Intracellular endosymbiotic bacteria are common and can play a crucial role for insect pathology. Therefore, such bacteria could be a potential key to our understanding of major losses of Western honey bees (Apis mellifera) colonies. However, the transmission and potential effects of endosymbiotic bacteria in A. mellifera and other Apis spp. are poorly understood. Here, we explore the prevalence and transmission of the genera Arsenophonus, Wolbachia, Spiroplasma and Rickettsia in Apis spp. Colonies of A. mellifera (N = 33, with 20 eggs from worker brood cells and 100 adult workers each) as well as mated honey bee queens of A. cerana, A. dorsata and A. florea (N = 12 each) were screened using PCR. While Wolbachia, Spiroplasma and Rickettsia were not detected, Arsenophonus spp. were found in 24.2% of A. mellifera colonies and respective queens as well as in queens of A. dorsata (8.3%) and A. florea (8.3%), but not in A. cerana The absence of Arsenophonus spp. from reproductive organs of A. mellifera queens and surface-sterilized eggs does not support transovarial vertical transmission. Instead, horizontal transmission is most likely.

A range of different genetic architectures underpin local adaptation in nature. Honey bees (Apis mellifera) in the Eastern African Mountains harbor high frequencies of two chromosomal inversions that likely govern adaptation to this high-elevation habitat. In the Americas, honey bees are hybrids of European and African ancestries and adaptation to latitudinal variation in climate correlates with the proportion of these ancestries across the genome. It is unknown which, if either, of these forms of genetic variation governs adaptation in honey bees living at high elevations in the Americas. Here, we performed whole-genome sequencing of 29 honey bees from both high- and low-elevation populations in Colombia. Analysis of genetic ancestry indicated that both populations were predominantly of African ancestry, but the East African inversions were not detected. However, individuals in the higher elevation population had significantly higher proportions of European ancestry, likely reflecting local adaptation. Several genomic regions exhibited particularly high differentiation between highland and lowland bees, containing candidate loci for local adaptation. Genes that were highly differentiated between highland and lowland populations were enriched for functions related to reproduction and sperm competition. Furthermore, variation in levels of European ancestry across the genome was correlated between populations of honey bees in the highland population and populations at higher latitudes in South America. The results are consistent with the hypothesis that adaptation to both latitude and elevation in these hybrid honey bees are mediated by variation in ancestry at many loci across the genome.

Host shifts of parasites are often causing devastating effects in the new hosts. The Varroa genus is known for a lineage of Varroa destructor that shifted to the Western honey bee, Apis mellifera, with disastrous effects on wild populations and the beekeeping industry. Despite this, the biology of Varroa spp. remains poorly understood in its native distribution range, where it naturally parasitizes the Eastern honey bee, Apis cerana. Here, we combined mitochondrial and nuclear DNA analyses with the assessment of mite reproduction to determine the population structure and host specificity of V. destructor and Varroa jacobsonii in Thailand, where both hosts and several Varroa species and haplotypes are sympatric. Our data confirm previously described mite haplogroups, and show three novel haplotypes. Multiple infestations of single host colonies by both mite species and introgression of alleles between V. destructor and V. jacobsonii suggest that hybridization occurs between the two species. Our results indicate that host specificity and population genetic structure in the genus Varroa is more labile than previously thought. The ability of the host shifted V. destructor haplotype to spillback to A. cerana and to hybridize with V. jacobsonii could threaten honey bee populations of Asia and beyond.

Viruses are omnipresent, yet the knowledge on drivers of viral prevalence in wild host populations is often limited. Biotic factors, such as sympatric managed host species, as well as abiotic factors, such as climatic variables, are likely to impact viral prevalence. Managed and wild bees, which harbor several multi-host viruses with a mostly fecal-oral between-species transmission route, provide an excellent system with which to test for the impact of biotic and abiotic factors on viral prevalence in wild host populations. Here we show on a continental scale that the prevalence of three broad host viruses: the AKI-complex (Acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus), Deformed wing virus, and Slow bee paralysis virus in wild bee populations (bumble bees and solitary bees) is positively related to viral prevalence of sympatric honey bees as well as being impacted by climatic variables. The former highlights the need for good beekeeping practices, including Varroa destructor management to reduce honey bee viral infection and hive placement. Furthermore, we found that viral prevalence in wild bees is at its lowest at the extreme ends of both temperature and precipitation ranges. Under predicted climate change, the frequency of extremes in precipitation and temperature will continue to increase and may hence impact viral prevalence in wild bee communities.

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee's susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.

Recent major losses of managed honeybee, Apis mellifera, colonies at a global scale have resulted in a multitude of research efforts to identify the underlying mechanisms. Numerous factors acting singly and/or in combination have been identified, ranging from pathogens, over nutrition to pesticides. However, the role of apiculture in limiting natural selection has largely been ignored. This is unfortunate, because honeybees are more exposed to environmental stressors compared to other livestock and management can severely compromise bee health. Here, we briefly review apicultural factors that influence bee health and focus on those most likely interfering with natural selection, which offers a broad range of evolutionary applications for field practice. Despite intense breeding over centuries, natural selection appears to be much more relevant for the health of managed A. mellifera colonies than previously thought. We conclude that sustainable solutions for the apicultural sector can only be achieved by taking advantage of natural selection and not by attempting to limit it.

Climate change and biological invasions are two major global environmental challenges. Both may interact, e.g. via altered impact and distribution of invasive alien species. Even though invasive species play a key role for compromising the health of honey bees, the impact of climate change on the severity of such species is still unknown. The small hive beetle (SHB, Aethina tumida, Murray) is a parasite of honey bee colonies. It is endemic to sub-Saharan Africa and has established populations on all continents except Antarctica. Since SHBs pupate in soil, pupation performance is governed foremost by two abiotic factors, soil temperature and moisture, which will be affected by climate change. Here, we investigated SHB invasion risk globally under current and future climate scenarios. We modelled survival and development time during pupation (=pupal performance) in response to soil temperature and soil moisture using published and novel experimental data. Presence data on SHB distribution were used for model validation. We then linked the model with global soil data in order to classify areas (resolution: 10 arcmin; i.e. 18.6 km at the equator) as unsuitable, marginal and suitable for SHB pupation performance. Under the current climate, the results show that many areas globally yet uninvaded are actually suitable, suggesting considerable SHB invasion risk. Future scenarios of global warming project a vehement increase in climatic suitability for SHB and corresponding potential for invasion, especially in the temperate regions of the Northern hemisphere, thereby creating demand for enhanced and adapted mitigation and management. Our analysis shows, for the first time, effects of global warming on a honey bee pest and will help areas at risk to prepare adequately. In conclusion, this is a clear case for global warming promoting biological invasion of a pest species with severe potential to harm important pollinator species globally.

Host shift of parasites may have devastating effects on the novel hosts. One remarkable example is that of the ectoparasitic mite Varroa destructor, which has shifted its host from Eastern honey bees (Apis cerana) to Western honey bees (Apis mellifera) and posed a global threat to apiculture.

The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. Several honeybee populations across Europe have well-documented adaptations of mite-resistant traits but little is known about host adaptations towards the virus infections vectored by the mite. The aim of this study was to assess and compare the possible contribution of adapted virus tolerance and/or resistance to the enhanced survival of four well-documented mite-resistant honeybee populations from Norway, Sweden, The Netherlands and France, in relation to unselected mite-susceptible honeybees. Caged adult bees and laboratory reared larvae, from colonies of these four populations, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical in all groups, but all mite-resistant honeybee populations had significantly higher survival rates compared to the mite-susceptible honeybees. These results suggest that adapted virus tolerance is an important component of survival mechanisms.

Eusocial insect colonies form superorganisms, in which nestmates cooperate and use social immunity to combat parasites. However, social immunity may fail in case of emerging diseases. This is the case for the ectoparasitic mite Varroa destructor, which switched hosts from the Eastern honeybee, Apis cerana, to the Western honey bee, Apis mellifera, and currently is the greatest threat to A. mellifera apiculture globally. Here, we show that immature workers of the mite's original host, A. cerana, are more susceptible to V. destructor infestations than those of its new host, thereby enabling more efficient social immunity and contributing to colony survival. This counterintuitive result shows that susceptible individuals can foster superorganism survival, offering empirical support to theoretical arguments about the adaptive value of worker suicide in social insects. Altruistic suicide of immature bees constitutes a social analogue of apoptosis, as it prevents the spread of infections by sacrificing parts of the whole organism, and unveils a novel form of transgenerational social immunity in honey bees. Taking into account the key role of susceptible immature bees in social immunity will improve breeding efforts to mitigate the unsustainably high colony losses of Western honey bees due to V. destructor infestations worldwide.

Multiple infections are common in honey bees, Apis mellifera, but the possible role of nutrition in this regard is poorly understood. Microsporidian infections, which are promoted by protein-fed, can negatively correlate with virus infections, but the role of protein nutrition for the microsporidian-virus interface is unknown. Here, we challenged naturally deformed wing virus - B (DWV-B) infected adult honey bee workers fed with or without pollen ( = protein) in hoarding cages, with the microsporidian Nosema ceranae. Bee mortality was recorded for 14 days and N. ceranae spore loads and DWV-B titers were quantified. Amongst the groups inoculated with N. ceranae, more spores were counted in protein-fed bees. However, N. ceranae infected bees without protein-diet had reduced longevity compared to all other groups. N. ceranae infection had no effect on protein-fed bee's longevity, whereas bees supplied only with sugar-water showed reduced survival. Our data also support that protein-feeding can have a significant negative impact on virus infections in insects. The negative correlation between N. ceranae spore loads and DWV-B titers was stronger expressed in protein-fed hosts. Proteins not only enhance survival of infected hosts, but also significantly shape the microsporidian-virus interface, probably due to increased spore production and enhanced host immunity.

Western honeybee populations, Apis mellifera, in Europe have been known to survive infestations of the ectoparasitic mite, Varroa destructor, by means of natural selection. Proposed mechanisms in literature have been focused on the management of this parasite, however literature remains scare on the differences in viral ecology between colonies that have adapted to V. destructor and those that are consistently treated for it. Samples were collected from both a mite-surviving and a sympatric mite-susceptible honeybee population in Norway. The prevalence and abundances of 10 viruses, vectored by the parasite or not, were investigated in adult host workers and pupae as well as in V. destructor mites. Here we show that the mite-vectored Deformed wing virus (DWV-A) is often lower in both abundance and prevalence in the mite-surviving population in tandem with lower phoretic mite infestations compared to the mite susceptible population. However, the non-mite-vectored Black queen cell virus (BQCV), had both a higher abundance and prevalence in the mite-surviving population compared to the susceptible population. The data therefore suggest that general adaptations to virus infections may be unlikely to explain colony survival. Instead, mechanisms suppressing mite reproduction and therefore the impact seem to be more important.

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.

Interspecies transmission of RNA viruses is a major concern for human and animal health. However, host-range, transmission routes and especially the possible impact of these viruses on alternative hosts are often poorly understood. Here, we investigated the role of the labyrinth spider, Agelena labyrinthica, as a potential alternative host of viruses commonly known from western honey bees, Apis mellifera. Field-collected spiders were screened for Acute bee paralysis virus (ABPV), Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus type A and B (DWV-B), Israeli acute paralysis virus, Lake Sinai virus and Sacbrood virus. In a laboratory experiment, labyrinth spiders were fed with ABPV and DWV-B infected honey bees or virus free control food. Our results show that natural infections of A. labyrinthica with these viruses are common in the field, as 62.5% of the samples were positive for at least one virus, supporting their wide host range. For DWV-B, the laboratory data indicate that foodborne transmission occurs and that high virus titres may reduce cocoon building, which would be the first report of clinical symptoms of DWV in Araneae. Since cocoons are tokens of fitness, virus transmission from honey bees might affect spider populations, which would constitute a concern for nature conservation.

Neonicotinoid insecticides can cause a variety of adverse sub-lethal effects in bees. In social species such as the honeybee, Apis mellifera, queens are essential for reproduction and colony functioning. Therefore, any negative effect of these agricultural chemicals on the mating success of queens may have serious consequences for the fitness of the entire colony. Queens were exposed to the common neonicotinoid pesticides thiamethoxam and clothianidin during their developmental stage. After mating, their spermathecae were dissected to count the number of stored spermatozoa. Furthermore, their worker offspring were genotyped with DNA microsatellites to determine the number of matings and the genotypic composition of the colony. Colonies providing the male mating partners were also inferred. Both neonicotinoid and control queens mated with drones originating from the same drone source colonies, and stored similar number of spermatozoa. However, queens reared in colonies exposed to both neonicotinoids experienced fewer matings. This resulted in a reduction of the genetic diversity in their colonies (i.e. higher intracolonial relatedness). As decreased genetic diversity among worker bees is known to negatively affect colony vitality, neonicotinoids may have a cryptic effect on colony health by reducing the mating frequency of queens.

Host-parasite co-evolution history is lacking when parasites switch to novel hosts. This was the case for Western honey bees (Apis mellifera) when the ectoparasitic mite, Varroa destructor, switched hosts from Eastern honey bees (Apis cerana). This mite has since become the most severe biological threat to A. mellifera worldwide. However, some A. mellifera populations are known to survive infestations, largely by suppressing mite population growth. One known mechanism is suppressed mite reproduction (SMR), but the underlying genetics are poorly understood. Here, we take advantage of haploid drones, originating from one queen from the Netherlands that developed Varroa-resistance, whole exome sequencing and elastic-net regression to identify genetic variants associated with SMR in resistant honeybees. An eight variants model predicted 88% of the phenotypes correctly and identified six risk and two protective variants. Reproducing and non-reproducing mites could not be distinguished using DNA microsatellites, which is in agreement with the hypothesis that it is not the parasite but the host that adapted itself. Our results suggest that the brood pheromone-dependent mite oogenesis is disrupted in resistant hosts. The identified genetic markers have a considerable potential to contribute to a sustainable global apiculture.

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.

Emerging infectious diseases are often the products of host shifts, where a pathogen jumps from its original host to a novel species. Viruses in particular cross species barriers frequently. Acute bee paralysis virus (ABPV) and deformed wing virus (DWV) are viruses described in honey bees (Apis mellifera) with broad host ranges. Ants scavenging on dead honey bees may get infected with these viruses via foodborne transmission. However, the role of black garden ants, Lasius niger and Lasius platythorax, as alternative hosts of ABPV and DWV is not known and potential impacts of these viruses have not been addressed yet. In a laboratory feeding experiment, we show that L. niger can carry DWV and ABPV. However, negative-sense strand RNA, a token of virus replication, was only detected for ABPV. Therefore, additional L. niger colonies were tested for clinical symptoms of ABPV infections. Symptoms were detected at colony (fewer emerging workers) and individual level (impaired locomotion and movement speed). In a field survey, all L. platythorax samples carried ABPV, DWV-A and -B, as well as the negative-sense strand RNA of ABPV. These results show that L. niger and L. platythorax are alternative hosts of ABPV, possibly acting as a biological vector of ABPV and as a mechanical one for DWV. This is the first study showing the impact of honey bee viruses on ants. The common virus infections of ants in the field support possible negative consequences for ecosystem functioning due to host shifts.

The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups.

Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies, Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia (N = 3 regions, N = 9 locations, N = 151 colonies), where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses (Sacbrood virus, Black queen cell virus, Deformed wing virus (DWV) and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.