Molecular characterizations of natalisin and its roles in modulating mating in the oriental fruit fly, Bactrocera dorsalis (Hendel)

Initially, natalisin (NTL) was identified from three holometabolous insect species, Drosophila melanogaster, Tribolium castaneum and Bombyx mori, and was documented to regulate reproductive behaviours in D. melanogaster and T. castaneum. In this study, we report the sequences of the NTL precursor and its receptor (NTLR) from an important agricultural pest, Bactrocera dorsalis (Hendel). NTLR is a typical G-protein coupled receptor and phylogenetic analysis showed that B. dorsalis NTLR was closely related to insect natalisin receptors from other species. A functional assay of NTLR transiently expressed in Chinese hamster ovary cells showed that it was activated by putative natalisin mature peptides in a concentration-dependent manner, with 50% effective concentrations (EC50) at nanomolar or micromolar levels. As indicated by quantitative real-time PCR, both NTL and NTLR had the highest expression in the central nervous system of B. dorsalis compared with the other tested tissues. Three pairs of adult brain neurones of B. dorsalis were identified with immunohistochemical antibody staining against D. melanogaster NTL4, and in situ hybridization with specific DNA probes. Moreover, RNA interference mediated by double-stranded RNA injection in adults provided evidence for the important roles of NTL in regulating both male and female mating frequencies in this fly.