Identification and manipulation of oviposition stimulating proteins in the migratory grasshopper Melanoplus sanguinipes

Abstract

Seminal fluid proteins (SFPs) control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and reproductive isolation between species. Targetting reproduction genes for species-specific crop protection, is becoming one main challenge for moving towards the use of RNAi technology as insect pest control agent. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. Genetic tools such as RNAi technology and the ability to generate massive amounts of genomics and proteomics data, makes it possible to identify these proteins and determine their biological function. Seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America, were characterized by combining high throughput using RNA sequencing and shotgun proteomic profiling. Transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory gland complex were identified using RNA-seq while proteomic profiling identified 353 proteins. Of special interest were two prostaglandin synthetases involved in the synthesis of prostaglandins, a group of bioactive lipid compounds derived enzymatically of arachidonic acid, a oviposition stimulating protein (OSP), a ejaculate serine protease (EJAC-SP), and angiotensin coverting enzyme (ACE), which are known to regulate female oviposition in several insects species. First, injections of 100ug prostaglandin E2 or F2α into the abdominal region of virgin females showed that females treated with F2α increased the oviposition rate in about 60% compared to the control group. dsRNAs-mediated RNAi were performed to validate the functional role of the OSP, EJAC-SP and ACE in the reproductive capability of M. Sanguinipes. Quantitative real time PCR showed a significant reduction in gene expression after injections of dsRNAs. Then, mating bioassays confirmed that ACE knocked down males resulted in females laying significantly (p0.05) fewer eggs when compared from matings with unexposed males. Females mated with EJAC-SP knocked down males also showed a marginal significance (p0.07) reduction in the number of eggs laid. This study provides new insights into the proteomic components of male ejaculate in M. Sanguinipes and specific male accessory gland proteins which could be potential targets for the future development of species-specific gene silencing biopesticides based on RNAi technology.