LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites.

Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur.

Evolutionarily diverse origins of deformed wing viruses in western honey bees.

Novel transmission routes can allow infectious diseases to spread, often with devastating consequences. Ectoparasitic varroa mites vector a diversity of RNA viruses, having switched hosts from the eastern to western honey bees (Apis cerana to Apis mellifera). They provide an opportunity to explore how novel transmission routes shape disease epidemiology. As the principal driver of the spread of deformed wing viruses (mainly DWV-A and DWV-B), varroa infestation has also driven global honey bee health declines. The more virulent DWV-B strain has been replacing the original DWV-A strain in many regions over the past two decades. Yet, how these viruses originated and spread remains poorly understood. Here, we use a phylogeographic analysis based on whole-genome data to reconstruct the origins and demography of DWV spread. We found that, rather than reemerging in western honey bees after varroa switched hosts, as suggested by previous work, DWV-A most likely originated in East Asia and spread in the mid-20th century. It also showed a massive population size expansion following the varroa host switch. By contrast, DWV-B was most likely acquired more recently from a source outside East Asia and appears absent from the original varroa host. These results highlight the dynamic nature of viral adaptation, whereby a vector's host switch can give rise to competing and increasingly virulent disease pandemics. The evolutionary novelty and rapid global spread of these host-virus interactions, together with observed spillover into other species, illustrate how increasing globalization poses urgent threats to biodiversity and food security.

The final frontier: ecological and evolutionary dynamics of a global parasite invasion.

Studying rapid biological changes accompanying the introduction of alien organisms into native ecosystems can provide insights into fundamental ecological and evolutionary theory. While powerful, this quasi-experimental approach is difficult to implement because the timing of invasions and their consequences are hard to predict, meaning that baseline pre-invasion data are often missing. Exceptionally, the eventual arrival of Varroa destructor (hereafter Varroa) in Australia has been predicted for decades. Varroa is a major driver of honeybee declines worldwide, particularly as vectors of diverse RNA viruses. The detection of Varroa in 2022 at over a hundred sites poses a risk of further spread across the continent. At the same time, careful study of Varroa's spread, if it does become established, can provide a wealth of information that can fill knowledge gaps about its effects worldwide. This includes how Varroa affects honeybee populations and pollination. Even more generally, Varroa invasion can serve as a model for evolution, virology and ecological interactions between the parasite, the host and other organisms.

Surveillance for Avocado Sunblotch Viroid Utilizing the European Honey Bee (Apis mellifera).

Avocado is one of the world's fastest growing tropical fruit industries, and the pathogen avocado sunblotch viroid (ASBVd) is a major threat to both production and access to international export markets. ASBVd is seed transmissible, with infection possible via either the male (pollen) or female gametes. Surveillance for ASBVd across commercial orchards is a major logistical task, particularly when aiming to meet the stringent standards of evidence required for a declaration of pest freedom. As with many fruit crops, insect pollination is important for high avocado yields, and honey bee (Apis mellifera) hives are typically moved into orchards for paid pollination services. Exploiting the foraging behavior of honey bees can provide a complementary strategy to traditional surveillance methods. High-throughput sequencing (HTS) of bee samples for plant viruses shows promise, but this surveillance method has not yet been tested for viroids or in a targeted plant biosecurity context. Here, we tested samples of bees and pollen collected from pollination hives in two ASBVd orchard locations, one in Australia, where only four trees in a block were known to be infected, and a second in South Africa, where the estimated incidence of infection was 10%. Using real-time RT-PCR and HTS (total RNA-seq and small RNA-seq), we demonstrated that ASBVd can be confidently detected in bees and pollen samples from hives within 100 m of infected trees. The potential for using this approach in ASBVd surveillance for improved orchard management and supporting market access is discussed.

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.

The first steps toward a global pandemic: Reconstructing the demographic history of parasite host switches in its native range.

Host switching allows parasites to expand their niches. However, successful switching may require suites of adaptations and also may decrease performance on the old host. As a result, reductions in gene flow accompany many host switches, driving speciation. Because host switches tend to be rapid, it is difficult to study them in real-time, and their demographic parameters remain poorly understood. As a result, fundamental factors that control subsequent parasite evolution, such as the size of the switching population or the extent of immigration from the original host, remain largely unknown. To shed light on the host switching process, we explored how host switches occur in independent host shifts by two ectoparasitic honey bee mites (Varroa destructor and V. jacobsoni). Both switched to the western honey bee (Apis mellifera) after being brought into contact with their ancestral host (Apis cerana), ~70 and ~12 years ago, respectively. Varroa destructor subsequently caused worldwide collapses of honey bee populations. Using whole-genome sequencing on 63 mites collected in their native ranges from both the ancestral and novel hosts, we were able to reconstruct the known temporal dynamics of the switch. We further found multiple previously undiscovered mitochondrial lineages on the novel host, along with the genetic equivalent of tens of individuals that were involved in the initial host switch. Despite being greatly reduced, some gene flow remains between mites adapted to different hosts. Our findings suggest that while reproductive isolation may facilitate the fixation of traits beneficial for exploiting the new host, ongoing genetic exchange may allow genetic amelioration of inbreeding effects.

Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees.

Chalkbrood infection caused by the fungus Ascosphaera apis currently has a significant impact on Australia's apicultural industry. We investigated the genetic variation of A. apis and colony and apiary level conditions to determine if an emerging, more virulent strain or specific conditions were responsible for the prevalence of the disease. We identified six genetically distinct strains of A. apis, four have been reported elsewhere and two are unique to Australia. Colonies and individual larvae were found to be infected with multiple strains of A. apis, neither individual strains, combinations of strains, or obvious colony or apiary characteristics were found to be predictive of hive infection levels. These results suggest that host genotype plays an important role in colony level resistance to chalkbrood infection in Australia.

Tolerance of Honey Bees to Varroa Mite in the Absence of Deformed Wing Virus.

The global spread of the parasitic mite Varroa destructor has emphasized the significance of viruses as pathogens of honey bee (Apis mellifera) populations. In particular, the association of deformed wing virus (DWV) with V. destructor and its devastating effect on honey bee colonies has led to that virus now becoming one of the most well-studied insect viruses. However, there has been no opportunity to examine the effects of Varroa mites without the influence of DWV. In Papua New Guinea (PNG), the sister species, V. jacobsoni, has emerged through a host-shift to reproduce on the local A. mellifera population. After initial colony losses, beekeepers have maintained colonies without chemicals for more than a decade, suggesting that this bee population has an unknown mite tolerance mechanism. Using high throughput sequencing (HTS) and target PCR detection, we investigated whether the viral landscape of the PNG honey bee population is the underlying factor responsible for mite tolerance. We found A. mellifera and A. cerana from PNG and nearby Solomon Islands were predominantly infected by sacbrood virus (SBV), black queen cell virus (BQCV) and Lake Sinai viruses (LSV), with no evidence for any DWV strains. V. jacobsoni was infected by several viral homologs to recently discovered V. destructor viruses, but Varroa jacobsoni rhabdovirus-1 (ARV-1 homolog) was the only virus detected in both mites and honey bees. We conclude from these findings that A. mellifera in PNG may tolerate V. jacobsoni because the damage from parasitism is significantly reduced without DWV. This study also provides further evidence that DWV does not exist as a covert infection in all honey bee populations, and remaining free of this serious viral pathogen can have important implications for bee health outcomes in the face of Varroa.

Effectiveness of control strategies for Varroa jacobsoni and Tropilaelaps mercedesae in Papua New Guinea.

Apiculture in the Pacific island country of Papua New Guinea (PNG) is under significant pressure from emerging parasitic mites, Varroa jacobsoni and Tropilaelaps mercedesae. Although numerous mite control products exist, beekeepers in PNG have limited resources and access to these products and their effectiveness under local conditions is untested. Here we determined the effectiveness of two brood manipulation strategies-queen caging and queen removal-for managing V. jacobsoni and T. mercedesae in comparison to the chemical miticide Bayvarol®. Our results found Bayvarol was the most effective control strategy for V. jacobsoni, maintaining high efficacy (> 90%) over 4 months with significantly reduced levels of V. jacobsoni compared to untreated control hives. In contrast, T. mercedesae were significantly reduced by the brood manipulation strategies over 2 months, but not significantly by Bayvarol compared to the controls. These results highlight that a combination of strategies is likely needed to effectively manage both mite pests in PNG. We discuss how these findings are relevant to informing best practice for honey bee biosecurity and how these strategies can be implemented to improve the effectiveness of mite management for PNG beekeepers.

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities.

Metagenomic analysis of Varroa-free Australian honey bees (Apis mellifera) shows a diverse Picornavirales virome.

The viral landscape of the honey bee (Apismellifera) has changed as a consequence of the global spread of the parasitic mite Varroa destructor and accompanying virulent strains of the iflavirus deformed wing virus (DWV), which the mite vectors. The presence of DWV in honey bee populations is known to influence the occurrence of other viruses, suggesting that the current known virome of A. mellifera may be undercharacterized. Here we tested this hypothesis by examining the honey bee virome in Australia, which is uniquely free of parasitic mites or DWV. Using a high-throughput sequencing (HTS) approach, we examined the RNA virome from nine pools of A. mellifera across Australia. In addition to previously reported honey bee viruses, several other insect viruses were detected, including strains related to aphid lethal paralysis virus (ALPV) and Rhopalosiphum padi virus (RhPV), which have recently been identified as infecting honey bees in the USA, as well as several other viruses recently found in Drosophila spp. A further 42 putative novel insect virus genomes spanning the order Picornavirales were assembled, which significantly increases the known viral diversity in A. mellifera. Among these novel genomes, we identified several that were similar (but different) to key A. mellifera viruses, such as DWV, that warrant further investigation. We propose that A. mellifera may be preferentially infected with viruses of the order Picornavirales and that a diverse population of these viruses may be representative of a Varroa-free landscape.

Absence of deformed wing virus and Varroa destructor in Australia provides unique perspectives on honeybee viral landscapes and colony losses.

Honeybee (Apis mellifera) health is threatened globally by the complex interaction of multiple stressors, including the parasitic mite Varroa destructor and a number of pathogenic viruses. Australia provides a unique opportunity to study this pathogenic viral landscape in the absence of V. destructor. We analysed 1,240A. mellifera colonies across Australia by reverse transcription-polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS). Five viruses were prevalent: black queen cell virus (BQCV), sacbrood virus (SBV), Israeli acute paralysis virus (IAPV) and the Lake Sinai viruses (LSV1 and LSV2), of which the latter three were detected for the first time in Australia. We also showed several viruses were absent in our sampling, including deformed wing virus (DWV) and slow bee paralysis virus (SBPV). Our findings highlight that viruses can be highly prevalent in A. mellifera populations independently of V. destructor. Placing these results in an international context, our results support the hypothesis that the co-pathogenic interaction of V. destructor and DWV is a key driver of increased colony losses, but additional stressors such as pesticides, poor nutrition, etc. may enable more severe and frequent colony losses to occur.

Population dynamics and diapause response of the springtail pest Sminthurus viridis (Collembola: Sminthuridae) in southeastern Australia.

The springtail, Sminthurus viridis (L.) (Collembola: Sminthuridae), is an important agricultural pest across southern Australia. We investigated the seasonal abundance patterns and summer diapause response of S. viridis in southeastern Australia by using field and shadehouse (a greenhouse that offers seedlings shade) experiments. Seasonal activity patterns of S. viridis were largely consistent with previous studies, with the pest active from autumn to spring. In addition, the timing and pattern of the summer-diapausing egg stage was established, with multiple generations probably producing diapause eggs. A strong relationship between soil moisture and temperature with autumn emergence also was observed. These results suggest that S. viridis autumn pest pressure can be predicted and indicate that late-season spraying strategies currently used for a sympatric agricultural pest are unlikely to be as effective against S. viridis.

Genetic structure and long-distance dispersal in populations of the wingless pest springtail, Sminthurus viridis (Collembola: Sminthuridae).

The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae) (L.) is a major pest of broadacre agriculture across southern Australia. Few molecular studies have been conducted on S. viridis and none have examined its population genetics, despite the importance for developing effective control strategies. Here, we characterize the genetic structure of Australian populations using three allozyme and eight microsatellite loci, as well as sequencing a fragment of the mitochondrial DNA cytochrome oxidase I gene. We found that S. viridis in Australia are diploid, sexually reproducing and exhibit significant population structure as a result of limited gene flow. Despite significant differentiation between populations, there was very low cytochrome oxidase subunit I (COI) gene sequence variation, indicating the presence of a single species in Australia. The observed structure only marginally complied with an 'isolation by distance' model with human-mediated long-distance dispersal likely occurring. Allozymes and microsatellites gave very similar FST estimates, although differences found for novel alternative estimates of differentiation suggest that the allozymes did not capture the full extent of the population structure. These results highlight that control strategies may need to vary for locally adapted S. viridis populations and strategies aimed at limiting the spread of any future pesticide resistance will need to manage the effects of human-mediated dispersal.