Identification, RNAi Knockdown, and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezygotic Phenotype in a Cricket

University of Texas Arlington, United States of America
PLoS ONE (Impact Factor: 3.23). 10/2009; 4(10):e7537. DOI: 10.1371/journal.pone.0007537
Source: PubMed


Postmating, prezygotic phenotypes, especially those that underlie reproductive isolation between closely related species, have been a central focus of evolutionary biologists over the past two decades. Such phenotypes are thought to evolve rapidly and be nearly ubiquitous among sexually reproducing eukaryotes where females mate with multiple partners. Because these phenotypes represent interplay between the male ejaculate and female reproductive tract, they are fertile ground for reproductive senescence--as ejaculate composition and female physiology typically change over an individual's life span. Although these phenotypes and their resulting dynamics are important, we have little understanding of the proteins that mediate these phenotypes, particularly for species groups where postmating, prezygotic traits are the primary mechanism of reproductive isolation. Here, we utilize proteomics, RNAi, mating experiments, and the Allonemobius socius complex of crickets, whose members are primarily isolated from one another by postmating, prezygotic phenotypes (including the ability of a male to induce a female to lay eggs), to demonstrate that one of the most abundant ejaculate proteins (a male accessory gland-biased protein similar to a trypsin-like serine protease) decreases in abundance over a male's reproductive lifetime and mediates the induction of egg-laying in females. These findings represent one of the first studies to identify a protein that plays a role in mediating both a postmating, prezygotic isolation pathway and reproductive senescence.

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    • "Among them, we have identified a gene (contig MS0081) that encodes a highly expressed trypsin-like serine protease, which is the putative orthologous protein of the ejaculate serine protease ejac-sp gene. Along with OSP, EJAC-SP is one of the most abundant proteins in the seminal fluid of the ground cricket Allonemobious socius, and is one of the few peptides known to mediate oviposition in orthop- terans [46]. Zinc-dependent metallopeptidases, including neprilysins and angiotensin-like converting enzymes, are important proteolysis regulators. "
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    ABSTRACT: Background: Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. 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. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the 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. Results: Using RNA sequencing, we characterized the transcripts of similar to 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Conclusions: Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species specific genetic control strategies.
    Preview · Article · Dec 2015 · BMC Genomics
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    • "There are many opportunities for PMPZ reproductive isolation, particularly in internal fertilizers where fertility is dependent on a complex array of processes occurring within the female reproductive environment. Understanding the precise mechanisms underlying PMPZ isolation is currently a topic of great interest amongst evolutionary biologists [3] [4] [5] [6] [7] [8]. "
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    ABSTRACT: Following mating and insemination, fertility is dependent on the successful execution of a complex array of morphological, physiological and molecular interactions between male and female proteins, cells and tissues. Many of these interacting components bear hallmarks of co-evolving systems and are suspected to contribute to postmating, prezygotic (PMPZ) reproductive barriers involved in the formation of new species. Although PMPZ reproductive isolation has historically been more difficult to study than precopulatory and postzygotic barriers, recent research has highlighted its potential role in speciation and revealed the potential impact of molecular investigation utilizing proteomic approaches. Proteomics, in conjunction with transcriptomic and evolutionary genomic studies, has been widely used to identify rapidly evolving male and female reproductive proteins. Increased access to high-throughput and quantitative proteomic techniques, as well as the ease of generating genomic and transcriptomic resources necessary for protein identification, can facilitate the extension of proteomics from traditional model species to systems of relevance to PMPZ phenotypes and hence greatly expand our understanding of how rapidly diverging molecular systems may contribute to PMPZ barriers. Here we review the influence proteomic analyses can have on our understanding of the function and evolution of the complex cellular and molecular processes governing postcopulatory male-female interactions and the study of PMPZ reproductive isolation, with the goal of expanding our understanding of the contribution of PMPZ processes to speciation.
    Full-text · Article · Oct 2015 · Journal of proteomics
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    • "The role of particular proteins in mediating specific behaviors can be tested experimentally, for example using RNAi technology (Marshall et al. 2009; Rogers et al. 2009; Marshall and DiRienzo 2012), recombinant proteins (Fleming et al. 2009), or enzymatic (Schippers et al. 2006; Rogers et al. 2009) or toxicological (Resende et al. 2013) assays. Recently developed MS-based assays (Aebersold et al. 2013) also offer increasing opportunities for the subsequent quantitative monitoring of individual proteins in large number of individuals (Plumel et al. 2013). "
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