Bradysia odoriphaga Yang et Zhang damages roots of 30 plant species, resulting in >50% yield loss. Heat stress can not only affect the survival but also affect the expression of heat shock proteins of B. odoriphaga. In this study, two small heat shock protein genes, Hsp21.9 and Hsp22.3, were cloned from B. odoriphaga. The full-length cDNA sequences of BoHsp21.9 and BoHsp22.3 were 749 and 941 bp in length and contained a 588 and 594 bp open reading frame (ORF), encoding a protein of 196 and 198 amino acids with a calculated molecular weight of 21.9 and 22.3 kDa and an isoelectric point of 6.84 and 6.91. Phylogenetic tree analysis showed that BoHsp21.9 and BoHsp22.3 clustered into one branch with flies. qRT-PCR analyses indicated that BoHsp21.9 and BoHsp22.3 were expressed in all tested developmental stages and body segments, especially induced by heat stress. RNAi-mediated silencing of BoHsp21.9 and BoHsp22.3 significantly decreased the survival rate of fourth-instar larvae when exposed to 38 °C. This is the first study on small heat shock proteins in B. odoriphaga, and BoHsp21.9, and BoHsp22.3 play important roles in the molecular mechanism of B. odoriphaga to theromotolerance.

In this study, PCR amplification, cloning and sequencing analysis were adopted to explore genetic diversity of microsporidia (LEP9557) infecting Athetis lepigone . The small subunit ribosomal DNA (SSU rDNA), internal transcribed spacer (ITS) and intergenic spacers (IGS) of ribosomal RNA (rRNA) were cloned from the strain and sequenced. By means of multiple sequence alignment, we found that the three gene regions had different levels of polymorphism. There was greater polymorphism in ITS (74 variable sites) and IGS (55.59%) regions than in the SSU rDNA (17 variable sites). Phylogenetic analysis was performed using Kimura 2-parameter with neighbor joining and the results showed that LEP9557 had a close relationship with Nosema bombycis . Sequences of each clone were submitted to Genbank (Accession number: MF150254 - MF150258 ). All of the results indicated the presence of genetic diversity in LEP9557, which established the foundation for identifying the phylogeny and relationships with other microsporidian strains, and had significant biological meaning for maintaining the survival and population continuity of the strain.

Bradysia odoriphaga is a devastating soil pest of Allium tuberosum (Chinese chive), and current control relies heavily on chemical insecticides. Cry39Ab1 toxins from Bacillus thuringiensis (Bt), which are highly toxic to B. odoriphaga, offer an environmentally friendly alternative. However, its mechanism of action remains unclear. In this study, we identified the involvement of vacuolar H⁺-ATPase subunit A (V-ATPase A) in Cry39Ab1 insecticidal activities. The full-length cDNA sequences of BoV-ATPase A was contained 1659 bp open reading frame (ORF), encoding a protein of 552 amino acids with a calculated molecular weight of 59 kDa and an isoelectric point of 9.11. Successful expression and purification of BoV-ATPase A (with GST and His tags) and Cry39Ab1 (with GST and His tags) proteins were achieved. GST pull-down assays demonstrated a direct interaction between recombinant BoV-ATPase A and activated Cry39Ab1 toxin in vitro. Heterologous expression of BoV-ATPase A in Cry-insensitive Sf9 cells conferred susceptibility to Cry39Ab1, resulting in a significant increase in cytolysis compared with control cells. Finally, RNAi-mediated knockdown of BoV-ATPase A in larvae significantly decreased their susceptibility to Cry39Ab1, as evidenced by a marked decrease in mortality. This is the first report that BoV-ATPase A is a key protein required for Cry39Ab1 toxicity, revealing its insecticidal mechanism and establishing BoV-ATPase A as a potential target for pest control.