Lactobacillus salivarius A3iob Reduces the Incidence of Varroa destructor and Nosema Spp. in Commercial Apiaries Located in the Northwest of Argentina.

Lactobacillus salivarius A3iob was administered to productive colonies belonging to commercial apiaries of small beekeepers (around 30-50 hives each one), from four departments of the province of Jujuy (Argentina): Yala, Tilquiza, El Carmen, and Los Alisos. The incidence of Varroa destructor and Nosema spp., before and after winter, was monitored during 2 years of study (2014-2015). Depending on the geographical location of each apiary and the application time, a monthly dose of the bacteria (105 CFU/mL) reduced the levels of varroasis between 50 and 80%. Interestingly, L. salivarius A3iob cells remitted the percentage of the mites to undetectable values in an apiary treated with flumethrin (at Yala, Yungas region).On the other hand, the spore levels of Nosema spp. in the lactobacilli-treated colonies also depended on the apiary and the year of application, but a significant decrease was mainly observed in the post-winter period. However, at Rivera (El Carmen's department), no significant changes were detected in both parameters.These results obtained after 2 years of work suggest that delivering L. salivarius A3iob cells to the bee colonies can become a new eco-friendly tool to cooperate with the control of these bees' pests.

Engineered symbionts activate honey bee immunity and limit pathogens.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

Pathogenicity of Serratia marcescens Strains in Honey Bees.

Although few honey bee diseases are known to be caused by bacteria, pathogens of adult worker bees may be underrecognized due to social immunity mechanisms. Specifically, infected adult bees typically abandon the hive or are removed by guards. Serratia marcescens, an opportunistic pathogen of many plants and animals, is often present at low abundance in the guts of honey bee workers and has recently been isolated from Varroa mites and from the hemolymph of dead and dying honey bees. However, the severity and prevalence of S. marcescens pathogenicity in honey bees have not been fully investigated. Here we characterized three S. marcescens strains isolated from the guts of honey bees and one previously isolated from hemolymph. In vivo tests confirmed that S. marcescens is pathogenic in workers. All strains caused mortality when a few cells were injected into the hemocoel, and the gut-isolated strains caused mortality when administered orally. In vitro assays and comparative genomics identified possible mechanisms of virulence of gut-associated strains. Expression of antimicrobial peptide and phenoloxidase genes was not elevated following infection, suggesting that these S. marcescens strains derived from honey bees can evade the immune response in their hosts. Finally, surveys from four locations in the United States indicated the presence of S. marcescens in the guts of over 60% of the worker bees evaluated. Taken together, these results suggest that S. marcescens is a widespread opportunistic pathogen of adult honey bees and that it may be highly virulent under some conditions such as perturbation of the normal gut microbiota or the presence of Varroa mites that puncture the integument, thereby enabling entry of bacterial cells.IMPORTANCE Recently, it has become apparent that multiple factors are responsible for honey bee decline, including climate change, pests and pathogens, pesticides, and loss of foraging habitat. Of the large number of pathogens known to infect honey bees, very few are bacteria. Because adult workers abandon hives when diseased, many of their pathogens may go unnoticed. Here we characterized the virulence of Serratia marcescens strains isolated from honey bee guts and hemolymph. Our results indicate that S. marcescens, an opportunistic pathogen of many plants and animals, including humans, is a virulent opportunistic pathogen of honey bees, which could contribute to bee decline. Aside from the implications for honey bee health, the discovery of pathogenic S. marcescens strains in honey bees presents an opportunity to better understand how opportunistic pathogens infect and invade hosts.

Bacterial communities associated with the ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae of the honey bee (Apis mellifera).

Varroa and Tropilaelaps mites have been reported as serious ectoparasites of the honey bee (Apis mellifera). In this study, bacterial communities associated with Varroa destructor and Tropilaelaps mercedesae from northern Thailand were determined, using both culture-dependent and culture-independent approaches. Adult female mites were collected from apiaries in Chiang Mai and Lampang provinces. Culturable bacteria were isolated from individual mites. On average, we observed approximately 1340 and 1140 CFU/mite in Varroa and Tropilaelaps, respectively. All isolates were assigned to the genus Enterococcus. Six samples of genomic DNA from 30-50 mites were extracted and subjected to pyrosequencing of bacterial 16S rRNA amplicons. The resulting 81 717 sequences obtained from Varroa were grouped into 429 operational taxonomic units. The most abundant bacteria in Varroa mites belonged to the family Enterobacteriaceae, especially the genera Arsenophonus, Enterobacter and Proteus. For Tropilaelaps mites, 84 075 sequences were obtained and clustered into 166 operational taxonomic units, within which the family Enterococcaceae (particularly the genus Enterococcus) was predominant. Localization of bacteria in the mites using fluorescence in situ hybridization with two universal bacterial probes revealed that these bacteria were in the cecum of the mites. Taxon-specific Enterobacteriaceae and Arsenophonus probes also confirmed their localization in the cecum of Varroa.

Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100µg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15µg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

Comparison of Varroa destructor and Worker Honeybee Microbiota Within Hives Indicates Shared Bacteria.

The ectoparasitic mite Varroa destructor is a major pest of the honeybee Apis mellifera. In a previous study, bacteria were found in the guts of mites collected from winter beehive debris and were identified using Sanger sequencing of their 16S rRNA genes. In this study, community comparison and diversity analyses were performed to examine the microbiota of honeybees and mites at the population level. The microbiota of the mites and honeybees in 26 colonies in seven apiaries in Czechia was studied. Between 10 and 50 Varroa females were collected from the bottom board, and 10 worker bees were removed from the peripheral comb of the same beehive. Both bees and mites were surface sterilized. Analysis of the 16S rRNA gene libraries revealed significant differences in the Varroa and honeybee microbiota. The Varroa microbiota was less diverse than was the honeybee microbiota, and the relative abundances of bacterial taxa in the mite and bee microbiota differed. The Varroa mites, but not the honeybees, were found to be inhabited by Diplorickettsia. The relative abundance of Arsenophonus, Morganella, Spiroplasma, Enterococcus, and Pseudomonas was higher in Varroa than in honeybees, and the Diplorickettsia symbiont detected in this study is specific to Varroa mites. The results demonstrated that there are shared bacteria between Varroa and honeybee populations but that these bacteria occur in different relative proportions in the honeybee and mite bacteriomes. These results support the suggestion of bacterial transfer via mites, although only some of the transferred bacteria may be harmful.

Isolation of oxalotrophic bacteria associated with Varroa destructor mites.

UNLABELLED: Bacteria associated with varroa mites were cultivated and genotyped by 16S RNA. Under our experimental conditions, the cultivable bacteria were few in number, and most of them proved to be fastidious to grow. Cultivation with seven different media under O2 /CO2 conditions and selection for colony morphology yielded a panel of species belonging to 13 different genera grouped in two different phyla, proteobacteria and actinobacteria. This study identified one species of actinobacteria that is a known commensal of the honey bee. Some isolates are oxalotrophic, a finding that may carry ramifications into the use of oxalic acid to control the number of phoretic mites in the managed colonies of honey bees. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalic acid, legally or brevi manu, is widely used to control phoretic Varroa destructor mites, a major drive of current honey bees' colony losses. Unsubstantiated by sanctioned research are rumours that in certain instances oxalic acid is losing efficacy, forcing beekeepers to increase the frequency of treatments. This investigation fathoms the hypothesis that V. destructor associates with bacteria capable of degrading oxalic acid. The data show that indeed oxalotrophy, a rare trait among bacteria, is common in bacteria that we isolated from V. destructor mites. This finding may have ramifications in the use of oxalic acid as a control agent.

Bacteria detected in the honeybee parasitic mite Varroa destructor collected from beehive winter debris.

AIMS: The winter beehive debris containing bodies of honeybee parasitic mite Varroa destructor is used for veterinary diagnostics. The Varroa sucking honeybee haemolymph serves as a reservoir of pathogens including bacteria. Worker bees can pick up pathogens from the debris during cleaning activities and spread the infection to healthy bees within the colony. The aim of this study was to detect entomopathogenic bacteria in the Varroa collected from the winter beehive debris. METHODS AND RESULTS: Culture-independent approach was used to analyse the mite-associated bacterial community. Total DNA was extracted from the samples of 10 Varroa female individuals sampled from 27 different sites in Czechia. The 16S rRNA gene was amplified using universal bacterial primers, cloned and sequenced, resulting in a set of 596 sequences representing 29 operational taxonomic units (OTU97). To confirm the presence of bacteria in Varroa, histological sections of the mites were observed. Undetermined bacteria were observed in the mite gut and fat tissue. CONCLUSION: Morganella sp. was the most frequently detected taxon, followed by Enterococcus sp., Pseudomonas sp., Rahnella sp., Erwinia sp., and Arsenophonus sp. The honeybee putative pathogen Spiroplasma sp. was detected at one site and Bartonella-like bacteria were found at four sites. PCR-based analysis using genus-specific primers enabled detection of the following taxa: Enterococcus, Bartonella-like bacteria, Arsenophonus and Spiroplasma. SIGNIFICANCE AND IMPACT OF THE STUDY: We found potentially pathogenic (Spiroplasma) and parasitic bacteria (Arsenophonus) in mites from winter beehive debris. The mites can be reservoirs of the pathogenic bacteria in the apicultures.

Towards a better understanding of Apis mellifera and Varroa destructor microbiomes: introducing 'phyloh' as a novel phylogenetic diversity analysis tool.

The study of diversity in biological communities is an intriguing field. Huge amount of data are nowadays available (provided by the innovative DNA sequencing techniques), and management, analysis and display of results are not trivial. Here, we propose for the first time the use of phylogenetic entropy as a measure of bacterial diversity in studies of microbial community structure. We then compared our new method (i.e. the web tool phyloh) for partitioning phylogenetic diversity with the traditional approach in diversity analyses of bacteria communities. We tested phyloh to characterize microbiome in the honeybee (Apis mellifera, Insecta: Hymenoptera) and its parasitic mite varroa (Varroa destructor, Arachnida: Parasitiformes). The rationale is that the comparative analysis of honeybee and varroa microbiomes could open new perspectives concerning the role of the parasites on honeybee colonies health. Our results showed a dramatic change of the honeybee microbiome when varroa occurs, suggesting that this parasite is able to influence host microbiome. Among the different approaches used, only the entropy method, in conjunction with phylogenetic constraint as implemented in phyloh, was able to discriminate varroa microbiome from that of parasitized honeybees. In conclusion, we foresee that the use of phylogenetic entropy could become a new standard in the analyses of community structure, in particular to prove the contribution of each biological entity to the overall diversity.

Efficacy of two fungus-based biopesticide against the honeybee ectoparasitic mite, Varroa destructor.

The varroa mite, Varroa destructor (Anderson and Trueman) (Acari: Varroidae), is known as the most serious ectoparasitic mite on honeybee, Apis mellifera (Hymenoptera: Apidae) in the world. Based on the spores of entomopathogenic fungi, two commercial preparations; Bioranza (Metarhizium anisopliae) and Biovar (Beauveria bassiana) were evaluated through application into the hives against varroa mite. Data showed significant differences between treatments with Bioranza and Biovar, the results were significant after 7 and 14 days post-treatment. Mean a daily fallen mite individual was significantly different between the hives before and after the applications of the two biopesticides and wheat flour. Also, mites' mortality was, significantly, different between the hives before and after treatments. There were significant differences between treatments with the two biopesticides in worker's body weight. Bioranza and Biovar did not infect the honeybee in larval, prepupal, pupal and adult stages. Scanning and transmission electron microscopy images showed spores and hyphae penetration through stigma and wounds on varroa. The results suggest that Bioranza and Biovar are potentially are effective biopesticides against V. destructor in honeybee colonies.

Entomopathogenic fungi as potential biocontrol agents of the ecto-parasitic mite, Varroa destructor, and their effect on the immune response of honey bees (Apis mellifera L.).

Three isolates of each of the entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Clonostachys rosea, were assessed for their pathogenicity to the honey bee parasitic mite, Varroa destructor. The fungi were applied to varroa mites by immersing them in a spore solution, and then the inoculated mites were placed on honey bee brood inside capped cells. At 7 days post inoculation (dpi), the three fungi caused significant varroa mortality compared to non-inoculated mites. In brood treated only with varroa mites, expression of the honey bee genes, hymenoptaecin and poly U binding factor 68 Kd (pUf68), decreased over time, while expression of blue cheese (BlCh) and single minded (SiMd) was not affected. In brood inoculated directly only with M. anisopliae or B. bassiana, the emerged adults showed reduced weight indicating infection by the fungi, which was confirmed by observation of hyphae in the brood. Fungal infection of the brood resulted in increased expression of hymenoptaecin, pUf68 and BlCh, but not SiMd. In brood treated with varroa mites that had been inoculated with the fungi, expression of hymenoptaecin, pUf68 and BlCh, but not SiMd, was even more up-regulated. While varroa mites can suppress gene expression in honey bee brood, varroa mites infected with entomopathogenic fungi induced their expression. This may be due to a low level of fungal infection of the bee, which negated the immunosuppression by the mites. Therefore, entomopathogenic fungi could reduce varroa mite damage to honey bee brood by both infecting the parasite and preventing varroa-associated suppression of honey bee immunity.

Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera.

BACKGROUND: The ectoparasitic mite Varroa destructor has emerged as the primary pest of domestic honey bees (Apis mellifera). Here we present an initial survey of the V. destructor genome carried out to advance our understanding of Varroa biology and to identify new avenues for mite control. This sequence survey provides immediate resources for molecular and population-genetic analyses of Varroa-Apis interactions and defines the challenges ahead for a comprehensive Varroa genome project. RESULTS: The genome size was estimated by flow cytometry to be 565 Mbp, larger than most sequenced insects but modest relative to some other Acari. Genomic DNA pooled from ~1,000 mites was sequenced to 4.3× coverage with 454 pyrosequencing. The 2.4 Gbp of sequencing reads were assembled into 184,094 contigs with an N50 of 2,262 bp, totaling 294 Mbp of sequence after filtering. Genic sequences with homology to other eukaryotic genomes were identified on 13,031 of these contigs, totaling 31.3 Mbp. Alignment of protein sequence blocks conserved among V. destructor and four other arthropod genomes indicated a higher level of sequence divergence within this mite lineage relative to the tick Ixodes scapularis. A number of microbes potentially associated with V. destructor were identified in the sequence survey, including ~300 Kbp of sequence deriving from one or more bacterial species of the Actinomycetales. The presence of this bacterium was confirmed in individual mites by PCR assay, but varied significantly by age and sex of mites. Fragments of a novel virus related to the Baculoviridae were also identified in the survey. The rate of single nucleotide polymorphisms (SNPs) in the pooled mites was estimated to be 6.2 × 10-5 per bp, a low rate consistent with the historical demography and life history of the species. CONCLUSIONS: This survey has provided general tools for the research community and novel directions for investigating the biology and control of Varroa mites. Ongoing development of Varroa genomic resources will be a boon for comparative genomics of under-represented arthropods, and will further enhance the honey bee and its associated pathogens as a model system for studying host-pathogen interactions.