Nosema ceranae (Opisthosporidia: Microsporidia) is an emergent intracellular parasite of the European honey bee (Apis mellifera) and causes serious Nosema disease which has been associated with worldwide honey bee colony losses. The only registered treatment for Nosema disease is fumagillin-b, and this has raised concerns about resistance and off-target effects. Fumagillin-B is banned from use in honey bee colonies in many countries, particularly in Europe. As a result, there is an urgent need for new and effective therapeutic options to treat Nosema disease in honey bees. An RNA interference (RNAi)-based approach can be a potent strategy for controlling diseases in honey bees. We explored the therapeutic potential of silencing the sequences of two N. ceranae encoded spore wall protein (SWP) genes by means of the RNAi-based methodology. Our study revealed that the oral ingestion of dsRNAs corresponding to SWP8 and SWP12 used separately or in combination could lead to a significant reduction in spore load, improve immunity, and extend the lifespan of N. ceranae-infected bees. The results from the work completed here enhance our understanding of honey bee host responses to microsporidia infection and highlight that RNAi-based therapeutics are a promising treatment for honey bee diseases.

RNA viruses that contain single-stranded RNA genomes of positive sense make up the largest group of pathogens infecting honey bees. Sacbrood virus (SBV) is one of the most widely distributed honey bee viruses and infects the larvae of honey bees, resulting in failure to pupate and death. Among all of the viruses infecting honey bees, SBV has the greatest number of complete genomes isolated from both European honey bees Apis mellifera and Asian honey bees A. cerana worldwide. To enhance our understanding of the evolution and pathogenicity of SBV, in this study, we present the first report of whole genome sequences of two U.S. strains of SBV. The complete genome sequences of the two U.S. SBV strains were deposited in GenBank under accession numbers: MG545286.1 and MG545287.1. Both SBV strains show the typical genomic features of the Iflaviridae family. The phylogenetic analysis of the single polyprotein coding region of the U.S. strains, and other GenBank SBV submissions revealed that SBV strains split into two distinct lineages, possibly reflecting host affiliation. The phylogenetic analysis based on the 5′UTR revealed a monophyletic clade with the deep parts of the tree occupied by SBV strains from both A. cerane and A. mellifera, and the tips of branches of the tree occupied by SBV strains from A. mellifera. The study of the cold stress on the pathogenesis of the SBV infection showed that cold stress could have profound effects on sacbrood disease severity manifested by increased mortality of infected larvae. This result suggests that the high prevalence of sacbrood disease in early spring may be due to the fluctuating temperatures during the season. This study will contribute to a better understanding of the evolution and pathogenesis of SBV infection in honey bees, and have important epidemiological relevance.

Sulfoxaflor is a novel sulfoximine insecticide which is widely used to control crop pests. Risk assessments have reported its high toxicity to pollinators. However, sulfoxaflor is not persistent in the environment and few studies have addressed its negative effects on larval and newly emerged honeybees at environmentally relevant concentrations. In the present study, the sublethal effects of a sulfoxaflor commercial product, Isoclast™ Active, were evaluated in the laboratory using larvae and newly emerged worker honeybees. The results of 96-h acute toxicity showed that Isoclast is moderately toxic to adult bees, and it could induce significant death and growth failure of larvae after continuous dietary intake. In addition, Isoclast induced significant changes in antioxidative (SOD, CAT), lipid peroxidation (POD, LPO, MDA), detoxification (GST, GR, GSH) and signal transduction-related (AChE, ACh) enzymes or products both in larvae and adult honey bees under residue levels. Here we firstly reported the lethal and sublethal effects of commercial sulfoxaflor to honeybees' larvae and young workers. All these findings revealed the potential risks of sulfoxaflor residue in environment to honey bees, and may also to other pollinators. This is a laboratory mimic studies, and further studies are still needed to investigate the risks and in-depth mechanisms of sulfoxaflor to bees in field.

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.

Vairimorpha (Nosema) ceranae is the most common eukaryotic gut pathogen in honey bees. Infection is typically chronic but may result in mortality. Gut microbiota is a factor that was recently noted for gut infectious disease development. Interestingly, studies identified positive, instead of negative, associations between core bacteria of honey bee microbiota and V. ceranae infection. To investigate the effects of the positive associations, we added isomaltooligosaccharide (IMO), a prebiotic sugar also found in honey, to enhance the positive associations, and we then investigated the infection and the gut microbiota alterations using qPCR and 16S rRNA gene sequencing. We found that infected bees fed IMO had significantly higher V. ceranae spore counts but lower mortalities. In microbiota comparisons, V. ceranae infections alone significantly enhanced the overall microbiota population in the honey bee hindgut and feces; all monitored core bacteria significantly increased in the quantities but not all in the population ratios. The microbiota alterations caused by the infection were enhanced with IMO, and these alterations were similar to the differences found in bees that naturally have longer lifespans. Although our results did not clarify the causations of the positive associations between the infections and microbiota, the associations seemed to sustain the host survival and benefit the pathogen. Enhancing indigenous gut microbe to control nosema disease may result in an increment of bee populations but not the control of the pathogen. This interaction between the pathogen and microbiota potentially enhances disease transmission and avoids the social immune responses that diseased bees die prematurely to curb the disease from spreading within colonies.

Homogeneous liquid–liquid extraction (HLLE) has attracted considerable interest in the sample preparation of multi-analyte analysis. In this study, HLLEs of multiple phenolic compounds in propolis, a polyphenol-enriched resinous substance collected by honeybees, were performed for improving the understanding of the differences in partition efficiencies in four acetonitrile–water-based HLLE methods, including salting-out assisted liquid–liquid extraction (SALLE), sugaring-out assisted liquid–liquid extraction (SULLE), hydrophobic-solvent assisted liquid–liquid extraction (HSLLE), and subzero-temperature assisted liquid–liquid extraction (STLLE). Phenolic compounds were separated in reversed-phase HPLC, and the partition efficiencies in different experimental conditions were evaluated. Results showed that less-polar phenolic compounds (kaempferol and caffeic acid phenethyl ester) were highly efficiently partitioned into the upper acetonitrile (ACN) phase in all four HLLE methods. For more-polar phenolic compounds (caffeic acid, p-coumaric acid, isoferulic acid, dimethoxycinnamic acid, and cinnamic acid), increasing the concentration of ACN in the ACN–H₂O mixture could dramatically improve the partition efficiency. Moreover, results indicated that NaCl-based SALLE, HSLLE, and STLLE with ACN concentrations of 50:50 (ACN:H₂O, v/v) could be used for the selective extraction of low-polarity phenolic compounds. MgSO₄-based SALLE in the 50:50 ACN–H₂O mixture (ACN:H₂O, v/v) and the NaCl-based SALLE, SULLE, and STLLE with ACN concentrations of 70:30 (ACN:H₂O, v/v) could be used as general extraction methods for multiple phenolic compounds.