to the Eastern Honeybee, Apis cerana.

The expansion of pathogen distribution may result in a new threat to the host. The braconid Syntretomorpha szaboi Papp is an obligate parasite that targets Apis cerana, the Eastern honeybee, engaging in endoparasitism by ovipositing eggs inside the host bee. Although S. szaboi has been documented in India and in various regions across China, its epidemiological data are notably lacking. In this study, we summarized the distribution of S. szaboi based on the available literature and described the symptoms of infested honeybee workers. We also investigated the infestation rate in 36 apiaries in Zhejiang Province, China, after a new occurrence of the parasite was reported in these regions in 2020. A rapid increase in infestation rate was found from the year 2021 to 2022, reaching 53.88% at the colony level of the sampled colonies in the Jinhua and Wenzhou apiaries. The infestation rate at an individual level in positive colonies reached an average of 26%. A monthly survey showed high seasonal variation in S. szaboi infestation, with the peak occurring from May to August. These results suggest that S. szaboi poses a great threat to A. cerana. Further research is needed to elucidate its epidemiology and pathology and to develop disease prevention and control strategies.

Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization

Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and the fitness of overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.

Phylogenomic analysis of Stylops reveals the evolutionary history of a Holarctic Strepsiptera radiation parasitizing wild bees

Holarctic Stylops is the largest genus of the enigmatic insect order Strepsiptera, twisted winged parasites. Members of Stylops are obligate endoparasites of Andrena mining bees and exhibit extreme sexual dimorphism typical of Strepsiptera. So far, molecular studies on Stylops have focused on questions on species delimitation. Here, we utilize the power of whole genome sequencing to infer the phylogeny of this morphologically challenging genus from thousands of loci. We use a species tree method, concatenated maximum likelihood analysis and Bayesian analysis with a relaxed clock model to reconstruct the phylogeny of 46 Stylops species, estimate divergence times, evaluate topological consistency across methods and infer the root position. Furthermore, the biogeographical history and coevolutionary patterns with host species are assessed. All methods recovered a well resolved topology with close to all nodes maximally supported and only a handful of minor topological variations. Based on the result, we find that included species can be divided into 12 species groups, seven of them including only Palaearctic species, three Nearctic and two were geographically mixed. We find a strongly supported root position between a clade formed by the spreta, thwaitesi and gwynanae species groups and the remaining species and that the sister group of Stylops is Eurystylops or Eurystylops + Kinzelbachus. Our results indicate that Stylops originated in the Western Palaearctic or Western Palaearctic and Nearctic in the early Neogene or late Paleogene, with four independent dispersal events to the Nearctic. Cophylogenetic analyses indicate that the diversification of Stylops has been shaped by both significant coevolution with the mining bee hosts and host-shifting. The well resolved and strongly supported phylogeny will provide a valuable phylogenetic basis for further studies into the fascinating world of Strepsipterans.

A comparative genomic analysis of Fructobacillus evanidus sp. nov. from bumble bees

The increase in studies on bee microbiomes is prompted by concerns over global pollinator declines. Bumble bees host core and non-core microbiota which may contribute to increased lifetime fitness. The presence of Fructobacillus in the gut microbiomes of bumble bee workers, or the replacement of core symbionts with Fructobacillus bacteria, has been considered a marker of dysbiosis. A phylogenomic analysis and functional genomic characterization of the genomes of 21 Fructobacillus isolates from bumble bees demonstrated that they represented four species, i.e. Fructobacillus cardui, Fructobacillus fructosus, Fructobacillus tropaeoli, and the novel species Fructobacillus evanidus sp. nov. Our results confirmed and substantiated the presence of two phylogenetically and functionally distinct Fructobacillus species clades that differ in genome size, percentage G + C content, the number of coding DNA sequences and metabolic characteristics. Clade 1 and clade 2 species differed in amino acid and, to a lesser extent, in carbohydrate metabolism, with F. evanidus and F. tropaeoli genomes featuring a higher number of complete metabolic pathways. While Fructobacillus genomes encoded genes that allow adhesion, biofilm formation, antibacterial activity and detoxification, other bacteria isolated from the bumble bee gut appeared better equipped to co-exist with the bumble bee host. The isolation and identification of multiple Fructobacillus species from several bumble bee gut samples in the present study also argued against a specific partnership between Fructobacillus species and their bumble bee hosts.

Dynamics and regulatory role of circRNAs in Asian honey bee larvae following fungal infection

Non-coding RNA (ncRNA) plays a vital part in the regulation of immune responses, growth, and development in plants and animals. Here, the identification, characteristic analysis, and molecular verification of circRNAs in Apis cerana cerana worker larval guts were conducted, followed by in-depth investigation of the expression pattern of larval circRNAs during Ascosphaera apis infection and exploration of the potential regulatory part of differentially expressed circRNAs (DEcircRNAs) in host immune responses. A total of 3178 circRNAs in the larval guts of A. c. cerana were identified, with a length distribution ranging from 15 to 96,007 nt. Additionally, 155, 95, and 86 DEcircRNAs were identified in the in the 4-, 5-, and 6-day-old larval guts following A. apis infection. These DEcircRNAs were predicted to target 29, 25, and 18 parental genes relevant to 12, 20, and 17 GO terms as well as 144, 114, and 61 KEGG pathways, including 5 cellular and 4 humoral immune pathways. Complex competing endogenous RNA (ceRNA) regulatory networks were detected as being formed among DEcircRNAs, DEmiRNAs, and DEmRNAs. The target DEmRNAs were engaged in 36, 47, and 47 GO terms as well as 331, 332, and 331 pathways, including 6 cellular and 6 humoral immune pathways. Further, 19 DEcircRNAs, 5 DEmiRNAs, and 3 mRNAs were included in the sub-networks relative to 3 antioxidant enzymes. Finally, back-splicing sites within 15 circRNAs and the difference in the 15 DEcircRNAs’ expression between uninoculated and A. apis–inoculated larval guts were confirmed based on molecular methods. These findings not only enrich our understanding of bee host–fungal pathogen interactions but also lay a foundation for illuminating the mechanism underlying the DEcircRNA-mediated immune defense of A. c. cerana larvae against A. apis invasion.Key points• The expression pattern of circRNAs was altered in the A. cerana worker larval guts following A. apis infection.• Back-splicing sites within 15 A. cerana circRNAs were verified using molecular approaches. DEcircRNAs potentially modulated immune responses and antioxidant enzymes in A. apis–challenged host guts.

Life-history stage determines the diet of ectoparasitic mites on their honey bee hosts

Ectoparasitic mites of the genera Varroa and Tropilaelaps have evolved to exclusively exploit honey bees as food sources during alternating dispersal and reproductive life history stages. Here we show that the primary food source utilized by Varroa destructor depends on the host life history stage. While feeding on adult bees, dispersing V. destructor feed on the abdominal membranes to access to the fat body as reported previously. However, when V. destructor feed on honey bee pupae during their reproductive stage, they primarily consume hemolymph, indicated by wound analysis, preferential transfer of biostains, and a proteomic comparison between parasite and host tissues. Biostaining and proteomic results were paralleled by corresponding findings in Tropilaelaps mercedesae, a mite that only feeds on brood and has a strongly reduced dispersal stage. Metabolomic profiling of V. destructor corroborates differences between the diet of the dispersing adults and reproductive foundresses. The proteome and metabolome differences between reproductive and dispersing V. destructor suggest that the hemolymph diet coincides with amino acid metabolism and protein synthesis in the foundresses while the metabolism of non-reproductive adults is tuned to lipid metabolism. Thus, we demonstrate within-host dietary specialization of ectoparasitic mites that coincides with life history of hosts and parasites.

Exposure to a fungicide for a field-realistic duration does not alter bumble bee fecal microbiota structure.

Social bees are frequently exposed to pesticides when foraging on nectar and pollen. Recent research has shown that pesticide exposure not only impacts social bee host health but can also alter the community structure of social bee gut microbiotas. However, most research on pesticide-bee gut microbiota interactions has been conducted in honey bees; bumble bees, native North American pollinators, have received less attention and, due to differences in their ecology, may be exposed to certain pesticides for shorter durations than honey bees. Here, we examine how exposure to the fungicide chlorothalonil for a short, field-realistic duration alters bumble bee fecal microbiotas (used as a proxy for gut microbiotas) and host performance. We expose small groups of Bombus impatiens workers (microcolonies) to field-realistic chlorothalonil concentrations for 5 days, track changes in fecal microbiotas during the exposure period and a recovery period, and compare microcolony offspring production between treatments at the end of the experiment. We also assess the use of fecal microbiotas as a gut microbiota proxy by comparing community structures of fecal and gut microbiotas. We find that chlorothalonil exposure for a short duration does not alter bumble bee fecal microbiota structure or affect microcolony production at any concentration but that fecal and gut microbiotas differ significantly in community structure. Our results show that, at least when exposure durations are brief and unaccompanied by other stressors, bumble bee microbiotas are resilient to fungicide exposure. Additionally, our work highlights the importance of sampling gut microbiotas directly, when possible.IMPORTANCEWith global pesticide use expected to increase in the coming decades, studies on how pesticides affect the health and performance of animals, including and perhaps especially pollinators, will be crucial to minimize negative environmental impacts of pesticides in agriculture. Here, we find no effect of exposure to chlorothalonil for a short, field-realistic period on bumble bee fecal microbiota community structure or microcolony production regardless of pesticide concentration. Our results can help inform pesticide use practices to minimize negative environmental impacts on the health and fitness of bumble bees, which are key native, commercial pollinators in North America. We also find that concurrently sampled bumble bee fecal and gut microbiotas contain similar microbes but differ from one another in community structure and consequently suggest that using fecal microbiotas as a proxy for gut microbiotas be done cautiously; this result contributes to our understanding of proxy use in gut microbiota research.

Zymography of proteases in honey bees (Apis Mellifera) infected with Nosema ceranae

Nosemosis is one of the most important honey bee diseases and is caused by two fungal species of the genus Nosema, i.e., Nosema apis and Nosema ceranae. Evidence suggests that in the coming years, Nosema ceranae, which is less sensitive to fumagillin, could replace Nosema apis as the predominant pathogenic Nosema. Identifying and developing drugs that target these microsporidia without having serious effects on host bee physiology is essential for controlling nosemosis, and this requires information about bee enzymes. Serine proteases, the major protease enzymes in insects, play an important role in the insect immune system. In the present study, some proteases of honey bees infected with N. ceranae were studied by zymography. PCR tests showed that all honey bee samples were infected with N. ceranae. Also, the protein pattern of infected honey bee tissue extracts was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands with molecular weights of 25, 35, 48, 63, 75, and 100 kDa were observed. Subsequently, the serine proteases in the bees? digestive tracts ranged in molecular weight between 23-35 kDa, and the molecular weight of cathepsins was 25 kDa by gelatin zymography. Investigation of bee proteases might be useful for acquiring more knowledge about the interaction between host and parasite and to access an effective and low-risk compound for nosemosis treatment.

Salivary Cystatin-L2-like of Varroa destructor Causes Lower Metabolism Activity and Abnormal Development in Apis mellifera Pupae

Varroa destructor injects a salivary secretion into honeybees during their feeding process. The salivary secretion plays a vital role in mite–bee interactions and is the main cause of honeybee illness. To determine the biological function of cystatin-L2-like, one of the components of V. destructor salivary secretion, its gene expression in mites during the reproductive phase and dispersal phase was quantified using RT-qPCR, respectively. Moreover, the E. coli-expressed and -purified cystatin was injected into the white-eyed honeybee pupae, and its effects on the survival, the weight of the newly emerged bee, and the transcriptome were determined. The results showed that cystatin was significantly upregulated in mites during the reproductive phase. Cystatin significantly shortened the lifespan of pupae and decreased the weight of the newly emerged bees. Transcriptome sequencing showed that cystatin upregulated 1496 genes and downregulated 1483 genes in pupae. These genes were mainly enriched in ATP synthesis, the mitochondrial respiratory chain, and cuticle structure and function. Cystatin comprehensively downregulated the metabolism of carbohydrates, fatty acids, and amino acids, and energy production in the pupae. The downregulation of metabolic activity could save more nutrients and energy for V. destructor, helping it to maximize its reproduction potential, implying that the mite could manipulate the metabolism of host bees through the injected salivary secretion. The results provide new insights into mite–bee interactions, providing a basis for related studies and applications.

Effects of dialkoxybenzenes against Varroa destructor and identification of 1-allyloxy-4-propoxybenzene as a promising acaricide candidate

The honey bee is responsible for pollination of a large proportion of crop plants, but the health of honey bee populations has been challenged by the parasitic mite Varroa destructor. Mite infestation is the main cause of colony losses during the winter months, which causes significant economic challenges in apiculture. Treatments have been developed to control the spread of varroa. However, many of these treatments are no longer effective due to acaricide resistance. In a search of varroa-active compounds, we tested the effect of dialkoxybenzenes on the mite. A structure–activity relationship revealed that 1-allyloxy-4-propoxybenzene is most active of a series of dialkoxybenzenes tested. We found that three compounds (1-allyloxy-4-propoxybenzene, 1,4-diallyloxybenzene and 1,4-dipropoxybenzene) cause paralysis and death of adult varroa mites, whereas the previously discovered compound, 1,3-diethoxybenzene, which alters host choice of adult mites in certain conditions, did not cause paralysis. Since paralysis can be caused by inhibition of acetylcholinesterase (AChE), a ubiquitous enzyme in the nervous system of animals, we tested dialkoxybenzenes on human, honey bee and varroa AChE. These tests revealed that 1-allyloxy-4-propoxybenzene had no effects on AChE, which leads us to conclude that 1-allyloxy-4-propoxybenzene does not exert its paralytic effect on mites through AChE. In addition to paralysis, the most active compounds affected the ability of the mites to find and remain at the abdomen of host bees provided during assays. A test of 1-allyloxy-4-propoxybenzene in the field, during the autumn of 2019 in two locations, showed that this compound has promise in the treatment of varroa infestations.

Report of amoebic disease in a colony of Western honey bees (Apis mellifera).

The amoeba Malpighamoeba mellificae is the etiologic agent of amoebic (amoeba) disease of Western honey bees (Apis mellifera). M. mellificae damages the Malpighian tubules, which is believed to weaken and kill the host bee. Here, the authors describe the detection of this organism in a honey bee colony in the Yukon Territory, Canada. The Malpighian tubules of 14% (7/50) of the adult worker bees were discolored dark brown. Fifteen bees screened using conventional polymerase chain reaction for the 18S gene of M. mellificae were positive for the pathogen. Histologically, the lumens of Malpighian tubules were packed with amoebae, causing dilation of the tubules and attenuation and loss of the tubular epithelium. This phylogenetic analysis places M. mellificae in a new clade, a sister group to the Entamoebidae. This work provides a foundation for further investigation into the distribution, prevalence, and pathology associated with M. mellificae infection.

Consequences of microsporidian prior exposure for virus infection outcomes and bumble bee host health.

Host-parasite interactions do not occur in a vacuum, but in connected multi-parasite networks that can result in co-exposures and coinfections of individual hosts. These can affect host health and disease ecology, including disease outbreaks. However, many host-parasite studies examine pairwise interactions, meaning we still lack a general understanding of the influence of co-exposures and coinfections. Using the bumble bee Bombus impatiens, we study the effects of larval exposure to a microsporidian Nosema bombi, implicated in bumble bee declines, and adult exposure to Israeli Acute Paralysis Virus (IAPV), an emerging infectious disease from honey bee parasite spillover. We hypothesize that infection outcomes will be modified by co-exposure or coinfection. Nosema bombi is a potentially severe, larval-infecting parasite, and we predict that prior exposure will result in decreased host resistance to adult IAPV infection. We predict double parasite exposure will also reduce host tolerance of infection, as measured by host survival. Although our larval Nosema exposure mostly did not result in viable infections, it partially reduced resistance to adult IAPV infection. Nosema exposure also negatively affected survival, potentially due to a cost of immunity in resisting the exposure. There was a significant negative effect of IAPV exposure on survivorship, but prior Nosema exposure did not alter this survival outcome, suggesting increased tolerance given the higher IAPV infections in the bees previously exposed to Nosema. These results again demonstrate that infection outcomes can be non-independentwhen multiple parasites are present, even when exposure to one parasite does not result in a substantial infection.