Ontogeny of immunity and natural viral infection in Apis mellifera drones and workers.

The most common viral diseases affecting honey bees (Apis mellifera) in Israel include deformed wing viruses (DWV-A and DWV-B) and acute paralysis viruses (ABPV and IAPV). These viruses are transmitted within and between colonies, both horizontally and vertically. All members of the colony contribute to this transmission, on the other hand individual and social immunity, particularly hygienic behaviour, may affect the outcome of the process. In this study, we evaluated the ontogeny of natural infections of DWV-A, DWV-B, ABPV and IAPV, their prevalence and loads, in workers and drones from high (H) and low (L) hygienic colonies. In parallel, we evaluated the expression of two immune genes: peptidoglycan recognition protein S2(PGRP-S2) and hymenoptaecin. The prevalence of DWV-B and IAPV increased with age and was higher in workers than in drones. ABPV was not detected in drones. The expression of both immune genes was significantly affected by age and sex. Drones from H colonies had higher expression of these genes. The increased expression of immune genes with drones' age, particularly in hygienic colonies, suggest additional value of honey bee breeding for hygienic behaviour for sustainable beekeeping.

Virus transmission via honey bee prey and potential impact on cocoon-building in labyrinth spiders (Agelena labyrinthica).

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.

Multiple benefits of breeding honey bees for hygienic behavior.

Honey bee colonies are prone to invasion by pests and pathogens. The combination of the parasitic mite Varroa destructor (Varroa) and the multiple viruses it vectors, is a major driver of colony losses. Breeding for hygienic behavior to reduce Varroa populations is considered a sustainable way to reduce the impact of Varroa on honey bee health. However, hygienic behavior may have a cost to the health of individual bees, both in terms of viral infection risk and immune function. To determine whether selection for hygienic behavior at the colony level is associated with trade-offs in honey bee viral infection and immune function, we compared Varroa populations, viral loads, and individual immune function between honey bee colonies that were bred for high and low hygienic behavior. Specifically, we measured Varroa infestation, Deformed wing virus DWV-A, DWV-B, Acute bee paralysis virus (ABPV), and Israeli acute paralysis virus IAPV viral genome levels in bee samples from artificially inseminated queens in our bi-directional selection program for hygienic behavior in Israel. In addition, we evaluated the expression of 12 genes from the Jak-STAT, Toll, IMD and RNAi immune pathways. We found significantly lower Varroa infestation and DWV loads in highly hygienic colonies than in colonies exhibiting low hygienic behavior. In addition, workers of the hygienic colonies had significantly higher expression of the immune genes PGRP-S2 and hymenoptaecin compared to workers from low hygienic colonies. These results indicate no trade-offs in breeding for hygienic behavior. Hygienic honey bees were associated with reduced Varroa populations and reduced DWV prevalence or load at the colony level. Individual immunity of hygienic bees was increased, which could also contribute to lower virus levels, although lower Varroa levels due to social immunity presumably contributed as well. In sum, we demonstrate multiple health benefits of breeding for honey bee hygiene.

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.

A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.).

With a growing number of parasites and pathogens experiencing large-scale range expansions, monitoring diversity in immune genes of host populations has never been so important because it can inform on the adaptive potential to resist the invaders. Population surveys of immune genes are becoming common in many organisms, yet they are missing in the honey bee (Apis mellifera L.), a key managed pollinator species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in a wide range of honey bee immune genes and developed a medium-density assay targeting a subset of these genes. Using a discovery panel of 123 whole-genomes, representing seven A. mellifera subspecies and three evolutionary lineages, 180 immune genes were scanned for SNPs in exons, introns (< 4 bp from exons), 3' and 5´UTR, and < 1 kb upstream of the transcription start site. After application of multiple filtering criteria and validation, the final medium-density assay combines 91 quality-proved functional SNPs marking 89 innate immune genes and these can be readily typed using the high-sample-throughput iPLEX MassARRAY system. This medium-density-SNP assay was applied to 156 samples from four countries and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that honey bee ancestry can be delineated from functional variation. In addition to allowing analysis of immunogenetic variation, this newly-developed SNP assay can be used for inferring genetic structure and admixture in the honey bee.

Metatranscriptome Analysis of Sympatric Bee Species Identifies Bee Virus Variants and a New Virus, Andrena-Associated Bee Virus-1.

Bees are important plant pollinators in agricultural and natural ecosystems. High average annual losses of honey bee (Apis mellifera) colonies in some parts of the world, and regional population declines of some mining bee species (Andrena spp.), are attributed to multiple factors including habitat loss, lack of quality forage, insecticide exposure, and pathogens, including viruses. While research has primarily focused on viruses in honey bees, many of these viruses have a broad host range. It is therefore important to apply a community level approach in studying the epidemiology of bee viruses. We utilized high-throughput sequencing to evaluate viral diversity and viral sharing in sympatric, co-foraging bees in the context of habitat type. Variants of four common viruses (i.e., black queen cell virus, deformed wing virus, Lake Sinai virus 2, and Lake Sinai virus NE) were identified in honey bee and mining bee samples, and the high degree of nucleotide identity in the virus consensus sequences obtained from both taxa indicates virus sharing. We discovered a unique bipartite + ssRNA Tombo-like virus, Andrena-associated bee virus-1 (AnBV-1). AnBV-1 infects mining bees, honey bees, and primary honey bee pupal cells maintained in culture. AnBV-1 prevalence and abundance was greater in mining bees than in honey bees. Statistical modeling that examined the roles of ecological factors, including floral diversity and abundance, indicated that AnBV-1 infection prevalence in honey bees was greater in habitats with low floral diversity and abundance, and that interspecific virus transmission is strongly modulated by the floral community in the habitat. These results suggest that land management strategies that aim to enhance floral diversity and abundance may reduce AnBV-1 spread between co-foraging bees.

Infection of a Lepidopteran Cell Line with Deformed Wing Virus.

Many attempts to develop a reliable cell cultured-based system to study honey bee virus infections have encountered substantial difficulties. We investigated the ability of a cell line from a heterologous insect to sustain infection by a honey bee virus. For this purpose, we infected the Lepidopteran hemocytic cell line (P1) with Deformed wing virus (DWV). The genomic copies of DWV increased upon infection, as monitored by quantitative RT-PCR. Moreover, a tagged-primer-based RT-PCR analysis showed the presence of DWV negative-sense RNA in the cells, indicating virus replication. However, the DWV from infected cells was mildly infectious to P1 cells. Similar results were obtained when the virus was injected into Apis mellifera pupae. Thus, though the virus yields from the infected cells appeared to be very low, we show for the first time that DWV can replicate in a heterologous cell line. Given the availability of many other insect cell lines, our study paves the way for future exploration in this direction. In the absence of adequate A. mellifera cell lines, exploring the ability of alternative cell lines to enable honey bee virus infections could provide the means to study and understand the viral infectious cycle at the cellular level and facilitate obtaining purified isolates of these viruses.

Bee Viruses: Routes of Infection in Hymenoptera.

Numerous studies have recently reported on the discovery of bee viruses in different arthropod species and their possible transmission routes, vastly increasing our understanding of these viruses and their distribution. Here, we review the current literature on the recent advances in understanding the transmission of viruses, both on the presence of bee viruses in Apis and non-Apis bee species and on the discovery of previously unknown bee viruses. The natural transmission of bee viruses will be discussed among different bee species and other insects. Finally, the research potential of in vivo (host organisms) and in vitro (cell lines) serial passages of bee viruses is discussed, from the perspective of the host-virus landscape changes and potential transmission routes for emerging bee virus infections.

Diversity and Global Distribution of Viruses of the Western Honey Bee, Apis mellifera.

In the past centuries, viruses have benefited from globalization to spread across the globe, infecting new host species and populations. A growing number of viruses have been documented in the western honey bee, Apis mellifera. Several of these contribute significantly to honey bee colony losses. This review synthetizes the knowledge of the diversity and distribution of honey-bee-infecting viruses, including recent data from high-throughput sequencing (HTS). After presenting the diversity of viruses and their corresponding symptoms, we surveyed the scientific literature for the prevalence of these pathogens across the globe. The geographical distribution shows that the most prevalent viruses (deformed wing virus, sacbrood virus, black queen cell virus and acute paralysis complex) are also the most widely distributed. We discuss the ecological drivers that influence the distribution of these pathogens in worldwide honey bee populations. Besides the natural transmission routes and the resulting temporal dynamics, global trade contributes to their dissemination. As recent evidence shows that these viruses are often multihost pathogens, their spread is a risk for both the beekeeping industry and the pollination services provided by managed and wild pollinators.

Foodborne Transmission and Clinical Symptoms of Honey Bee Viruses in Ants Lasius spp.

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.

Foodborne Transmission of Deformed Wing Virus to Ants (Myrmica rubra).

Virus host shifts occur frequently, but the whole range of host species and the actual transmission pathways are often poorly understood. Deformed wing virus (DWV), an RNA virus described from honeybees (Apis mellifera), has been shown to have a broad host range. Since ants are often scavenging on dead honeybees, foodborne transmission of these viruses may occur. However, the role of the ant Myrmica rubra as an alternative host is not known and foodborne transmission to ants has not been experimentally addressed yet. Here, we show with a 16-week feeding experiment that foodborne transmission enables DWV type-A and -B to infect M. rubra and that these ants may serve as a virus reservoir. However, the titers of both plus- and minus-sense viral RNA strands decreased over time. Since the ants were fed with highly virus-saturated honeybee pupae, this probably resulted in initial viral peaks, then approaching lower equilibrium titers in infected individuals later. Since DWV infections were also found in untreated field-collected M. rubra colonies, our results support the wide host range of DWV and further suggest foodborne transmission as a so far underestimated spread mechanism.

New Viruses from the Ectoparasite Mite Varroa destructor Infesting Apis mellifera and Apis cerana.

Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana(A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V.destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m.syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m.ligustica and A. m.syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the first report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites.

Nosema ceranae in Apis mellifera: a 12 years postdetection perspective.

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat.

Protein nutrition governs within-host race of honey bee pathogens.

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.

Presence of Apis Rhabdovirus-1 in Populations of Pollinators and Their Parasites from Two Continents.

The viral ecology of bee communities is complex, where viruses are readily shared among co-foraging bee species. Additionally, in honey bees (Apis mellifera), many viruses are transmitted - and their impacts exacerbated - by the parasitic Varroa destructor mite. Thus far, the viruses found to be shared across bee species and transmitted by V. destructor mites are positive-sense single-stranded RNA viruses. Recently, a negative-sense RNA enveloped virus, Apis rhabdovirus-1 (ARV-1), was found in A. mellifera honey bees in Africa, Europe, and islands in the Pacific. Here, we describe the identification - using a metagenomics approach - of ARV-1 in two bee species (A. mellifera and Bombus impatiens) and in V. destructor mites from populations collected in the United States and Israel. We confirmed the presence of ARV-1 in pools of A. mellifera, B. impatiens, and V. destructor from Israeli and U.S. populations by RT-PCR and found that it can reach high titers in individual honey bees and mites (107-108 viral genomic copies per individual). To estimate the prevalence of ARV-1 in honey bee populations, we screened 104 honey bee colonies across Israel, with 21 testing ARV-1-positive. Tagged-primer-mediated RT-PCR analysis detected the presence of the positive-sense ARV-1 RNA in A. mellifera and V. destructor, indicating that ARV-1 replicates in both hosts. This is the first report of the presence of ARV-1 in B. impatiens and of the replication of a rhabdovirus in A. mellifera and V. destructor. Our data suggest that Varroa mites could act as an ARV-1 vector; however, the presence of ARV-1 in B. impatiens (which are not parasitized by Varroa) suggests that it may not require the mite for transmission and ARV-1 may be shared among co-foraging bee species. Given that ARV-1 is found in non-Apis bee species, and because "ARV" is used for the Adelaide River virus, we propose that this virus should be called bee rhabdovirus 1 and abbreviated BRV-1. These results greatly expand our understanding of the diversity of viruses that can infect bee communities, though further analysis is required to determine how infection with this virus impacts these different hosts.

Two novel viruses associated with the Apis mellifera pathogenic mite Varroa destructor.

Varroa destructor infestation of Apis mellifera colonies carries and/or promotes replication of honey bee viruses like the Deformed wing virus, the Varroa destructor virus-1, the Acute bee paralysis virus, the Israeli acute bee paralysis virus and the Kashmir bee virus that have been well described and characterized; but viruses exclusively associated with Varroa were not found. To look for viruses that may associate with- or infect V. destructor we performed deep sequencing (RNA-seq) of RNA extracted from honey bees and mites in Varroa-infested untreated colonies. Comparative bioinformatic analysis of the two separate contig-assemblies generated from the sequences' reads annotated using Blastx enabled identification of new viruses unique to Varroa and absent in A. mellifera: an Iflavirus and a virus with homology to Ixodes scapularis associated virus 2, that we named Varroa destructor virus 2 (VDV-2) and 3(VDV-3), respectively. We validated these findings sequencing the mite- and honey bee-viromes and in separate mites and honey bees randomly sampled. The complete genomes of VDV-2 and VDV-3 bear 9576 nucleotides and 4202 nucleotides, respectively. Phylogenetic analysis of VDV-3 suggests that it belongs to a new group of viruses. Our results open venues for investigating the pathogenicity of these V. destructor viruses.

Using the Baculovirus/Insect Cell System to Study Apoptosis.

Apoptosis is a physiological program of cell suicide conserved in invertebrates and vertebrates. Apoptosis is crucial to the normal development of organisms and in tissue homeostasis by promoting elimination of unwanted cells, including damaged or virus-infected cells. Due to the importance of programmed cell death for the survival of the organism, a tight regulation is exerted at various activation levels of the cell-death machinery. The utilization of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to identify genes that inhibit the apoptotic process will be described using a transfection-based approach, illustrated by identification of the p49 gene.

Dynamics of the presence of israeli acute paralysis virus in honey bee colonies with colony collapse disorder.

The determinants of Colony Collapse Disorder (CCD), a particular case of collapse of honey bee colonies, are still unresolved. Viruses including the Israeli acute paralysis virus (IAPV) were associated with CCD. We found an apiary with colonies showing typical CCD characteristics that bore high loads of IAPV, recovered some colonies from collapse and tested the hypothesis if IAPV was actively replicating in them and infectious to healthy bees. We found that IAPV was the dominant pathogen and it replicated actively in the colonies: viral titers decreased from April to September and increased from September to December. IAPV extracted from infected bees was highly infectious to healthy pupae: they showed several-fold amplification of the viral genome and synthesis of the virion protein VP3. The health of recovered colonies was seriously compromised. Interestingly, a rise of IAPV genomic copies in two colonies coincided with their subsequent collapse. Our results do not imply IAPV as the cause of CCD but indicate that once acquired and induced to replication it acts as an infectious factor that affects the health of the colonies and may determine their survival. This is the first follow up outside the US of CCD-colonies bearing IAPV under natural conditions.