Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies.

In this study, we investigated the prevalence of honeybee viruses in Varroa destructor and Varroa underwoodi infesting Apis cerana colonies in China. Deformed wing virus (DWV) was the most prevalent virus in these two mite species, followed by Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Chronic bee paralysis virus (CBPV), Apis mellifera filamentous virus (AmFV) and Sacbrood virus (SBV) in V. destructor, while in V. underwoodi, it was followed by CBPV, AmFV, BQCV, IAPV and KBV. In addition, multiple viruses were commonly detectable in both mite species.

An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum.

BACKGROUND: Schistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum. METHODS: We optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4',6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male ß-alanyl-tryptamine (BATT) was conducted using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Both m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone ß-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum's reproductive and oviposition processes. CONCLUSIONS: We developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production.

Host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system.

Cophylogeny has been identified between gut bacteria and their animal host and is highly relevant to host health, but little research has extended to gut bacteriophages. Here we use bee model to investigate host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system. Through metagenomic sequencing upon different bee species, the gut phageome revealed a more variable composition than the gut bacteriome. Nevertheless, the bacteriome and the phageome showed a significant association of their dissimilarity matrices, indicating a reciprocal interaction between the two kinds of communities. Most of the gut phages were host generalist at the viral cluster level but host specialist at the viral OTU level. While the dominant gut bacteria Gilliamella and Snodgrassella exhibited matched phylogeny with bee hosts, most of their phages showed a diminished level of cophylogeny. The evolutionary rates of the bee, the gut bacteria and the gut phages showed a remarkably increasing trend, including synonymous and non-synonymous substitution and gene content variation. For all of the three codiversified tripartite members, however, their genes under positive selection and genes involving gain/loss during evolution simultaneously enriched the functions into metabolism of nutrients, therefore highlighting the tripartite coevolution that results in an enhanced ecological fitness for the whole holobiont.

Sacbrood Virus: A Growing Threat to Honeybees and Wild Pollinators.

Sacbrood virus (SBV) is one of the many viruses that infect both the Western honeybee (Apis mellifera) and the Eastern honeybee (Apis cerana). Recently, the interspecies transmission of SBV has been discovered, especially among wild pollinators. This newly discovered evolutionary occurrence regarding SBV indicates a much wider host range than previously believed, causing further concern about the future sustainability of agriculture and the resilience of ecosystems. Over the past few decades, vast numbers of studies have been undertaken concerning SBV infection in honeybees, and remarkable progress has been made in our understanding of the epidemiology, pathogenesis, transmission, and manifestations of SBV infection in honeybees and other pollinators. Meanwhile, some methods, including Chinese medicine, have been established to control and prevent sacbrood disease in A. cerana in Asian countries. In this review, we summarize the existing knowledge of SBV and address the gaps in the knowledge within the existing literature in the hope of providing future directions for the research and development of management strategies for controlling the spread of this deadly disease.

Genetic divergence and functional convergence of gut bacteria between the Eastern honey bee Apis cerana and the Western honey bee Apis mellifera.

Introduction: The functional relevance of intra-species diversity in natural microbial communities remains largely unexplored. The guts of two closely related honey bee species, Apis cerana and A. mellifera, are colonised by a similar set of core bacterial species composed of host-specific strains, thereby providing a good model for an intra-species diversity study. Objectives: We aim to assess the functional relevance of intra-species diversity of A. cerana and A. mellifera gut microbiota. Methods: Honey bee workers were collected from four regions of China. Their gut microbiomes were investigated by shotgun metagenomic sequencing, and the bacterial compositions were compared at the species level. A cross-species colonisation assay was conducted, with the gut metabolomes being characterised by LC-MS/MS. Results: Comparative analysis showed that the strain composition of the core bacterial species was host-specific. These core bacterial species presented distinctive functional profiles between the hosts. However, the overall functional profiles of the A. cerana and A. mellifera gut microbiomes were similar; this was further supported by the consistency of the honey bees' gut metabolome, as the gut microbiota of different honey bee species showed rather similar metabolic profiles in the cross-species colonisation assay. Moreover, this experiment also demonstrated that the gut microbiota of A. cerana and A. mellifera could cross colonise between the two honey bee species. Conclusion: Our findings revealed functional differences in most core gut bacteria between the guts of A. cerana and A. mellifera, which may be associated with their inter-species diversity. However, the functional profiles of the overall gut microbiomes between the two honey bee species converge, probably as a result of the overlapping ecological niches of the two species. Our findings provide critical insights into the evolution and functional roles of the mutualistic microbiota of honey bees and reveal that functional redundancy could stabilise the gene content diversity at the strain-level within the gut community.

A New Strain of Virus Discovered in China Specific to the Parasitic Mite Varroa destructor Poses a Potential Threat to Honey Bees.

The ectoparasitic mite, Varroa destructor, feeds directly on honey bees and serves as a vector for transmitting viruses among them. The Varroa mite causes relatively little damage to its natural host, the Eastern honey bee (Apis cerana) but it is the most devastating pest for the Western honey bee (Apis mellifera). Using Illumina HiSeq sequencing technology, we conducted a metatranscriptome analysis of the microbial community associated with Varroa mites. This study led to the identification of a new Chinese strain of Varroa destructor virus-2 (VDV-2), which is a member of the Iflaviridae family and was previously reported to be specific to Varroa mites. A subsequent epidemiological investigation of Chinese strain of VDV-2 (VDV-2-China) showed that the virus was highly prevalent among Varroa populations and was not identified in any of the adult workers from both A. mellifera and A.cerana colonies distributed in six provinces in China, clearly indicating that VDV-2-China is predominantly a Varroa-adapted virus. While A. mellifera worker pupae exposed to less than two Varroa mites tested negative for VDV-2-China, VDV-2-China was detected in 12.5% of the A. mellifera worker pupae that were parasitized by more than 10 Varroa mites, bringing into play the possibility of a new scenario where VDV-2 could be transmitted to the honey bees during heavy Varroa infestations. Bioassay for the VDV-2-China infectivity showed that A. cerana was not a permissive host for VDV-2-China, yet A. mellifera could be a biological host that supports VDV-2-China's replication. The different replication dynamics of the virus between the two host species reflect their variation in terms of susceptibility to the virus infection, posing a potential threat to the health of the Western honey bee. The information gained from this study contributes to the knowledge concerning genetic variabilities and evolutionary dynamics of Varroa-borne viruses, thereby enhancing our understanding of underlying molecular mechanisms governing honey bee Varroosis.

The Pass-on Effect of Tetracycline-Induced Honey Bee (Apis mellifera) Gut Community Dysbiosis.

Gut microbial community plays an important role in the regulation of insect health. Antibiotic treatment is powerful to fight bacterial infections, while it also causes collateral damage to gut microbiome, which may have long-lasting consequences for host health. However, current studies on honey bees mainly focus on the impact of direct exposure to antibiotics on individual bees, and little is known about the impact of social transmission of antibiotic-induced gut community disorder in honey bee colonies. In order to provide insight into the potential pass-on effect of antibiotic-induced dysbiosis, we colonized newly emerged germ-free workers with either normal or tetracycline-treated gut community and analyzed the gut bacteria composition. We also treated workers with low dosage of tetracycline to evaluate its impact on honey bee gut microbiota. Our results showed that the tetracycline-treated gut community caused disruption of gut community in their receivers, while the direct exposure to the low dosage of tetracycline had no significant effect. In addition, no significant difference was observed on the mortality rate of A. mellifera workers with different treatments. These results suggest that though the residue of antibiotic treatment may not have direct effect on honey bee gut community, the gut microbiota dysbiosis caused by high dosage of antibiotic treatment has a cascade effect on the gut community of the nestmates in honeybee colonies.

Population genetics and host specificity of Varroa destructor mites infesting eastern and western honeybees.

In a globalized world, parasites are often brought in contact with new potential hosts. When parasites successfully shift host, severe diseases can emerge at a large cost to society. However, the evolutionary processes leading to successful shifts are rarely understood, hindering risk assessment, prevention, or mitigation of their effects. Here, we screened populations of Varroa destructor, an ectoparasitic mite of the honeybee genus Apis, to investigate their genetic structure and reproductive potential on new and original hosts. From the patterns identified, we deduce the factors that influenced the macro- and microevolutionary processes that led to the structure observed. Among the mite variants identified, we found two genetically similar populations that differed in their reproductive abilities and thus in their host specificity. These lineages could interbreed, which represents a threat due to the possible increased virulence of the parasite on its original host. However, interbreeding was unidirectional from the host-shifted to the nonshifted native mites and could thus lead to speciation of the former. The results improve our understanding of the processes affecting the population structure and evolution of this economically important mite genus and suggest that introgression between shifted and nonshifted lineages may endanger the original host. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-020-01322-7.

Seasonal variation of viral infections between the eastern honey bee (Apis cerana) and the western honey bee (Apis mellifera).

It is a widespread practice in China to keep colonies of both the western honey bee, Apis mellifera, and the eastern honey bee, Apis cerana, in close proximity. However, this practice increases opportunities for spillover of parasites and pathogens between the two host bee species, impacting spatial and temporal patterns in the occurrence and prevalence of the viruses that adversely affect bee health. We conducted a 1-year large-scale survey to assess the current status of viral infection in both A. mellifera and A. cerana in China. Our study focused on multiple aspects of viral infections in honey bees, including infection rate, viral load, seasonal variation, regional variation, and phylogenetic relationships of the viruses within the same species found in this study and other parts of the world. The survey showed that the black queen cell virus (BQCV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV) were common in both A. mellifera and A. cerana, and infection dynamics of BQCV, DWV, and SBV between bee species or seasons were significantly different. DWV was the most common virus in A. mellifera, and its infection rate and load in A. mellifera were higher than those in A. cerana, which reflects the high susceptibility of A. mellifera to Varroa destructor infestation. The infection rate and viral load of SBV were higher in A. cerana than in A. mellifera, indicating that SBV poses a greater threat to A. cerana than to A. mellifera. Our results also suggested that there was no geographical variation in viral dynamics in A. mellifera and A. cerana. Phylogenetic analyses of BQCV, DWV, IAPV, and SBV suggested the cross-regional and cross-species spread of these viruses. This study provides important insights into the complex relationships between viruses and their hosts in different seasons and regions, which will be important for developing effective disease management strategies to improve bee health.

Apis cerana gut microbiota contribute to host health though stimulating host immune system and strengthening host resistance to Nosema ceranae.

Gut microbial communities play vital roles in the modulation of many insects' immunity, including Apis mellifera. However, little is known about the interaction of Apis cerana gut bacteria and A. cerana immune system. Here in this study, we conducted a comparison between germ-free gut microbiota deficient (GD) workers and conventional gut community (CV) workers, to reveal the possible impact of gut microbiota on the expression of A. cerana antimicrobial peptides and immune regulate pathways. We also test whether A. cerana gut microbiota can strengthen host resistance to Nosema ceranae. We find that the expression of apidaecin, abaecin and hymenoptaecin were significantly upregulated with the presence of gut bacteria, and JNK pathway was activated; in the meanwhile, the existence of gut bacteria inhibited the proliferation of Nosema ceranae. These demonstrated the essential role of A. cerana gut microbiota to host health and provided critical insight into the honeybee host-microbiome interaction.

Reproduction of ectoparasitic mites in a coevolved system: Varroa spp.-Eastern honey bees, Apis cerana.

Parasite host shifts can impose a high selective pressure on novel hosts. Even though the coevolved systems can reveal fundamental aspects of host-parasite interactions, research often focuses on the new host-parasite relationships. This holds true for two ectoparasitic mite species, Varroa destructor and Varroa jacobsonii, which have shifted hosts from Eastern honey bees, Apis cerana, to Western honey bees, Apis mellifera, generating colony losses of these pollinators globally. Here, we study infestation rates and reproduction of V. destructor and V. jacobsonii haplotypes in 185 A. cerana colonies of six populations in China and Thailand to investigate how coevolution shaped these features. Reproductive success was mostly similar and low, indicating constraints imposed by hosts and/or mite physiology. Infestation rates varied between mite haplotypes, suggesting distinct local co-evolutionary scenarios. The differences in infestation rates and reproductive output between haplotypes did not correlate with the virulence of the respective host-shifted lineages suggesting distinct selection scenarios in novel and original host. The occasional worker brood infestation was significantly lower than that of drone brood, except for the V. destructor haplotype (Korea) from which the invasive lineage derived. Whether mites infesting and reproducing in atypical intraspecific hosts (i.e., workers and queens) actually predisposes for and may govern the impact of host shifts on novel hosts should be determined by identifying the underlying mechanisms. In general, the apparent gaps in our knowledge of this coevolved system need to be further addressed to foster the adequate protection of wild and managed honey bees from these mites globally.