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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., (© 2021. The Author(s).)

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.

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.

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 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., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

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.

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.

Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A country-wide screen for viral pathogens in Israeli apiaries revealed significant incidence of deformed wing virus (DWV) and Varroa destructor-1 virus (VDV-1). To understand these viruses' possible involvement in deformed wing syndrome of honey bees, we studied their replication in symptomatically and asymptomatically infected bees qualitatively and quantitatively, using RT-PCR, quantitative real-time RT-PCR, and immunodetection of the major viral capsid protein VP1. We found, for the first time, replication of VDV-1 and/or a VDV-1-DWV recombinant virus in the heads of recently emerged symptomatic bees. These viruses replicated to high copy numbers, yielding the major viral capsid VP1 processed for subsequent assembly of viral particles. Our results clearly distinguished between symptomatic and asymptomatic bees infected with VDV-1 and VDV-1-DWV and suggest the hypothesis that VDV-1, in addition to DWV, may be involved in inducing the deformed wing pathology. Thus VDV-1-DWV recombination may yield virulent strains able to cause overt infections in Varroa-infested bee colonies., (Copyright © 2011 Elsevier Inc. All rights reserved.)