Caught in the act: the invasion of a viral vector changes viral prevalence and titre in native honeybees and bumblebees.

Novel transmission routes change pathogen landscapes and may facilitate disease emergence. The varroa mite is a virus vector that switched to western honeybees at the beginning of the last century, leading to hive mortality, particularly in combination with RNA viruses. A recent invasion of varroa on the French island of Ushant introduced vector-mediated transmission to one of the last varroa-naive native honeybee populations and caused rapid changes in the honeybee viral community. These changes were characterized by a drastic increase in deformed wing virus type B prevalence and titre in honeybees, as well as knock-on effects in bumblebees, particularly in the year following the invasion. Slow bee paralysis virus also appeared in honeybees and bumblebees, with a 1 year delay, while black queen cell virus declined in honeybees. This study highlights the rapid and far-reaching effects of vector-borne transmission that can extend beyond the directly affected host species, and that the direction of the effect depends on the pathogen's virulence.

Land-use-associated stressors interact to reduce bumblebee health at the individual and colony level.

In agricultural landscapes, bees face a variety of stressors, including insecticides and poor-quality food. Although both stressors individually have been shown to affect bumblebee health negatively, few studies have focused on stressor interactions, a scenario expected in intensively used agricultural landscapes. Using the bumblebee Bombus terrestris, a key pollinator in agricultural landscapes, we conducted a fully factorial laboratory experiment starting at nest initiation. We assessed the effects of food quality and insecticides, alone and in interaction, on health traits at various levels, some of which have been rarely studied. Pollen with a diluted nutrient content (low quality) reduced ovary size and delayed colony development. Wing asymmetry, indicating developmental stress, was increased during insecticide exposure and interactions with poor food, whereas both stressors reduced body size. Both stressors and their interaction changed the workers' chemical profile and reduced worker interactions and the immune response. Our findings suggest that insecticides combined with nutritional stress reduce bumblebee health at the individual and colony levels, thus possibly affecting colony performance, such as development and reproduction, and the stability of plant-pollinator networks. The synergistic effects highlight the need of combining stressors in risk assessments and when studying the complex effects of anthropogenic stressors on health outcomes.

Lake Sinai virus is a diverse, globally distributed but not emerging multi-strain honeybee virus.

Domesticated honeybees and wild bees are some of the most important beneficial insects for human and environmental health, but infectious diseases pose a serious risk to these pollinators, particularly following the emergence of the ectoparasitic mite Varroa destructor as a viral vector. The acquisition of this novel viral vector from the Asian honeybee Apis ceranae has fundamentally changed viral epidemiology in its new host, the western honeybee A. mellifera. While the recently discovered Lake Sinai Viruses (LSV) have been associated with weak honeybee colonies, they have not been associated with vector-borne transmission. By combining a large-scale multi-year survey of LSV in Chinese A. mellifera and A. cerana honeybee colonies with globally available LSV-sequence data, we investigate the global epidemiology of this virus. We find that globally distributed LSV is a highly diverse multi-strain virus, which is predominantly associated with the western honeybee A. mellifera. In contrast to the vector-borne deformed wing virus, LSV is not an emerging disease. Instead, demographic reconstruction and strong global and local population structure indicates that it is a highly variable multi-strain virus in a stable association with its main host, the western honeybee. Prevalence patterns in China suggest a potential role for migratory beekeeping in the spread of this pathogen, demonstrating the potential for disease transmission with the man-made transport of beneficial insects.

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970's Egypt.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

Complex relationship between amino acids, fitness and food intake in Bombus terrestris.

The ratio of amino acids to carbohydrates (AA:C) that bumble bees consume has been reported to affect their survival. However, it is unknown how dietary AA:C ratio affects other bumble bee fitness traits (e.g., fecundity, condition) and possible trade-offs between them. Moreover, while individual AAs affect phenotype in many species, the effects of AA blend on bumble bee fitness and food intake are unclear. We test how the AA:C ratio that bumble bees (Bombus terrestris) consume affects their condition (abdomen lipid and dry mass), survival following food removal, and ovarian activation. We then compare ovarian activation and food intake in bees fed identical AA:C ratios, but where the blend of AAs in diets differ, i.e., diets contained the same 10 AAs in an equimolar ratio or in the same ratio as in bee collected pollen. We found that AA:C ratio did not significantly affect survival following food removal or ovarian activation; however, high AA intake increased body mass, which is positively correlated with multiple fitness traits in bumble bees. AA blend (i.e., equimolar versus pollen) did not significantly affect overall ovarian activation or consumption of each experimental diet. However, there was an interaction between AA mix and dietary AA:C ratio affecting survival during the feeding experiment, and signs that there may have been weak, interactive effects of AA mix and AA:C ratio on food consumption. These results suggest that the effect of total AA intake on bumble bee phenotype may depend on the blend of individual AAs in experimental diets. We suggest that research exploring how AA blend affects bumble bee performance and dietary intake is warranted, and highlight that comparing research on bee nutrition is complicated by even subtle variation in experimental diet composition.

Viral adaptations to vector-borne transmission can result in complex host-vector-pathogen interactions.

Research Highlight: Norton, A. M., Remnant, E. J., Tom, J., Buchmann, G., Blacquiere, T., & Beekman, M. (2021). Adaptation to vector-based transmission in a honeybee virus. Journal of Animal Ecology, 90, https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2656.13493. In their paper on the adaptation to vector-based transmission via the mite Varroa destructor in a honeybee virus, Norton et al. study how high versus low levels of a viral vector affect viral load and potential competition between two strains of Deformed Wing Virus, an important highly virulent bee virus with the potential to spill-over into other pollinators and bee-associated insect species. This paper addresses two very timely issues, on the one hand on viral evolutionary ecology in response to vector-borne transmission, and on the other hand providing much needed information on an important honey bee pathogen. Using a complex natural system, this study shows that vector-borne transmission, and the control of the vector, can select for complex host-pathogen-vector interactions and that adaptations to changing transmission landscapes in fast evolving pathogens can create conditions for emerging pathogens to transition to endemic diseases.

OneHealth implications of infectious diseases of wild and managed bees.

The OneHealth approach aims to further our understanding of the drivers of human, animal and environmental health, and, ultimately, to improve them by combining approaches and knowledge from medicine, biology and fields beyond. Wild and managed bees are essential pollinators of crops and wild flowers. Their health thus directly impacts on human and environmental health. At the same time, these bee species represent highly amenable and relevant model organisms for a OneHealth approach that aims to study fundamental epidemiological questions. In this review, we focus on how infectious diseases of wild and managed bees can be used as a OneHealth model system, informing fundamental questions on ecological immunology and disease transmission, while addressing how this knowledge can be used to tackle the issues facing pollinator health.

Dose-dependent effects of antibiotic intake on Bombus Terrestris (Linnaeus, 1758) dietary intake, survival and parasite infection prevalence.

Diseases may contribute to the widespread declines seen in many bee species. The gut bacteria of bees may serve as one defence against disease, by preventing pathogen colonisation. However, exposure to antibiotics on forage or in the hive may disrupt bee gut bacteria and remove this protective effect. A number of studies show that high antibiotic doses reduce bee health but the effects of field-realistic antibiotic doses remain unclear. Here, we test how Bombus terrestris (Linnaeus, 1758) is affected by multiple field-realistic concentrations of the antibiotic oxytetracycline, which is sometimes used to protect flowering crops from bacterial infections. We measured survival, feeding behaviour and the likelihood of developing infection with the gut parasitic trypanosome Crithidia bombi Lipa & Triggiani, 1988 following oral inoculation with a range of antibiotic doses. Rising antibiotic concentrations were associated with reduced survival and food consumption, and an increased likelihood of becoming infected with C. bombi. These effects were seen at antibiotic concentrations that are applied to crops and so may be encountered by foraging bees in the field. These results support the hypothesis that field-realistic antibiotic doses have lethal and sub-lethal effects on B. terrestris and highlight the importance of improving our understanding of how field-realistic antibiotic doses affect pollinators.

Persistent effects of management history on honeybee colony virus abundances.

Infectious diseases are a major threat to both managed and wild pollinators. One key question is how the movement or transplantation of honeybee colonies under different management regimes affects honeybee disease epidemiology. We opportunistically examined any persistent effect of colony management history following relocation by characterising the virus abundances of honeybee colonies from three management histories, representing different management histories: feral, low-intensity management, and high-intensity "industrial" management. The colonies had been maintained for one year under the same approximate 'common garden' condition. Colonies in this observational study differed in their virus abundances according to management history, with the feral population history showing qualitatively different viral abundance patterns compared to colonies from the two managed population management histories; for example, higher abundance of sacbrood virus but lower abundances of various paralysis viruses. Colonies from the high-intensity management history exhibited higher viral abundances for all viruses than colonies from the low-intensity management history. Our results provide evidence that management history has persistent impacts on honeybee disease epidemiology, suggesting that apicultural intensification could be majorly impacting on pollinator health, justifying much more substantial investigation.

Host density drives viral, but not trypanosome, transmission in a key pollinator.

Supplemental feeding of wildlife populations can locally increase the density of individuals, which may in turn impact disease dynamics. Flower strips are a widely used intervention in intensive agricultural systems to nutritionally support pollinators such as bees. Using a controlled experimental semi-field design, we asked how density impacts transmission of a virus and a trypanosome parasite in bumblebees. We manipulated bumblebee density by using different numbers of colonies within the same area of floral resource. In high-density compartments, slow bee paralysis virus was transmitted more quickly, resulting in higher prevalence and level of infection in bumblebee hosts. By contrast, there was no impact of density on the transmission of the trypanosome Crithidia bombi, which may reflect the ease with which this parasite is transmitted. These results suggest that agri-environment schemes such as flower strips, which are known to enhance the nutrition and survival of bumblebees, may also have negative impacts on pollinators through enhanced disease transmission. Future studies should assess how changing the design of these schemes could minimize disease transmission and thus maximise their health benefits to wild pollinators.

Contrasting impacts of a novel specialist vector on multihost viral pathogen epidemiology in wild and managed bees.

Typically, pathogens infect multiple host species. Such multihost pathogens can show considerable variation in their degree of infection and transmission specificity, which has important implications for potential disease emergence. Transmission of multihost pathogens can be driven by key host species and changes in such transmission networks can lead to disease emergence. We study two viruses that show contrasting patterns of prevalence and specificity in managed honeybees and wild bumblebees, black queen cell virus (BQCV) and slow bee paralysis virus (SBPV), in the context of the novel transmission route provided by the virus-vectoring Varroa destructor. Our key result is that viral communities and RNA virus genetic variation are structured by location, not host species or V. destructor presence. Interspecific transmission is pervasive with the same viral variants circulating between pollinator hosts in each location; yet, we found virus-specific host differences in prevalence and viral load. Importantly, V. destructor presence increases the prevalence in honeybees and, indirectly, in wild bumblebees, but in contrast to its impact on deformed wing virus (DWV), BQCV and SBPV viral loads are not increased by Varroa presence, and do not show genetic evidence of recent emergence. Effective control of Varroa in managed honeybee colonies is necessary to mitigate further disease emergence, and alleviate disease pressure on our vital wild bee populations. More generally, our results highlight the over-riding importance of geographical location to the epidemiological outcome despite the complexity of multihost-parasite interactions.

Knock-on community impacts of a novel vector: spillover of emerging DWV-B from Varroa-infested honeybees to wild bumblebees.

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock-on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re-emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV-B overtaking DWV-A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock-on effects.

A novel approach to wildlife transcriptomics provides evidence of disease-mediated differential expression and changes to the microbiome of amphibian populations.

Ranaviruses are responsible for a lethal, emerging infectious disease in amphibians and threaten their populations throughout the world. Despite this, little is known about how amphibian populations respond to ranaviral infection. In the United Kingdom, ranaviruses impact the common frog (Rana temporaria). Extensive public engagement in the study of ranaviruses in the UK has led to the formation of a unique system of field sites containing frog populations of known ranaviral disease history. Within this unique natural field system, we used RNA sequencing (RNA-Seq) to compare the gene expression profiles of R. temporaria populations with a history of ranaviral disease and those without. We have applied a RNA read-filtering protocol that incorporates Bloom filters, previously used in clinical settings, to limit the potential for contamination that comes with the use of RNA-Seq in nonlaboratory systems. We have identified a suite of 407 transcripts that are differentially expressed between populations of different ranaviral disease history. This suite contains genes with functions related to immunity, development, protein transport and olfactory reception among others. A large proportion of potential noncoding RNA transcripts present in our differentially expressed set provide first evidence of a possible role for long noncoding RNA (lncRNA) in amphibian response to viruses. Our read-filtering approach also removed significantly more bacterial reads from libraries generated from positive disease history populations. Subsequent analysis revealed these bacterial read sets to represent distinct communities of bacterial species, which is suggestive of an interaction between ranavirus and the host microbiome in the wild.

Vertically transmitted rhabdoviruses are found across three insect families and have dynamic interactions with their hosts.

A small number of free-living viruses have been found to be obligately vertically transmitted, but it remains uncertain how widespread vertically transmitted viruses are and how quickly they can spread through host populations. Recent metagenomic studies have found several insects to be infected with sigma viruses (Rhabdoviridae). Here, we report that sigma viruses that infect Mediterranean fruit flies (Ceratitis capitata), Drosophila immigrans, and speckled wood butterflies (Pararge aegeria) are all vertically transmitted. We find patterns of vertical transmission that are consistent with those seen in Drosophila sigma viruses, with high rates of maternal transmission, and lower rates of paternal transmission. This mode of transmission allows them to spread rapidly in populations, and using viral sequence data we found the viruses in D. immigrans and C. capitata had both recently swept through host populations. The viruses were common in nature, with mean prevalences of 12% in C. capitata, 38% in D. immigrans and 74% in P. aegeria We conclude that vertically transmitted rhabdoviruses may be widespread in a broad range of insect taxa, and that these viruses can have dynamic interactions with their hosts.

Condition-dependent virulence of slow bee paralysis virus in Bombus terrestris: are the impacts of honeybee viruses in wild pollinators underestimated?

Slow bee paralysis virus (SBPV)-previously considered an obligate honeybee disease-is now known to be prevalent in bumblebee species. SBPV is highly virulent in honeybees in association with Varroa mites, but has been considered relatively benign otherwise. However, condition-dependent pathogens can appear asymptomatic under good, resource abundant conditions, and negative impacts on host fitness may only become apparent when under stressful or resource-limited conditions. We tested whether SBPV expresses condition-dependent virulence in its bumblebee host, Bombus terrestris, by orally inoculating bees with SBPV and recording longevity under satiated and starvation conditions. SBPV infection resulted in significant virulence under starvation conditions, with infected bees 1.6 times more likely to die at any given time point (a median of 2.3 h earlier than uninfected bees), whereas there was no effect under satiated conditions. This demonstrates clear condition-dependent virulence for SBPV in B. terrestris. Infections that appear asymptomatic in non-stressful laboratory assays may nevertheless have significant impacts under natural conditions in the wild. For multi-host pathogens such as SBPV, the use of sentinel host species in laboratory assays may further lead to the underestimation of pathogen impacts on other species in nature. In this case the impact of 'honeybee viruses' on wild pollinators may be underestimated, with detrimental effects on conservation and food security. Our results highlight the importance of multiple assays and multiple host species when testing for virulence, in order for laboratory studies to accurately inform conservation policy and mitigate disease impacts in wild pollinators.

Flies on the move: an inherited virus mirrors Drosophila melanogaster's elusive ecology and demography.

Vertically transmitted parasites rely on their host's reproduction for their transmission, leading to the evolutionary histories of both parties being intimately entwined. Parasites can thus serve as a population genetic magnifying glass for their host's demographic history. Here, we study the fruitfly Drosophila melanogaster's vertically transmitted sigma virus DMelSV. The virus has a high mutation rate and low effective population size, allowing us to reconstruct at a fine scale how the combined forces of the movement of flies and selection on the virus have shaped its migration patterns. We found that the virus is likely to have spread to Europe from Africa, mirroring the colonization route of Drosophila. The North American DMelSV population appears to be the result of a recent single immigration from Europe, invading together with its host in the late 19th century. Across Europe, DMelSV migration rates are low and populations are highly genetically structured, likely reflecting limited fly movement. Despite being intolerant of extreme cold, viral diversity suggests that fly populations can persist in harsh continental climates and that recolonization from the warmer south plays a minor role. In conclusion, studying DMelSV can provide insights into the poorly understood ecology of D. melanogaster, one of the best-studied organisms in biology.

Land-use affects pollinator-specific resource availability and pollinator foraging behaviour.

Land-use management is a key factor causing pollinator declines in agricultural grasslands. This decline can not only be directly driven by land-use (e.g., habitat loss) but also be indirectly mediated through a reduction in floral resource abundance and diversity, which might in turn affect pollinator health and foraging. We conducted surveys of the abundance of flowering plant species and behavioural observations of two common generalist pollinator species, namely the bumblebee Bombus lapidarius and the syrphid fly Episyrphus balteatus, in managed grasslands of variable land-use intensity (LUI) to investigate whether land-use affects (1) resource availability of the pollinators, (2) their host plant selection and (3) pollinator foraging behaviour. We have found that the floral composition of plant species that were used as resource by the investigated pollinator species depends on land-use intensity and practices such as mowing or grazing. We have also found that bumblebees, but not syrphid flies, visit different plants depending on LUI or management type. Furthermore, LUI indirectly changed pollinator behaviour via a reduction in plot-level flower diversity and abundance. For example, bumblebees show longer flight durations with decreasing flower cover indicating higher energy expenditure when foraging on land-use intensive plots. Syrphid flies were generally less affected by local land use, showing how different pollinator groups can differently react to land-use change. Overall, we show that land-use can change resource composition, abundance and diversity for pollinators, which can in turn affect pollinator foraging behaviour and potentially contribute to pollinator decline in agricultural grasslands.

Conservation measures or hotspots of disease transmission? Agri-environment schemes can reduce disease prevalence in pollinator communities.

Insects are under pressure from agricultural intensification. To protect pollinators, conservation measures such as the EU agri-environment schemes (AES) promote planting wildflowers along fields. However, this can potentially alter disease ecology by serving as transmission hubs or by diluting infections. We tested this by measuring plant-pollinator interactions and virus infections (DWV-A, DWV-B and ABPV) across pollinator communities in agricultural landscapes over a year. AES had a direct effect on DWV-B, reducing prevalence and load in honeybees, with a tentative general dilution effect on load in early summer. DWV-A prevalence was reduced both under AES and with increasing niche overlap between competent hosts, likely via a dilution effect. By contrast, AES had no impact on ABPV, its prevalence driven by the proportion of bumblebees in the community. Epidemiological differences were also reflected in the virus phylogenies, with DWV-B showing recent rapid expansion, while DWV-A and ABPV showed slower growth rates and geographical population structure. Phylogenies indicate that all three viruses freely circulate across their host populations. Our study illustrates how complex interactions between environmental, ecological and evolutionary factors may influence wildlife disease dynamics. Supporting pollinator nutrition can mitigate the transmission of important bee diseases, providing an unexpected boost to pollinator conservation. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Increasing flower species richness in agricultural landscapes alters insect pollinator networks: Implications for bee health and competition.

Ecological restoration programs are established to reverse land degradation, mitigate biodiversity loss, and reinstate ecosystem services. Following recent agricultural intensification that led to a decrease in flower diversity and density in rural areas and subsequently to the decline of many insects, conservation measures targeted at pollinators have been established, including sown wildflower strips (WFS) along field margins. Historically successful in establishing a high density of generalist bees and increasing pollinator diversity, the impact of enhanced flower provision on wider ecological interactions and the structure of pollinator networks has been rarely investigated. Here, we tested the effects of increasing flower species richness and flower density in agricultural landscapes on bee-plant interaction networks. We measured plant species richness and flower density and surveyed honeybee and bumblebee visits on flowers across a range of field margins on 10 UK farms that applied different pollinator conservation measures. We found that both flower species richness and flower density significantly increased bee abundance, in early and late summer, respectively. At the network level, we found that higher flower species richness did not significantly alter bee species' generality indices, but significantly reduced network connectance and marginally reduced niche overlap across honeybees and bumblebee species, a proxy for insect competition. While higher connectance and niche overlap is believed to strengthen network robustness and often is the aim for the restoration of pollinator networks, we argue that carefully designed WFS may benefit bees by partitioning their foraging niche, limiting competition for resources and the potential for disease transmission via shared floral use. We also discuss the need to extend WFS and their positive effects into spring when wild bee populations are established.

A second generation genetic map of the bumblebee Bombus terrestris (Linnaeus, 1758) reveals slow genome and chromosome evolution in the Apidae.

BACKGROUND: The bumblebee Bombus terrestris is an ecologically and economically important pollinator and has become an important biological model system. To study fundamental evolutionary questions at the genomic level, a high resolution genetic linkage map is an essential tool for analyses ranging from quantitative trait loci (QTL) mapping to genome assembly and comparative genomics. We here present a saturated linkage map and match it with the Apis mellifera genome using homologous markers. This genome-wide comparison allows insights into structural conservations and rearrangements and thus the evolution on a chromosomal level. RESULTS: The high density linkage map covers ~ 93% of the B. terrestris genome on 18 linkage groups (LGs) and has a length of 2'047 cM with an average marker distance of 4.02 cM. Based on a genome size of ~ 430 Mb, the recombination rate estimate is 4.76 cM/Mb. Sequence homologies of 242 homologous markers allowed to match 15 B. terrestris with A. mellifera LGs, five of them as composites. Comparing marker orders between both genomes we detect over 14% of the genome to be organized in synteny and 21% in rearranged blocks on the same homologous LG. CONCLUSIONS: This study demonstrates that, despite the very high recombination rates of both A. mellifera and B. terrestris and a long divergence time of about 100 million years, the genomes' genetic architecture is highly conserved. This reflects a slow genome evolution in these bees. We show that data on genome organization and conserved molecular markers can be used as a powerful tool for comparative genomics and evolutionary studies, opening up new avenues of research in the Apidae.