The fungus Ascosphaera apis, an obligate fungal pathogen of honey bee brood, causes chalkbrood disease in honey bee larvae worldwide. Biological characteristics of the fungal pathogen and the molecular interactions between A. apis and honey bees have been studied extensively. However, little is known about the effects of A. apis infection on antioxidant enzyme activities and metabolic profiles of the gut of honey bee larvae. In this study, sandwich enzyme-linked immunosorbent assay and LC-MS based untargeted metabolomic analysis were employed to determine the changes in the specific activities of antioxidant enzymes and the metabolomic profiles in gut tissues of A. apis-infected larvae (105 A. apis spores per larva) and controls. Results showed that specific activities of superoxide dismutase, catalase and glutathione S-transferase were significantly higher in the guts of the control larvae than in the guts of the A. apis-infected larvae. The metabolomic data revealed that levels of 28 and 52 metabolites were significantly higher and lower, respectively, in the guts of A. apis-infected larvae than in the guts of control larvae. The 5-oxo-ETE level in the infected larvae was two times higher than that in the control larvae. Elevated 5-oxo-ETE levels may act as a potential metabolic biomarker for chalkbrood disease diagnosis, suggesting that A. apis infection induced obvious oxidative stress in the honey bee larvae. The levels of metabolites such as taurine, docosahexaenoic acid, and L-carnitine involved in combating oxidative stress were significantly decreased in the gut of A. apis-infected larvae. Overall, our results suggest that A. apis infection may compromise the ability of infected larvae to cope with oxidative stress, providing new insight into changing patterns of physiological responses to A. apis infection in honey bee larvae by concurrent use of conventional biochemical assays and untargeted metabolomics.

Symbiotic bacteria could increase the nutrient provision, regulate the physiological state, and promote immunity in their insect host. Honeybee larvae harbor plenty of bacteria in their gut, but their functions are not well studied. To determine their effect on honeybee larvae, the 1-day-old larvae were grafted on to 24-well plates from the comb and artificially reared in the lab. They were treated with penicillin–streptomycin to remove the gut symbiotic bacteria. Then, the 5-day-old larvae and the newly emerged adults were weighted. The developmental periods to pupae and eclosion were investigated, respectively. The bacterial amount, expression of developmental regulation genes (ecr and usp), nutrient metabolism genes (ilp1, ilp2, hex 70a, hex 70b, hex 70c, and hex 110), and immunity genes (apidaecin, abaecin, defensin-1, and hymenoptaecin) were determined by qRT-PCR. The result showed that the antibiotics-treated larvae have significantly lower body weights in the 5-day-old larvae and the emerged bees. The expression of ilp2 and hex 70c in 5-day-old larvae was down-regulated. The usp was down-regulated in 5-day-old larvae, but increased in 7-day-old larvae, which disturbed the normal developmental process and caused the extension of eclosion. Moreover, antibiotics treatment significantly decreased the expression of apidaecin and abaecin in 5-day-old larvae, and defensin-1 and hymenoptaecin in 7-day-old larvae, respectively. These results showed that antibiotics could weaken the nutrient metabolism, disturb the development process, and decrease the immune competence of honeybee larvae, indicating the vital roles of gut bacteria in bee larvae fitness, so the antibiotics should be avoided to control microbial disease in honeybee larvae.

The Varroa destructor is an ectoparasitic mite and the most serious biotic threat to honey bee and apiculture worldwide. Genetic types of V. destructor determine its ability of transmission and pathogenicity. Population genetics tools could supply useful information for comprehensive management of the mite. In this study, transcriptome information of V. destructor was analyzed for mining more polymorphic microsatellite markers for the population genetics investigation with further experimental verified. A total of 83,711 unigenes were assembled with the N50 length of 1,826 bp and the GC content of 40.03%. A total of 27,775 potential microsatellite loci were identified in 18,563 unigenes. The di-nucleotide and mono-nucleotide were most abundant repeat motifs and the most dominant di-nucleotides and mono-nucleotide repeat motifs were A/T and AT/TA. Forty-two of sixty randomly selected microsatellite loci were successfully amplified. Six of them were confirmed to be polymorphic in five V. destructor geographical populations. Our result showed transcriptomic data provide valuable resources for molecular marker development, and these novel microsatellite loci would be valuable in facilitating population genetic and evolutionary of V. destructor and related species.