The presence of identical deformed wing virus sequence variants in co-occurring Apis species in Northern Thailand may represent a potential epidemiological threat to native honey bees of Southeast Asia.

Widespread native honey bee species in South and East Asia (Apis cerana, Apis dorsata and Apis florea) and the imported western honey bee (Apis mellifera) share habitats and potentially also share pathogens. Chief among the threats facing A. mellifera in Europe and North America is deformed wing virus (DWV), including its two principal genotypes: A and B (DWV-A and DWV-B respectively). Though DWV-A has been recorded in Asia's native Apis species, it is not known if DWV-B, or both DWV-A and DWV-B, are currently widespread in Asia and, if so, whether viral transmission is primarily intraspecific or interspecific. This study aims to fill these knowledge gaps by (i) determining the DWV genotype in four co-occurring Apis host species using qPCR and (ii) inferring viral transmission between them using nucleotide sequences of DWV from Apis host species collected at three independent localities in Northern Thailand. We found DWV-A and -B in all four Apis species, the exotic A. mellifera and the native A. cerana, A. dorsata and A. florea. That DWV-A sequences were identical across Apis species at the same locality, with a similar pattern for DWV-B sequences, suggests that DWV's epidemiology is largely driven by ongoing interspecific transmission (spillover) of DWV across co-occurring native and exotic Apis species. Both genotypes of DWV represent a serious threat to Asia's exotic and native honey bee species.

Experimental cross species transmission of a major viral pathogen in bees is predominantly from honeybees to bumblebees.

Cross-species transmission of a pathogen from a reservoir to a recipient host species, spillover, can have major impacts on biodiversity, domestic species and human health. Deformed wing virus (DWV) is a panzootic RNA virus in honeybees that is causal in their elevated colony losses, and several correlative field studies have suggested spillover of DWV from managed honeybees to wild bee species such as bumblebees. Yet unequivocal demonstration of DWV spillover is lacking, while spillback, the transmission of DWV from a recipient back to the reservoir host, is rarely considered. Here, we show in fully crossed laboratory experiments that the transmission of DWV (genotype A) from honeybees to bumblebees occurs readily, yet we neither detected viral transmission from bumblebees to honeybees nor onward transmission from experimentally infected to uninoculated bumblebees. Our results support the potential for viral spillover from honeybees to other bee species in the field when robbing resources from heterospecific nests or when visiting the same flowers. They also underscore the importance of studies on the virulence of DWV in wild bee species so as to evaluate viral impact on individual and population fitness as well as viral adaption to new host species.

Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan.

The essential ecosystem service of pollination is provided largely by insects, which are considered threatened by diverse biotic and abiotic global change pressures. RNA viruses are one such pressure, and have risen in prominence as a major threat for honey bees (Apis mellifera) and global apiculture, as well as a risk factor for other bee species through pathogen spill-over between managed honey bees and sympatric wild pollinator communities. Yet despite their potential role in global bee decline, the prevalence of honey bee-associated RNA viruses in wild bees is poorly known from both geographic and taxonomic perspectives. We screened members of pollinator communities (honey bees, bumble bees and other wild bees belonging to four families) collected from apple orchards in Georgia, Germany and Kyrgyzstan for six common honey bee-associated RNA virus complexes encompassing nine virus targets. The Deformed wing virus complex (DWV genotypes A and B) had the highest prevalence across all localities and host species and was the only virus complex found in wild bee species belonging to all four studied families. Based on amplification of negative-strand viral RNA, we found evidence for viral replication in wild bee species of DWV-A/DWV-B (hosts: Andrena haemorrhoa and several Bombus spp.) and Black queen cell virus (hosts: Anthophora plumipes, several Bombus spp., Osmia bicornis and Xylocopa spp.). Viral amplicon sequences revealed that DWV-A and DWV-B are regionally distinct but identical in two or more bee species at any one site, suggesting virus is shared amongst sympatric bee taxa. This study demonstrates that honey bee associated RNA viruses are geographically and taxonomically widespread, likely infective in wild bee species, and shared across bee taxa.

Nursing Honeybee Behavior and Sensorial-Related Genes Are Altered by Deformed Wing Virus Variant A.

Insect behavior is coordinated mainly by smell through the diverse odor-binding proteins (OBP) that allow them to identify and recognize their environment. Sensory information collected through smell is then analyzed and interpreted in the brain, allowing for correct insect functioning. The behavior of honeybees (Apis mellifera L.) can be affected by different pathogens, such as deformed wing virus (DWV). In particular, the DWV variant A (DWV-A) is capable of altering olfactory sensitivity and reducing the gene expression of different OBPs, including those associated with nursing behavior. The DWV is also capable of replicating itself in the sensory lobes of the brain, further compromising the processing of sensory information. This study evaluated the behavioral response of nurse honeybees exposed to a pheromone compound and the alterations in the gene expression of the pre- and post-synaptic neuronal genes neuroxins-1 and neurogilin-1 in the bee heads and OBP proteins in the antennae of nurse bees inoculated with DWV-A. The behavioral response of nurse bees exposed to the larval pheromone compound benzyl alcohol was analyzed using a Y-tube olfactometer. The viral load, the gene expression of OBP5 and OBP11 in antennae, and neuroxins-1 and neurogilin-1 in the bee heads were analyzed via qPCR. High viral loads significantly reduced the ability of 10- and 15-day-old nurse honeybees to choose the correct pheromone compound. Also, the gene expression of OBP5, OBP11, neuroxin-1, and neurogilin-1 in nurse honeybees decreased when they were highly infected with DWV-A. These results suggest that a DWV-A infection can disturb information processing and cause nursing honeybees to reduce their activity inside the hive, altering internal cohesion.

Interaction between Thiamethoxam and Deformed Wing Virus Type A on Wing Characteristics and Expression of Immune and Apoptosis Genes in Apis mellifera.

Honey bees are economically important insects for crop pollination. They play a significant role as pollinators of wild plants and agricultural crops and produce economical products, such as honey, royal jelly, wax, pollen, propolis, and venom. Despite their ecological and economical importance, the global honey bee population is in decline due to factors including pathogens, parasites, intensive agriculture, and pesticides. Moreover, these factors may be interlinked and exacerbate the loss of honey bees. This study aimed to investigate the interaction between a pesticide, thiamethoxam, and deformed wing virus type A (DWV-A) to honey bees and the effects on survival rate, wing characteristics, and expression of immune and apoptosis genes in Apis mellifera. We described the potential interaction between thiamethoxam and DWV-A on honey bee wing characteristics, DWV-A loads, and the expressions of immune (defensin, abaecin, and hymenoptaecin) and apoptosis genes (buffy, apaf1, caspase3-like, caspase8-like, and caspase9-like). Honey bee larvae were fed with three different thiamethoxam doses (0.001, 1.4, and 14.3 ng/µL of the diet). Then, thiamethoxam-treated white-eyed pupae were injected with 107 copy numbers/honey bee of the DWV-A genome. The interaction between thiamethoxam and DWV-A caused a high mortality rate, crippled wings in newly emerged adult honey bees (100%), and resulted in induced expression of hymenoptaecin gene compared to the control group, while downregulation of caspase8-like, caspase9-like genes compared to the DWV injection group. Therefore, the potential interaction between thiamethoxam and DWV-A might have a deleterious effect on honey bee lifespan. The results from this study could be used as a tool to combat DWV-A infection and mitigate pesticide usage to alleviate the decrease in the honey bee population.

Variant A of the Deformed Wings Virus Alters the Olfactory Sensitivity and the Expression of Odorant Binding Proteins on Antennas of Apis mellifera.

Insects have a highly sensitive sense of smell, allowing them to perform complex behaviors, such as foraging and peer recognition. Their sense of smell is based on the recognition of ligands and is mainly coordinated by odorant-binding proteins (OBPs). In Apis mellifera, behavior can be affected by different pathogens, including deformed wing virus (DWV) and its variants. In particular, it has been shown that variant A of DWV (DWV-A) is capable of altering the ultra-cellular structure associated with olfactory activity. In this study was evaluated olfactory sensitivity and the expression of OBP genes in honey bees inoculated with DWV-A. Electroantennographic analyses (EAG) were carried out to determine the olfactory sensitivity to the essential oils Eucalyptus globulus and Mentha piperita. The expression of nine antenna-specific OBP genes and DWV-A load in inoculated bees was also quantified by qPCR. We observed an inverse relationship between viral load and olfactory sensitivity and the expression of some OBP proteins. Thus, high viral loads reduced olfactory sensitivity to essential oils and the gene expression of the OBP2, OBP5, OBP11, and OBP12 proteins on the antennas of middle- and forager-age bees. These results suggest that DWV-A could have negative effects on the processes of aroma perception by worker bees, affecting their performance in tasks carried out in and outside the colony.

Honey Bee Virus Transmission via Hive Products.

The global trade of honey bee hive products has raised concern about pathogen transmission. However, the efficacy of hive products as virus vehicles is poorly understood. Here, we investigated the transmission capacity of hive products for Deformed wing virus genotype A (DWV-A) in a fully-crossed hoarding cage experiment and estimated the transmission risk by screening commercial products. Western honey bee workers were provided with honey, pollen and wax either contaminated with high (~2 × 109), medium (~1.7 × 108), low (~8 × 106) or zero (control) DWV-A genome copies. For 10 days, mortality was monitored. Then, virus titers were quantified in bee heads and 38 commercial products using RT-qPCR. For honey and pollen, a positive association between DWV-A concentration and mortality was observed. High concentrations always resulted in infections, medium ones in 47% of cases and low ones in 20% of cases. No significant difference was observed between the tested products. In commercial honey and pollen, 7.7 × 102-1.8 × 105 and 1.4 × 103-1.3 × 104 DWV-A copies per gram were found, respectively. The results show that DWV-A transmission via hive products is feasible. The risk of introducing novel viruses and/or strains should be considered in trade regulations by including virus analyses for health certificates of hive products.