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

ABSTRACT 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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Available from: Diana Huestis, Sep 25, 2015
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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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    ABSTRACT: Proteomics refers to the study of the protein complement expressed by a genome and aims to understand protein expression, regulation, function, and interactions. Expression proteomics affords an unbiased image of the proteins potentially associated with or responsible for specific behaviors without requiring previous knowledge of the nature of these molecules. Recent technological advances in mass spectrometry, bioinformatics, and genome sequencing have made proteomics accessible to the study of non-model species and to different fields of biological research. In this review, we call the attention of behavioral ecologists to proteomic technologies and we highlight the great potential they offer for interdisciplinary research by 1) pointing out the advantages of the large-scale study of proteins, 2) suggesting research topics best tackled by this approach, and 3) indicating some of the techniques available for the identification and quantification of proteins. We also show how proteomic approaches can help formulate and test hypotheses on the mechanisms underlying behavior and develop experimental tools which allow the manipulation of behavior.
    Behavioral Ecology 06/2014; 26(1). DOI:10.1093/beheco/aru096 · 3.18 Impact Factor
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    • "This protein is a trypsin‐like serine protease (Simmons et al. 2013) similar to those reported from the accessory glands of the crickets Gryllus pennsylvannicus (Andrés et al. 2006), Allenemobius socius and A. fasciatus (Marshall et al. 2011). In A. socius RNA interference knockdown has shown that ejaculate serine protease mediates the induction of egg laying in mated females (Marshall et al. 2009). Serine proteases are also involved in sperm function and fertility in invertebrates and vertebrates alike (Green and Summers 1980; Inaba et al. 1993; Kodama et al. 2002). "
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    ABSTRACT: The ejaculates of most internally fertilizing species consists of both sperm and seminal fluid proteins. Seminal fluid proteins have been studied largely in relation to their post-mating effects on female reproductive physiology, and predominantly in genomically well-characterized species. Seminal fluids can also play important roles in sperm maturation and performance. In the field cricket Teleogryllus oceanicus the viability of ejaculated sperm increases as males age, as does their competitive fertilization success. Here, using quantitative proteomics and quantitative real-time PCR, we document ontogenetic changes in seminal fluid protein abundance and in seminal fluid gene expression. We identified at least nine proteins that changed in abundance in the seminal fluid of crickets as they aged. Gene expression was quantified for five seminal fluid protein genes, and in four of these gene expression changed as males aged. These ontogenetic changes were associated with a general increase in the size of the male accessory glands. Several of the seminal fluid proteins that we have identified are novel, and some have BLAST matches to proteins implicated in sperm function. Our data suggest that age related changes in competitive fertilization success may be dependent on seminal fluid chemistry.
    Evolution & Development 03/2014; 16(2):101-9. DOI:10.1111/ede.12068 · 2.72 Impact Factor
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    • "RNAi knockdown of ejac-sp in male crickets led to reduced levels of the protein in the ejaculate, which correlated with reduced numbers of eggs laid by females mated to these males. ejac-sp is also a candidate speciation gene, as it shows a high level of divergence due to positive selection between two related cricket species (Marshall et al., 2009, 2011). In D. melanogaster, a predicted trypsin-like serine protease in the seminal fluid, seminase (CG10586), is also required for normal induction of egg-laying in mated females (LaFlamme et al., 2012). "
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    ABSTRACT: Proteins in the seminal fluid of animals with internal fertilization affect numerous responses in mated females that impact both male and female fertility. Among these proteins is the highly represented class of proteolysis regulators (proteases and their inhibitors). Though proteolysis regulators have now been identified in the seminal fluid of all animals in which proteomic studies of the seminal fluid have been conducted (as well as several other species in which they have not), a unified understanding of the importance of proteolysis to male fertilization success and other reproductive processes has not yet been achieved. In this review, we provide an overview of the identification of proteolysis regulators in the seminal fluid of humans and Drosophila melanogaster, the two species with the most comprehensively known seminal fluid proteomes. We also highlight reports demonstrating the functional significance of specific proteolysis regulators in reproductive and post-mating processes. Finally, we make broad suggestions for the direction of future research into the roles of both active seminal fluid proteolysis regulators and their inactive homologs, another significant class of seminal fluid proteins. We hope that this review aids researchers in pursuing a coordinated study of the functional significance of proteolysis regulators in semen. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 02/2013; 80(2). DOI:10.1002/mrd.22130 · 2.53 Impact Factor
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