RNA interference (RNAi) has been developed and used as an emerging strategy for pest management. Here, an entomopathogen Bacillus thuringiensis (Bt) was used to express the dsRNA for the control of Plutella xylostella. A vector containing a 325-bp fragment of the conserved region of P. xylostella arginine kinase gene (PxAK) flanking in two ends with the promoter Pro3α was developed and transferred into Bt 8010 and BMB171, and consequently engineered Bt strains 8010AKi and BMB171AKi expressing dsRNA of PxAK were developed. The two engineered Bt strains were separately mixed with Bt 8010 in a series of ratios, and then fed to the P. xylostella larvae. We found that 8010:8010AKi of 9:1 and 8010:BMB171AKi of 7:3 caused a higher mortality than Bt 8010. PxAK expression levels in the individuals treated with the mixtures, 8010AKi and BMB171Aki, were lower than that in the control. The intrinsic rate of increase (r) and net reproductive rate (R₀) of the population treated with 8010:8010AKi of 9:1 were lower than those of the population treated with Bt 8010 or 8010AKi. We developed a Bt-mediated insect RNAi for the control of P. xylostella and demonstrated a practical approach to integrating the entomopathogen with RNAi technique for the pest management.

DsRNA-degrading enzymes (dsRNases) have been recognized as important factors in reducing RNA interference (RNAi) efficiency in different insect species. However, dsRNases in Plutella xylostella are still unknown. We identified the full-length cDNAs of PxdsRNase1, PxdsRNase2, PxdsRNase3, and PxdsRNase4. Gene expression profile showed that PxdsRNase1 was mainly expressed in the hemolymph; and that PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. The expression of PxCht (Chitinase of P. xylostella) in P. xylostella larvae injected with the mixture of dsPxCht (dsRNA of PxCht) and dsPxdsRNase1 (dsRNA of PxdsRNase1), dsPxdsRNase2 (dsRNA of PxdsRNase2), or dsPxdsRNase3 (dsRNA of PxdsRNase3) was significantly higher than that in the larvae injected with the mixture of dsGFP (dsRNA of green fluorescent protein gene, GFP) and dsPxCht; the transcription level of PxCht in the larvae feeding on the mixture of dsPxCht and dsPxdsRNase1, dsPxdsRNase2, or dsPxdsRNase3 was significantly higher than that in the larvae feeding on the mixture of dsPxCht and dsGFP. The recombinant protein of PxdsRNase1 degraded dsRNA rapidly, PxdsRNase3 cleaved dsRNA without complete degradation, and PxdsRNase2 could not degrade dsRNA in vitro. These results suggested that PxdsRNases1, PxdsRNases2, and PxdsRNases3 were involved in the dsRNA degradation to reduce RNAi efficiency with different mechanisms.

Gametocyte-specific factor 1 (Gtsf1) plays a pivotal role in the gonadal development by transposon silencing via the piRNA pathway in Drosophila melanogaster and Bombyx mori, and is implicated in the female sex determination in B. mori. However, its function remains elusive in other insect species, including Plutella xylostella, a highly destructive pest in cruciferous crops. In this study, we characterized PxGtsf1, encoding a 27.84 kDa protein containing two conserved U11-48K-like CHHC zinc-finger domains. PxGtsf1 was predominantly expressed in male pupae, males and testes. Using CRISPR/Cas9 technique, we successfully generated a homozygous PxGtsf1-knockout strain (△PxGtsf1) with 11 bp deletion. △PxGtsf1 females exhibited normal morphology in terms of external genitalia, body weight, ovariole morphology, ovariole length, oocytes number, and internal ovarian structure, and maintained the expression levels of genes related to female sex determination and fertility. In contrast, △PxGtsf1 males displayed reduced testis size and enlarged eupyrene sperm bundles, whereas apyrene sperm bundles appeared normal. Wild-type (WT) or △PxGtsf1 females mated with △PxGtsf1 males had significantly smaller bursa copulatrix distention area compared to those mated with WT males. Furthermore, WT or △PxGtsf1 females mated with △PxGtsf1 males produced significantly fewer eggs with lower hatching rates than those mated with WT males. Co-immunoprecipitation assays revealed that PxGtsf1 specifically interacts with PxPiwi. Small RNA sequencing further showed that PxGtsf1 knockout resulted in transposon dysregulation in the testes without affecting overall piRNA abundance, suggesting that PxGtsf1 functions in piRNA-mediated transposon regulation rather than global piRNA biogenesis. PxGtsf1 is first reported to be specifically required for male fertility in Lepidoptera insects but is dispensable for female sex determination., PxGtsf1 regulates transposon homeostasis without affecting global piRNA biogenesis. These findings provide new insights into the diverse functions of Gtsf1 in Lepidopteran insects.