Ascosphaera apis is the causative agent of honey bee chalkbrood disease, and spores are the only known source of infections. Interference with sporulation is therefore a promising way to manage A. apis. The versicolorin reductase gene (StcU-2) is a ketoreductase protein related to sporulation and melanin biosynthesis. To study the StcU-2 gene in ascospore production of A. apis, CRISPR/Cas9 was used, and eight hygromycin B antibiotic-resistant transformants incorporating enhanced green fluorescent protein (EGFP) were made and analyzed. PCR amplification, gel electrophoresis, and sequence analysis were used for target gene editing analysis and verification. The CRISPR/Cas9 editing successfully knocked out the StcU-2 gene in A. apis. StcU-2 mutants had shown albino and non-functional spore-cyst development and lost effective sporulation. In conclusion, editing of StcU-2 gene has shown direct relation with sporulation and melanin biosynthesis of A. apis; this effective sporulation reduction would reduce the spread and pathogenicity of A. apis to managed honey bee. To the best of our knowledge, this is the first time CRISPR/Cas9-mediated gene editing has been efficiently performed in A. apis, a fungal honey bee brood pathogen, which offers a comprehensive set of procedural references that contributes to A. apis gene function studies and consequent control of chalkbrood disease.

Insect pollinators provide major pollination services for wild plants and crops. Honeybee viruses can cause serious damage to honeybee colonies. However, viruses of other wild pollinating insects have yet to be fully explored. In the present study, we used RNA sequencing to investigate the viral diversity of 50 species of wild pollinating insects. A total of 3 pathogenic honeybee viruses, 8 previously reported viruses, and 26 novel viruses were identified in sequenced samples. Among these, 7 novel viruses were shown to be closely related to honeybee pathogenic viruses, and 4 were determined to have potential pathogenicity for their hosts. The viruses detected in wild insect pollinators were mainly from the order Picornavirales and the families Orthomyxoviridae, Sinhaliviridae, Rhabdoviridae, and Flaviviridae. Our study expanded the species range of known insect pollinator viruses, contributing to future efforts to protect economic honeybees and wild pollinating insects.

Tissue phenotypic plasticity facilitates rapid adaptation of organisms to biotic and/or abiotic pressure. The reproductive capacity of honey bee workers (Apis mellifera) is plastic and responsive to pheromones produced by broods and the queen. Egg laying workers (ELWs), which could reactivate their ovaries and lay haploid eggs upon queen lost, have been commonly discussed from many aspects. However, it remains unclear whether midgut homeostasis in ELWs is affected during plastic changes. Here, we found that the expression of nutrition- and autophagy-related genes was up-regulated in the midguts of ELWs, compared with that in nurse workers (NWs) by RNA-sequencing. Furthermore, the area and number of autophagosomes were increased, along with significantly increased cell death in the midguts of ELWs. Moreover, cell cycle progression in the midguts of ELWs was increased compared with that in NWs. Consistent with the up-regulation of nutrition-related genes, the body and midgut sizes, and the number of intestinal proliferation cells of larvae reared with royal jelly (RJ) obviously increased more than those reared without RJ in vitro. Finally, cell proliferation was dramatically suppressed in the midguts of ELWs when autophagy was inhibited. Altogether, our data suggested that autophagy was induced and required to sustain cell proliferation in ELWs’ midguts, thereby revealing the critical role of autophagy played in the intestines during phenotypic plasticity changes.

In this article, we report the pathogenicity of a new strain of fungus, Rhizopus oryzae to honeybee larvae, isolated from the chalkbrood-diseased mummies of honeybee larvae and pupae collected from apiaries in China. Based on morphological observation and internal transcribed spacer (ITS) region analyses, the isolated pathogenic fungus was identified as R. oryzae. Koch’s postulates were performed to determine the cause-and-effect pathogenicity of this isolate fungus. The in vitro pathogenicity of this virulent fungus in honeybees was tested by artificially inoculating worker larvae in the lab. The pathogenicity of this new fungus for honeybee larvae was both conidial-concentration and exposure-time dependent; its highly infectious and virulent effect against the larvae was observed at 1 × 10⁵ conidia/larva in vitro after 96 h of challenge. Using probit regression analysis, the LT₅₀ value against the larvae was 26.8 h at a conidial concentration of 1 × 10⁵ conidia/larva, and the LC₅₀ was 6.2 × 10³ conidia/larva. These results indicate that the new isolate of R. oryzae has considerable pathogenicity in honeybee larvae. Additionally, this report suggests that pathogenic phytofungi may harm their associated pollinators. We recommend further research to quantify the levels, mechanisms, and pathways of the pathogenicity of this novel isolated pathogen for honeybee larvae at the colony level.

Chronic bee paralysis virus (CBPV) is a bipartite positive-sense RNA virus that causes significant colony losses in honey bees, yet its tissue-level infection dynamics remain poorly characterized. We performed tissue-resolved virome analysis of two naturally infected Apis mellifera apiaries during a CBPV outbreak in Shandong, China. The apiaries shared a queen source and overwintering site but were separated by over 80 km at the time of sampling and developed contrasting disease patterns. Symptomatic foragers showing characteristic clinical signs of CBPV infection, including trembling, flightlessness, crawling, and hairless dark cuticle, and asymptomatic foragers were collected. Head, thorax, and midgut tissues were then dissected and used for RNA sequencing, yielding 36 RNA-seq libraries. In symptomatic bees, CBPV dominated the virome, with viral loads highest in heads and lowest in midguts. The RNA1/RNA2 copy ratio revealed a conserved tissue-specific gradient, shifting from RNA2 predominance in heads and thoraces to RNA1 enrichment in midguts (p = 0.024 for each apiary). This directional imbalance was validated by multiple controls and conserved across two genetically distinguishable CBPV populations, implicating the host tissue environment as the primary driver. In asymptomatic bees, the two apiaries harbored markedly different covert viromes, with CBPV dominant in the heads and thoraces of one apiary and near-exclusive Lake Sinai virus dominance in the other. These findings reveal that CBPV segment stoichiometry is modulated in a tissue-dependent manner in vivo and that viral genetic composition and covert virome composition can diverge notably between epidemiologically connected apiaries, providing additional insight into the infection biology of CBPV under natural conditions.

Chronic bee paralysis virus (CBPV) is a serious pathogen of honey bees and has expanded across major beekeeping regions in recent years. Understanding viral connectivity between apiaries is therefore essential for disease control. However, consensus-based phylogenetic analysis has limited power to resolve epidemiological links between closely managed colonies. To address this, we applied virome sequencing and intra-host single-nucleotide variant (iSNV) profiling to investigate shared variation and possible viral exchange between two apiaries across two consecutive years. One apiary was translocated ~80 km away each spring and returned each winter to within ~1 km of a stationary apiary. This seasonal translocation mirrors the long-distance movements and winter aggregations common in commercial beekeeping operations in China and the United States, providing a natural microcosm of the migratory practices that drive large-scale viral connectivity. Parallel 16S rRNA gene profiling of the midgut microbiota revealed no detectable association between bacterial community composition and CBPV infection status. iSNV analysis identified extensive shared low-frequency variants between the two apiaries across both years, including formerly private variants that became detectable in both sites after winter co-location. In contrast, consensus phylogenies placed two co-located apiaries in separate clades, suggesting that standard surveillance may underestimate cryptic viral connectivity. We cannot rule out the possibility that the observed shared iSNVs reflect shared ancestry, earlier contact, or common external sources. Furthermore, the direction of viral exchange cannot be determined from observational data with limited replication and uneven sequencing depth. These results show that finer-scale iSNV profiling highlights patterns of viral connectivity in managed bee systems, providing a model for high-resolution surveillance of RNA viruses in mobile managed host populations where conventional methods underestimate connectivity, though validation in controlled experimental settings is needed to establish causal transmission pathways.