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.