Seminal Fluid Protein Genes Of The Brown Planthopper, Nilaparvata Lugens And Functional Analysis Of A Neuropeptide-ion Transport Peptide Gene

Seminal fluid proteins (SFPs) are produced mainly in accessory gland of male insects and transferred to females during mating, and induce numerous physiological and post-mating behavioral changes in females. Proteomic research of insect SFPs has not been implemented in any hemipterous species. Here, high-throughput mass spectrometry proteomic and transcriptomic analyses were apllied to characterize seminal fluid protein composition in the brown planthopper, Nilaparvata lugens, an economically important hemipterous pest. From the proteomic analysis,94 putative secreted SFPs were identified, of which 84 proteins possess a signal peptide, and the expression level of these proteins was provided by transcriptome. With the comparison between N. lugens and other insect’s SFPs, an apparent expansion of N. lugens seminal fluid trypsins and carboxylesterases was observed. The number of N. lugens seminal fluid trypsins (n=20) was at least twice than those found in other insects.6 carboxylesterases were deteced in N. lugens seminal fluid, while seminal fluid carboxylesterases was rarely detected in other insects. Eleven new insect SFPs were identified, including mesencephalic astrocyte-derived neurotrophic factor, selenoprotein, EGF domain containing proteins and a neuropeptide ion transport-like peptide. This work represented the first characterisation of putative SFPs in a hemipeteran species and identified new insect SFPs. Our result provides a foundation for future studies to investigate the functions of SFPs in N. lugens and is an important addition to available data for comparative studies of SFPs in insects.16 N. lugens seminal fluid trypsins were eliminated by RNAi, but this large loss of seminal fluid trypsins showed no obvious influence in female fecundity. Combining proteomics analysis and RNAi results, we predicted that N. lugens induced female post-mating behavior in a different molecular mechanism as Drosophila melanogaster.Ion transport peptide (ITP) and its alternatively spliced homologous ITP-like (ITPL) products play important roles in various insect developmental processes. We found for the first time that alternative 5’UTR was used by NLITPL (NLITPL-1,2,3 and 4) to control spatio-temporal expression in Nilaparvatalugens, as evidenced by RT-qPCR. By using alternative 5’UTR, NLITPL-1 was expressed exclusively in the male reproductive system, where it synthesized the NLITPL seminal fluid protein. And interestingly, NUTPLs-3 and -4 were expressed exclusively in the integument, indicating a specialized function for NLITPL during ecdysis and eclosion. The functions of NLITP and NLITPL were investigated using RNA interference.No apparent phenotypes were observed when expression of NLITPLs was suppressed. However, when NLITP expression was suppressed, the insects failed to expand their wings, indicating that NLITP is a neuropeptide associated with wing expansion in addition to bursicon. Additionally, in contrary to bursicon, the insects showed increased melanization when NLITP was eliminated by RNAi. Unlike in previous studies of ITP/ITPL in other species, NLITP was very important in controlling N. lugens postecdysial behaviors but not critical during ecdysis. Thus, the functions of ITP and ITPL are more complex in insects than previously thought.