Molecular characterization and functional analysis of daf‐16‐2b gene in the pine wood nematode, Bursaphelenchus xylophilus

The Forkhead box O transcription factor DAF‐16 is an important downstream transcription factor in the well‐conserved insulin signalling (IIS) pathway in Caenorhabditis elegans. The nuclear localization of DAF‐16 can activate a series of genes that mediate oxidative stress, heat shock, xenobiotic responses, innate immunity, metabolism and autophagy. In this study, a Bxdaf‐16‐2b gene encoding 510 amino acids was cloned from Bursaphelenchus xylophilus. Sequence analysis showed that the BxDAF‐16‐2b protein shared the highest similarity value (92%) with the DAF16‐2 (accession no. ADD84716.1) of B. xylophilus. Using in situ hybridization, Bxdaf‐16‐2b was shown to be constitutively expressed in the circum‐pharyngeal nerve rings and intestines of males, and in the head neurons and the ovaries of females. Quantitative RT‐PCR showed that the highest gene expression was found in females, followed by juveniles, males and eggs. Bxdaf‐16‐2b expression was up‐regulated after treatment with the botanical pesticides rotenone and matrine. RNA interference showed that the expression of Bxdaf‐16‐2b gene was reduced by dsRNA, and the silencing of Bxdaf‐16‐2b suppressed the expression of several IIS pathway genes. Bursaphelenchus xylophilus showed increased mortality at sublethal doses of rotenone and matrine when subjected to Bxdaf‐16‐2b gene silencing. Thus, Bxdaf‐16‐2b may participate in the stress response of B. xylophilus and play important roles in the immune system. This study paves the way towards design of new drug targets for non‐polluting control of nematodes.