Virus replication in the honey bee parasite, Varroa destructor.

IMPORTANCE: The parasitic mite Varroa destructor is a significant driver of worldwide colony losses of our most important commercial pollinator, the Western honey bee Apis mellifera. Declines in honey bee health are frequently attributed to the viruses that mites vector to honey bees, yet whether mites passively transmit viruses as a mechanical vector or actively participate in viral amplification and facilitate replication of honey bee viruses is debated. Our work investigating the antiviral RNA interference response in V. destructor demonstrates that key viruses associated with honey bee declines actively replicate in mites, indicating that they are biological vectors, and the host range of bee-associated viruses extends to their parasites, which could impact virus evolution, pathogenicity, and spread.

Deformed wing virus genotypes A and B do not elicit immunologically different responses in naïve honey bee hosts.

Iflavirus aladeformis (Picornavirales: Iflaviridae), commonly known as deformed wing virus(DWV), in association with Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae), is a leading factor associated with honey bee (Apis mellifera L. [Hymenoptera: Apidae]) deaths. The virus and mite have a near global distribution, making it difficult to separate the effect of one from the other. The prevalence of two main DWV genotypes (DWV-A and DWV-B) has changed over time, leading to the possibility that the two strains elicit a different immune response by the host. Here, we use a honey bee population naïve to both the mite and the virus to investigate if honey bees show a different immunological response to DWV genotypes. We examined the expression of 19 immune genes by reverse transcription quantitative PCR (RT-qPCR) and analysed small RNA after experimental injection with DWV-A and DWV-B. We found no evidence that DWV-A and DWV-B elicit different immune responses in honey bees. RNA interference genes were up-regulated during DWV infection, and small interfering RNA (siRNA) responses were proportional to viral loads yet did not inhibit DWV accumulation. The siRNA response towards DWV was weaker than the response to another honey bee pathogen, Triatovirus nigereginacellulae (Picornavirales: Dicistroviridae; black queen cell virus), suggesting that DWV is comparatively better at evading host antiviral defences. There was no evidence for the production of virus-derived Piwi-interacting RNAs (piRNAs) in response to DWV. In contrast to previous studies, and in the absence of V. destructor, we found no evidence that DWV has an immunosuppressive effect. Overall, our results advance our understanding of the immunological effect that DWV in isolation elicits in honey bees.

Adaptation to vector-based transmission in a honeybee virus.

Global pollinator declines as a result of emerging infectious diseases are of major concern. Managed honeybees Apis mellifera are susceptible to numerous parasites and pathogens, many of which appear to be transmissible to sympatric non-Apis taxa. The ectoparasitic mite Varroa destructor is considered to be the most significant threat to honeybees due to its role in vectoring RNA viruses, particularly Deformed wing virus (DWV). Vector transmission of DWV has resulted in the accumulation of high viral loads in honeybees and is often associated with colony death. DWV has two main genotypes, A and B. DWV-A was more prevalent during the initial phase of V. destructor establishment. In recent years, the global prevalence of DWV-B has increased, suggesting that DWV-B is better adapted to vector transmission than DWV-A. We aimed to determine the role vector transmission plays in DWV genotype prevalence at a colony level. We experimentally increased or decreased the number of V. destructor mites in honeybee colonies, and tracked DWV-A and DWV-B loads over a period of 10 months. Our results show that the two DWV genotypes differ in their response to mite numbers. DWV-A accumulation in honeybees was positively correlated with mite numbers yet DWV-A was largely undetected in the absence of the mite. In contrast, colonies had high loads of DWV-B even when mite numbers were low. DWV-B loads persisted in miticide-treated colonies, indicating that this genotype has a competitive advantage over DWV-A irrespective of mite numbers. Our findings suggest that the global increase in DWV-B prevalence is not driven by selective pressure by the vector. Rather, DWV-B is able to persist in colonies at higher viral loads relative to DWV-A in the presence and absence of V. destructor. The interplay between V. destructor and DWV genotypes within honeybee colonies may have broad consequences upon viral diversity in sympatric taxa as a result of spillover.

Accumulation and Competition Amongst Deformed Wing Virus Genotypes in Naïve Australian Honeybees Provides Insight Into the Increasing Global Prevalence of Genotype B.

Honeybee colony deaths are often attributed to the ectoparasitic mite Varroa destructor and deformed wing virus (DWV), vectored by the mite. In the presence of V. destructor both main genotypes (DWV-A and DWV-B) have been correlated with colony loss. Studies show that DWV-B is the most prevalent genotype in the United Kingdom and Europe. More recently DWV-B has increased in prevalence in the United States. The increasing prevalence of DWV-B at the expense of DWV-A suggests that competition exists between the genotypes. Competition may be due to disparities in virulence between genotypes, differences in fitness, such as rate of replication, or a combination of factors. In this study we investigated if DWV genotypes differ in their rate of accumulation in Australian honeybees naïve to both V. destructor and DWV, and if viral load was associated with mortality in honeybee pupae. We singly and co-infected pupae with DWV-A, DWV-B, and a recombinant strain isolated from a V. destructor tolerant bee population. We monitored viral accumulation throughout pupation, up to 192 h post-injection. We found significant differences in accumulation, where DWV-A accumulated to significantly lower loads than DWV-B and the DWV-recombinant. We also found evidence of competition, where DWV-B loads were significantly reduced in the presence of DWV-A, but still accumulated to the highest loads overall. In contrast to previous studies, we found significant differences in virulence between pupae injected with DWV-A and DWV-B. The average mortality associated with DWV-B (0.4% ± 0.33 SE) and DWV-recombinant (2.2% ± 0.83 SE) injection were significantly less than observed for DWV-A (11% ± 1.2 SE). Our results suggest that a higher proportion of DWV-B infected pupae will emerge into adults, compared to DWV-A. Overall, our data suggest that low mortality in pupae and the ability of DWV-B to accumulate to higher loads relative to DWV-A even during co-infection may favor vector transmission by V. destructor, and may thus be contributing factors to the increasing prevalence of DWV-B globally.

Quantitation of Dihydroxyacetone in Australian Leptospermum Nectar via High-Performance Liquid Chromatography.

The nonperoxide antibacterial activity of New Zealand ma̅nuka honey originates from dihydroxyacetone (DHA) within Leptospermum scoparium nectar. This study determined if DHA was present within the nectar of four Australian Leptospermum species: L. laevigatum, L. polygalifolium, L. trinervium, and L. whitei. A rapid and convenient new method was developed, which quantitated DHA/sugar ratios (ppm). The DHA and sugars were derivatized with o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride and analyzed via RP-HPLC with diode array detection at two wavelengths (200 and 243 nm). DHA was detected in all L. whitei and L. polygalifolium samples, where DHA/sugar ratios ranged from 10169 to 24199 ppm and from 9321 to 20174 ppm, respectively. DHA was undetected in any of the L. laevigatum and L. trinervium samples, and nectar activity was <100 ppm. The results of this study have implications for the Australian beekeeping industry, as the findings indicated that not all species of Leptospermum will produce active honey.