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Title: Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket

Author
item MARSHALL, JEREMY - Kansas State University
item HUESTIS, DIANA - Kansas State University
item HIROMASA, YASUAKI - Kansas State University
item OPPERT, CRIS - University Of Tennessee
item MARSHALL, SUSAN - Kansas State University
item TOMICH, JOHN - Kansas State University
item Oppert, Brenda

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2009
Publication Date: 10/23/2009
Citation: Marshall, J.L., Huestis, D.L., Hiromasa, Y., Oppert, C., Marshall, S.A., Tomich, J., Oppert, B.S. 2009. Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket. PLoS One 4(10): e7537-e7546.

Interpretive Summary: Understanding how insects reproduce is important in the development of new control products. In a proteomic study to identify all proteins in the male cricket ejaculate, one protein was found to be related to a serine protease. Using RNA interference, the genetic transcript encoding the cricket protease was eliminated, or “knocked-out”. This is the first demonstration that specific proteins can be knocked-down/knocked out in insect reproductive organs. The results have implications in understanding how insects reproduce as well as potential targets for insect control.

Technical Abstract: Male ejaculate proteins, including both sperm and seminal fluid proteins, play an important role in mediating reproductive biology. The function of ejaculate proteins can include enabling sperm-egg interactions, enhancing sperm storage, mediating female attractiveness, and even regulating female life-span. Although such proteins are clearly important, within internal fertilizing organisms it is often difficult to assess the function and significance of individual ejaculate proteins. To better assess such functions and significance, we propose combining an organismal system that has internal fertilization yet produces an external ejaculate with RNAi technology to manipulate the abundance of specific ejaculate proteins. We found that injecting adult male crickets (Allonemobius socius) in the abdomen with gene-specific dsRNA is a successful technique to knockdown the abundance of the target protein in the male ejaculate. Moreover, this significant knockdown appears to last throughout the reproductive lifespan of the cricket. To our knowledge, this is the first study to document the knockdown of an individual ejaculate protein by injecting adult males in the abdomen with gene-specific dsRNA. The ability to knockdown specific ejaculate proteins, in combination with organismal systems that have internal fertilization yet produce external ejaculates, will greatly enhance our ability to assess the function and importance of male reproductive proteins in mediating a range of reproductive processes – including successful fertilization, sperm competition, reproductive physiologies, and male-female reproductive conflicts.