Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.     

Virus infections in Brazilian honey bees

This work describes the first molecular-genetic evidence for viruses in Brazilian honey bee samples. Three different bee viruses, Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), and Deformed wing virus (DWV) were identified during a screening of RNAs from 1920 individual adult bees collected in a region of southeastern Brazil that has recently shown unusual bee declines. ABPV was detected in 27.1% of colony samples, while BQCV and DWV were found in 37% and 20.3%, respectively. These levels are substantially lower than the frequencies found for these viruses in surveys from other parts of the world. We also developed and validated a multiplex RT-PCR assay for the simultaneous detection of ABPV, BQCV, and DWV in Brazil.     

Molecular and phylogenetic characterization of honey bee viruses,Nosema microsporidia,protozoan parasites,and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana‐infecting SBV, and relatively homogenous populations of IAPV and A. meliifera‐infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast‐encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.     

Multiple virus infections in the honey bee and genome divergence of honey bee viruses

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.     

Pollen diets and niche overlap of honey bees and native bees in protected areas

The decline of both managed and wild bee populations has been extensively reported for over a decade now, with growing concerns amongst the scientific community. Also, evidence is growing that both managed and feral honey bees may exacerbate threats to wild bees. In Australia, there are over 1600 native bee species and introduced European honey bees (Apis mellifera) have established throughout most landscapes. There is a major gap in knowledge of the interactions between honey bees and native bees in Australian landscapes, especially floral resource use.Here we report on the pollen diets of wild bees in protected areas of coastal heathland, an ecosystem characterised by mass flowering in late winter and spring. We sampled bees within three sites and DNA metabarcoding was used to compare the pollen diets of honey bees and native bees. We recorded 2, 772 bees in total, with 13 genera and 18 described species identified. Apis mellifera was the most common species across all locations, accounting for 42% of all bees collected. Native bee genera included eusocial Tetragonula (stingless bees) (37%), and semi-social Exoneura and Braunsapis (19.8% combined). Metabarcoding data revealed both Tetragonula and honey bees have wide foraging patterns, and the bipartite network overall was highly generalised (H₂’ = 0.24). Individual honey bees carried pollen of 7–29 plant species, and significantly more species than all other bees. We found niche overlap in the diets of honey bees and native bees generally (0.42), and strongest overlap with stingless bees (0.70) and species of Braunsapis (0.62). A surprising finding was that many species carried pollen from Restionaceae and Cyperaceae, families generally considered to be predominantly wind-pollinated in Australia. Our study showed introduced honey bee use of resources overlaps with that of native bees in protected heathlands, but there are clear differences in their diet preferences.     

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.     

First molecular detection of a viral pathogen in Ugandan honey bees

Ugandan honey bees (Apis mellifera L.) produce honey, and are key pollinators within commercial crops and natural ecosystems. Real-time RT-PCR was used to screen immature and adult bees collected from 63 beekeeping sites across Uganda for seven viral pathogens. No samples tested positive for Chronic bee paralysis virus, Sacbrood virus, Deformed wing virus, Acute bee paralysis virus, Apis iridescent virus or Israeli acute paralysis virus. However, Black queen cell virus (BQCV) was found in 35.6% of samples. It occurred in adults and larvae, and was most prevalent in the Western highlands, accounting for over 40% of positive results nationally.     

New evidence that deformed wing virus and black queen cell virus are multi-host pathogens

The host-range breadth of pathogens can have important consequences for pathogens' long term evolution and virulence, and play critical roles in the emergence and spread of the new diseases. Black queen cell virus (BQCV) and Deformed wing virus (DWV) are the two most common and prevalent viruses in European honey bees, Apis mellifera. Here we provide the evidence that BQCV and DWV infect wild species of honey bees, Apis florea and Apis dorsata. Phylogenetic analyses suggest that these viruses might have moved from A. mellifera to wild bee species and that genetic relatedness as well as the geographical proximity of host species likely play an important role in host range of the viruses. The information obtained from this present study can have important implication for understanding the population structure of bee virus as well as host-virus interactions.     

Admixture increases diversity in managed honey bees: Reply to De la Rúa et al. (2013)

De la Rúa et al. (2013) express some concerns about the conclusions of our recent study showing that management increases genetic diversity of honey bees (Apis mellifera) by promoting admixture (Harpur et al. 2012). We provide a brief review of the literature on the population genetics of A. mellifera and show that we utilized appropriate sampling methods to estimate genetic diversity in the focal populations. Our finding of higher genetic diversity in two managed A. mellifera populations on two different continents is expected to be the norm given the large number of studies documenting admixture in honey bees. Our study focused on elucidating how management affects genetic diversity in honey bees, not on how to best manage bee colonies. We do not endorse the intentional admixture of honey bee populations, and we agree with De la Rúa et al. (2013) that native honey bee subspecies should be conserved.     

The novel insecticides flupyradifurone and sulfoxaflor do not act synergistically with viral pathogens in reducing honey bee (Apis mellifera) survival but sulfoxaflor modulates host immunocompetence

The decline of insect pollinators threatens global food security. A major potential cause of decline is considered to be the interaction between environmental stressors, particularly between exposure to pesticides and pathogens. To explore pesticide–pathogen interactions in an important pollinator insect, the honey bee, we used two new nicotinic acetylcholine receptor agonist insecticides (nACHRs), flupyradifurone (FPF) and sulfoxaflor (SULF), at sublethal and field-realistic doses in a fully crossed experimental design with three common viral honey bee pathogens, Black queen cell virus (BQCV) and Deformed wing virus (DWV) genotypes A and B. Through laboratory experiments in which treatments were administered singly or in combination to individual insects, we recorded harmful effects of FPF and pathogens on honey bee survival and immune gene expression. Though we found no evidence of synergistic interactions among stressors on either honey bee survival or viral load, the combined treatment SULF and DWV-B led to a synergistic upregulation of dicer-like gene expression. We conclude that common viral pathogens pose a major threat to honey bees, while co-exposure to these novel nACHR insecticides does not significantly exacerbate viral impacts on host survival in the laboratory.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Orchard pollination in Capitol Reef National Park,Utah, USA. Honey bees or native bees?

Capitol Reef National Park in central Utah, USA surrounds 22 managed fruit orchards started over a century ago by Mormon pioneers. Honey bees are imported for pollination, although the area in which the Park is embedded has over 700 species of native bees, many of which are potential orchard pollinators. We studied the visitation of native bees to apple, pear, apricot, and sweet cherry over 2 years. Thirty species of bees visited the flowers but, except for pear flowers, most were uncommon compared to honey bees. Evidence that honey bees prevented native bees from foraging on orchard crop flowers was equivocal: generally, honey bee and native bee visitation rates to the flowers were not negatively correlated, nor were native bee visitation rates positively correlated with distance of orchards from honey bee hives. Conversely, competition was tentatively suggested by much larger numbers of honey bees than natives on the flowers of apples, apricots and cherry; and by the large increase of native bees on pears, where honey bee numbers were low. At least one-third of the native bee species visiting the flowers are potential pollinators, including cavity-nesting species such as Osmia lignaria propinqua, currently managed for small orchard pollination in the US, plus several fossorial species, including one rosaceous flower specialist (Andrena milwaukiensis). We suggest that gradual withdrawal of honey bees from the Park would help conserve native bee populations without decreasing orchard crop productivity, and would serve as a demonstration of the commercial value of native pollinators.     

Genetic evidence for coinfection of honey bees by acute bee paralysis and Kashmir bee viruses

Nucleotide sequence analyses were used to identify acute bee paralysis virus (ABPV) and Kashmir bee virus (KBV) isolated from a single honey bee colony. Most of the bees in this colony carried KBV. Some individual bees also carried ABPV, a coexistence not yet seen between these two viruses. Implications of coinfection on viral efficacy are discussed, along with a restriction enzyme assay that can be used to discriminate between these two widespread viruses.     

Sudden deaths and colony population decline in Greek honey bee colonies

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor,Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow up in-depth survey across all Greek regions is required to provide context to these preliminary findings.     

Effects of Imidacloprid and Varroa destructor on survival and health of European honey bees,Apis mellifera

There has been growing concern over declines in populations of honey bees and other pollinators which are a vital part to our food security. It is imperative to identify factors responsible for accelerated declines in bee populations and develop solutions for reversing bee losses. While exact causes of colony losses remain elusive, risk factors thought to play key roles are ectoparasitic mites Varroa destructor and neonicotinoid pesticides. The present study aims to investigate effects of a neonicotinoid pesticide Imidacloprid and Varroa mites individually on survivorship, growth, physiology, virus dynamics and immunity of honey bee workers. Our study provides clear evidence that the exposure to sublethal doses of Imidacloprid could exert a significantly negative effect on health and survival of honey bees. We observed a significant reduction in the titer of vitellogenin (Vg), an egg yolk precursor that regulates the honey bees development and behavior and often are linked to energy homeostasis, in bees exposed to Imidacloprid. This result indicates that sublethal exposure to neonicotinoid could lead to increased energy usage in honey bees as detoxification is a energy‐consuming metabolic process and suggests that Vg could be a useful biomarker for measuring levels of energy stress and sublethal effects of pesticides on honey bees. Measurement of the quantitative effects of different levels of Varroa mite infestation on the replication dynamic of Deformed wing virus (DWV), an RNA virus associated with Varroa infestation, and expression level of immune genes yields unique insights into how honey bees respond to stressors under laboratory conditions.     

Viral Infection Affects Sucrose Responsiveness and Homing Ability of Forager Honey Bees,Apis mellifera L.

Honey bee health is mainly affected by Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 10⁷ copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.     

The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni

The prevalence of nine honey bee viruses in samples of dead adult bees from Apis mellifera colonies in the Netherlands and Germany infested with the parasitic mite Varroa jacobsoni was compared with virus incidence in uninfested colonies in Britain. In colonies with low mite populations the viruses present and their incidence during the year were similar to the results obtained from British colonies. However, in marked contrast with findings in Britain, acute paralysis virus (APV) was the primary cause of adult bee mortality in German honey bee colonies severely infested with V. jacobsoni. Dead brood from unsealed and sealed infested cells from German colonies with high mite populations also contained much APV. The evidence suggests that V. jacobsoni activates APV replication in adult bees by its feeding behaviour and transmits virus from adult honey bees to pupae. In addition, adult bees, in which APV is multiplying, transmit the virus to unsealed brood in the larval food.     

Honey bees and wild pollinators differ in their preference for and use of introduced floral resources

Introduced plants may be important foraging resources for honey bees and wild pollinators, but how often and why pollinators visit introduced plants across an entire plant community is not well understood. Understanding the importance of introduced plants for pollinators could help guide management of these plants and conservation of pollinator habitat. We assessed how floral abundance and pollinator preference influence pollinator visitation rate and diversity on 30 introduced versus 24 native plants in central New York. Honey bees visited introduced and native plants at similar rates regardless of floral abundance. In contrast, as floral abundance increased, wild pollinator visitation rate decreased more strongly for introduced plants than native plants. Introduced plants as a group and native plants as a group did not differ in bee diversity or preference, but honey bees and wild pollinators preferred different plant species. As a case study, we then focused on knapweed (Centaurea spp.), an introduced plant that was the most preferred plant by honey bees, and that beekeepers value as a late‐summer foraging resource. We compared the extent to which honey bees versus wild pollinators visited knapweed relative to coflowering plants, and we quantified knapweed pollen and nectar collection by honey bees across 22 New York apiaries. Honey bees visited knapweed more frequently than coflowering plants and at a similar rate as all wild pollinators combined. All apiaries contained knapweed pollen in nectar, 86% of apiaries contained knapweed pollen in bee bread, and knapweed was sometimes a main pollen or nectar source for honey bees in late summer. Our results suggest that because of diverging responses to floral abundance and preferences for different plants, honey bees and wild pollinators differ in their use of introduced plants. Depending on the plant and its abundance, removing an introduced plant may impact honey bees more than wild pollinators.     

Management increases genetic diversity of honey bees via admixture

The process of domestication often brings about profound changes in levels of genetic variation in animals and plants. The honey bee, Apis mellifera, has been managed by humans for centuries for both honey and wax production and crop pollination. Human management and selective breeding are believed to have caused reductions in genetic diversity in honey bee populations, thereby contributing to the global declines threatening this ecologically and economically important insect. However, previous studies supporting this claim mostly relied on population genetic comparisons of European and African (or Africanized) honey bee races; such conclusions require reassessment given recent evidence demonstrating that the honey bee originated in Africa and colonized Europe via two independent expansions. We sampled honey bee workers from two managed populations in North America and Europe as well as several old-world progenitor populations in Africa, East and West Europe. Managed bees had highly introgressed genomes representing admixture between East and West European progenitor populations. We found that managed honey bees actually have higher levels of genetic diversity compared with their progenitors in East and West Europe, providing an unusual example whereby human management increases genetic diversity by promoting admixture. The relationship between genetic diversity and honey bee declines is tenuous given that managed bees have more genetic diversity than their progenitors and many viable domesticated animals.     

Acaricide treatment affects viral dynamics in Varroa destructor-infested honey bee colonies via both host physiology and mite control

Honey bee (Apis mellifera) colonies are declining, and a number of stressors have been identified that affect, alone or in combination, the health of honey bees. The ectoparasitic mite Varroa destructor, honey bee viruses that are often closely associated with the mite, and pesticides used to control the mite population form a complex system of stressors that may affect honey bee health in different ways. During an acaricide treatment using Apistan (plastic strips coated with tau-fluvalinate), we analyzed the infection dynamics of deformed wing virus (DWV), sacbrood virus (SBV), and black queen cell virus (BQCV) in adult bees, mite-infested pupae, their associated Varroa mites, and uninfested pupae, comparing these to similar samples from untreated control colonies. Titers of DWV increased initially with the onset of the acaricide application and then slightly decreased progressively coinciding with the removal of the Varroa mite infestation. This initial increase in DWV titers suggests a physiological effect of tau-fluvalinate on the host's susceptibility to viral infection. DWV titers in adult bees and uninfested pupae remained higher in treated colonies than in untreated colonies. The titers of SBV and BQCV did not show any direct relationship with mite infestation and showed a variety of possible effects of the acaricide treatment. The results indicate that other factors besides Varroa mite infestation may be important to the development and maintenance of damaging DWV titers in colonies. Possible biochemical explanations for the observed synergistic effects between tau-fluvalinate and virus infections are discussed.