The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

Trypanosomatids infecting honey bees have been poorly studied with molecular methods until recently. After the description of Crithidia mellificae (Langridge and McGhee, 1967) it took about forty years until molecular data for honey bee trypanosomatids became available and were used to identify and describe a new trypanosomatid species from honey bees, Lotmaria passim (Evans and Schwarz, 2014). However, an easy method to distinguish them without sequencing is not yet available. Research on the related bumble bee parasites Crithidia bombi and Crithidia expoeki revealed a fragment length polymorphism in the internal transcribed spacer 1 (ITS1), which enabled species discrimination. In search of fragment length polymorphisms for differential diagnostics in honey bee trypanosomatids, we studied honey bee trypanosomatid cell cultures of C. mellificae and L. passim. This research resulted in the identification of fragment length polymorphisms in ITS1 and ITS1-2 markers, which enabled us to develop a diagnostic method to differentiate both honey bee trypanosomatid species without the need for sequencing. However, the amplification success of the ITS1 marker depends probably on the trypanosomatid infection level. Further investigation confirmed that L. passim is the dominant species in Belgium, Japan and Switzerland. We found C. mellificae only rarely in Belgian honey bee samples, but not in honey bee samples from other countries. C. mellificae was also detected in mason bees (Osmia bicornis and Osmia cornuta) besides in honey bees. Further, the characterization and comparison of additional markers from L. passim strain SF (published as C. mellificae strain SF) and a Belgian honey bee sample revealed very low divergence in the 18S rRNA, ITS1-2, 28S rRNA and cytochrome b sequences. Nevertheless, a variable stretch was observed in the gp63 virulence factor.

Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).

Pathogens and parasites represent significant threats to the health and well-being of honeybee species that are key pollinators of agricultural crops and flowers worldwide. We conducted a nationwide survey to determine the occurrence and prevalence of pathogens and parasites in Asian honeybees, Apis cerana, in China. Our study provides evidence of infections of A. cerana by pathogenic Deformed wing virus (DWV), Black queen cell virus (BQCV), Nosema ceranae, and C. bombi species that have been linked to population declines of European honeybees, A. mellifera, and bumble bees. However, the prevalence of DWV, a virus that causes widespread infection in A. mellifera, was low, arguably a result of the greater ability of A. cerana to resist the ectoprasitic mite Varroa destructor, an efficient vector of DWV. Analyses of microbial communities from the A. cerana digestive tract showed that Nosema infection could have detrimental effects on the gut microbiota. Workers infected by N. ceranae tended to have lower bacterial quantities, with these differences being significant for the Bifidobacterium and Pasteurellaceae bacteria groups. The results of this nationwide screen show that parasites and pathogens that have caused serious problems in European honeybees can be found in native honeybee species kept in Asia. Environmental changes due to new agricultural practices and globalization may facilitate the spread of pathogens into new geographic areas. The foraging behavior of pollinators that are in close geographic proximity likely have played an important role in spreading of parasites and pathogens over to new hosts. Phylogenetic analyses provide insights into the movement and population structure of these parasites, suggesting a bidirectional flow of parasites among pollinators. The presence of these parasites and pathogens may have considerable implications for an observed population decline of Asian honeybees.

Two species of Spiroplasma (Mollicutes) bacteria were isolated from and described as pathogens of the European honey bee, Apis mellifera, ~30 years ago but recent information on them is lacking despite global concern to understand bee population declines. Here we provide a comprehensive survey for the prevalence of these two Spiroplasma species in current populations of honey bees using improved molecular diagnostic techniques to assay multiyear colony samples from North America (U.S.A.) and South America (Brazil). Significant annual and seasonal fluctuations of Spiroplasma apis and Spiroplasma melliferum prevalence in colonies from the U.S.A. (n = 616) and Brazil (n = 139) occurred during surveys from 2011 through 2013. Overall, 33% of U.S.A. colonies and 54% of Brazil colonies were infected by Spiroplasma spp., where S. melliferum predominated over S. apis in both countries (25% vs. 14% and 44% vs. 38% frequency, respectively). Colonies were co-infected by both species more frequently than expected in both countries and at a much higher rate in Brazil (52%) compared to the U.S.A. (16.5%). U.S.A. samples showed that both species were prevalent not only during spring, as expected from prior research, but also during other seasons. These findings demonstrate that the model of honey bee spiroplasmas as springtime-restricted pathogens needs to be broadened and their role as occasional pathogens considered in current contexts.

Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models.

RNA viruses impact honey bee health and contribute to elevated colony loss rates worldwide. Deformed wing virus (DWV) and the closely related Varroa destructor virus-1 (VDV1), are the most widespread honey bee viruses. VDV1 is known to cause high rates of overwintering colony losses in Europe, however it was unknown in the United States (US). Using next generation sequencing, we identified VDV1 in honey bee pupae in the US. We tested 603 apiaries the US in 2016 and found that VDV1 was present in 66.0% of them, making it the second most prevalent virus after DWV, which was present in 89.4% of the colonies. VDV1 had the highest load in infected bees (7.45*1012 ± 1.62*1012 average copy number ± standard error) compared to other tested viruses, with DWV second (1.04*1012 ± 0.53*1012). Analysis of 75 colonies sourced in 2010 revealed that VDV1 was present in only 2 colonies (2.7%), suggesting its recent spread. We also detected newly emerged recombinants between the US strains of VDV1 and DWV. The presence of these recombinants poses additional risk, because similar VDV1-DWV recombinants constitute the most virulent honeybee viruses in the UK.

Olfaction is key to many insects. Odorant receptors (ORs) stand among the key chemosensory receptors mediating the detection of pheromones and kairomones. Small hive beetles (SHBs), Aethina tumida, are parasites of social bee colonies and olfactory cues are especially important for host finding. However, how interactions with their hosts may have shaped the evolution of ORs in the SHB remains poorly understood. Here, for the first time, we analyzed the evolution of SHB ORs through phylogenetic and positive selection analyses. We then tested the expression of selected OR genes in antennae, heads, and abdomens in four groups of adult SHBs: colony odor-experienced/-naive males and females. The results show that SHBs experienced both OR gene losses and duplications, thereby providing a first understanding of the evolution of SHB ORs. Additionally, three candidate ORs potentially involved in host finding and/or chemical communication were identified. Significantly different downregulations of ORs between the abdomens of male and female SHBs exposed to colony odors may reflect that these expression patterns might also reflect other internal events, e.g., oviposition. Altogether, these results provide novel insights into the evolution of SHB ORs and provide a valuable resource for analyzing the function of key genes, e.g., for developing biological control. These results will also help in understanding the chemosensory system in SHBs and other beetles.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities.

Microsporidia comprise a phylum of single cell, intracellular parasites and represent the earliest diverging branch in the fungal kingdom. The microsporidian parasite Nosema ceranae primarily infects honey bee gut epithelial cells, leading to impaired memory, suppressed host immune responses and colony collapse under certain circumstances. As the genome of N. ceranae is challenging to assembly due to very high genetic diversity and repetitive region, the genome was re-sequenced using long reads. We present a robust 8.8 Mbp genome assembly of 2,280 protein coding genes, including a high number of genes involved in transporting nutrients and energy, as well as drug resistance when compared with sister species Nosema apis. We also describe the loss of the critical protein Dicer in approximately half of the microsporidian species, giving new insights into the availability of RNA interference pathway in this group. Our results provided new insights into the pathogenesis of N. ceranae and a blueprint for treatment strategies that target this parasite without harming honey bees. The unique infectious apparatus polar filament and transportation pathway members can help to identify treatments to control this parasite.

The temperature dependence of infection reflects changes in performance of parasites and hosts. High temperatures often mitigate infection by favoring heat-tolerant hosts over heat-sensitive parasites. Honey bees exhibit endothermic thermoregulation-rare among insects-that can favor resistance to parasites. However, viruses are heavily host-dependent, suggesting that viral infection could be supported-not threatened-by optimum host function. To understand how temperature-driven changes in performance of viruses and hosts shape infection, we compared the temperature dependence of isolated viral enzyme activity, three honey bee traits, and infection of honey bee pupae. Viral enzyme activity varied <2-fold over a > 30 °C interval spanning temperatures typical of ectothermic insects and honey bees. In contrast, honey bee performance peaked at high (≥ 35 °C) temperatures and was highly temperature-sensitive. Although these results suggested that increasing temperature would favor hosts over viruses, the temperature dependence of pupal infection matched that of pupal development, falling only near pupae's upper thermal limits. Our results reflect the host-dependent nature of viruses, suggesting that infection is accelerated-not curtailed-by optimum host function, contradicting predictions based on relative performance of parasites and hosts, and suggesting tradeoffs between infection resistance and host survival that limit the viability of bee 'fever'.

The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.

Recent losses in honey bee colonies are unusual in their severity, geographical distribution, and, in some cases, failure to present recognized characteristics of known disease. Domesticated honey bees face numerous pests and pathogens, tempting hypotheses that colony collapses arise from exposure to new or resurgent pathogens. Here we explore the incidence and abundance of currently known honey bee pathogens in colonies suffering from Colony Collapse Disorder (CCD), otherwise weak colonies, and strong colonies from across the United States. Although pathogen identities differed between the eastern and western United States, there was a greater incidence and abundance of pathogens in CCD colonies. Pathogen loads were highly covariant in CCD but not control hives, suggesting that CCD colonies rapidly become susceptible to a diverse set of pathogens, or that co-infections can act synergistically to produce the rapid depletion of workers that characterizes the disorder. We also tested workers from a CCD-free apiary to confirm that significant positive correlations among pathogen loads can develop at the level of individual bees and not merely as a secondary effect of CCD. This observation and other recent data highlight pathogen interactions as important components of bee disease. Finally, we used deep RNA sequencing to further characterize microbial diversity in CCD and non-CCD hives. We identified novel strains of the recently described Lake Sinai viruses (LSV) and found evidence of a shift in gut bacterial composition that may be a biomarker of CCD. The results are discussed with respect to host-parasite interactions and other environmental stressors of honey bees.

The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes.

Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species.

The Asian honeybee Apis cerana is one of two bee species that have been commercially kept with immense economic value. Here we present the analysis of genomic sequence and transcriptomic exploration for A. cerana as well as the comparative genomic analysis of the Asian honeybee and the European honeybee A. mellifera. The genome and RNA-seq data yield new insights into the behavioral and physiological resistance to the parasitic mite Varroa the evolution of antimicrobial peptides, and the genetic basis for labor division in A. cerana. Comparison of genes between the two sister species revealed genes specific to A. cerana, 54.5% of which have no homology to any known proteins. The observation that A. cerana displayed significantly more vigilant grooming behaviors to the presence of Varroa than A. mellifera in conjunction with gene expression analysis suggests that parasite-defensive grooming in A. cerana is likely triggered not only by exogenous stimuli through visual and olfactory detection of the parasite, but also by genetically endogenous processes that periodically activates a bout of grooming to remove the ectoparasite. This information provides a valuable platform to facilitate the traits unique to A. cerana as well as those shared with other social bees for health improvement.

It has become increasingly clear that gut bacteria play vital roles in the development, nutrition, immunity, and overall fitness of their eukaryotic hosts. We conducted the present study to investigate the effects of gut microbiota disruption on the honey bee's immune responses to infection by the microsporidian parasite Nosema ceranae. Newly emerged adult workers were collected and divided into four groups: Group I-no treatment; Group II-inoculated with N. ceranae, Group III-antibiotic treatment, and Group IV-antibiotic treatment after inoculation with N. ceranae. Our study showed that Nosema infection did not cause obvious disruption of the gut bacterial community as there was no significant difference in the density and composition of gut bacteria between Group I and Group II. However, the elimination of gut bacteria by antibiotic (Groups III and IV) negatively impacted the functioning of the honey bees' immune system as evidenced by the expression of genes encoding antimicrobial peptides abaecin, defensin1, and hymenoptaecin that showed the following ranking: Group I > Group II > Group III > Group IV. In addition, significantly higher Nosema levels were observed in Group IV than in Group II, suggesting that eliminating gut bacteria weakened immune function and made honey bees more susceptible to Nosema infection. Based on Group IV having displayed the highest mortality rate among the four experimental groups indicates that antibiotic treatment in combination with stress, associated with Nosema infection, significantly and negatively impacts honey bee survival. The present study adds new evidence that antibiotic treatment not only leads to the complex problem of antibiotic resistance but can impact honey bee disease resistance. Further studies aimed at specific components of the gut bacterial community will provide new insights into the roles of specific bacteria and possibly new approaches to improving bee health.

We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture.

The recent sequencing of the Anopheles gambiae genome showcases the genetic breadth of insects and a trend towards sequencing organisms directly involved with human welfare. We describe traits in other insect species that make them important candidates for genomics projects, and review several recent workshops aimed at uniting researchers working with insect species to efficiently address problems in medicine, biotechnology, and agriculture.

Honey bees, Apis mellifera, face many parasites and pathogens and consequently rely on a diverse set of individual and group-level defenses to prevent disease. One route by which honey bees and other insects might combat disease is through the shielding effects of their microbial symbionts. Bees carry a diverse assemblage of bacteria, very few of which appear to be pathogenic. Here we explore the inhibitory effects of these resident bacteria against the primary bacterial pathogen of honey bees, Paenibacillus larvae.