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Seminal fluid protein depletion and replenishment in the fruit fly, Drosophila melanogaster: an ELISA-based method for tracking individual ejaculates

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Abstract

In many species, seminal fluid proteins (SFPs) affect female post-mating behavioral patterns, including sperm storage, egg laying, feeding, and remating. Yet, few studies have investigated the patterns of allocation, depletion, and replenishment of SFPs in male animals, despite the importance of these proteins to male and female reproductive success. To investigate such SFP dynamics, it is necessary to have a sensitive method for quantifying SFP levels in males and mated females. We developed such a method by adapting the enzyme-linked immunosorbent assay (ELISA) using anti-SFP antibodies. Here, we first use two Drosophila melanogaster SFPs (ovulin and sex peptide) to demonstrate that ELISAs provide accurate measures of SFP levels. We find that, consistent with previous data from Western blotting or immunofluorescence studies, levels of both ovulin and sex peptide decline in the mated female with time since mating, but they do so at different rates. We then use ELISAs to show that males become depleted of SFPs with repeated matings, but that previously mated males are able to transfer “virgin” levels of SFPs after 3 days of sexual inactivity. Finally, we demonstrate that ELISAs can detect SFPs from wild-caught D. melanogaster males and, thus, potentially can be used to track mating patterns in the wild. This method of measuring SFP dynamics can be used in a wide range of species to address questions related to male reproductive investment, female mating history, and variation in female post-mating behavioral changes.

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Acknowledgments

We thank Lisa Daley and Judy Appleton for providing training in ELISAs and Tom Giardina, Andrew Orapallo, Dustin Rubinstein, and Shawna Tonick for helping with the experiments. We are grateful to Andrew Clark for statistical insights and for the use of his plate reader. Eric Kubli generously provided sex peptide antiserum. Frank Avila, Lauren Cator, Michelle Helinski, Mari Kimura, Brooke LaFlamme, Lisa McGraw, and Peter Piermarini provided valuable insights for the writing of this manuscript. We thank Dennis Hartley for access to Little Tree Orchards for our studies of wild D. melanogaster. The research was funded by a Ruth L. Kirschstein National Research Service Award Post-Doctoral Fellowship (1F32GM074361) to L.K.S., a National Science Foundation grant (DEB-0746915) to A.C.F, and a National Institutes of Health grant (HD38921) to M.F.W.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, 421 Biotechnology Building, Ithaca, NY, 14853, USA

    Laura K. Sirot, Norene A. Buehner & Mariana F. Wolfner

  2. Biological Sciences Department, Binghamton University, Vestal Parkway East, P.O. Box 6000, Binghamton, NY, 13902, USA

    Anthony C. Fiumera

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  1. Laura K. Sirot
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  2. Norene A. Buehner
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  4. Mariana F. Wolfner
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Correspondence to Laura K. Sirot.

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Communicated by N. Wedell

Norene A. Buehner and Anthony C. Fiumera contributed equally to this work.

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Sirot, L.K., Buehner, N.A., Fiumera, A.C. et al. Seminal fluid protein depletion and replenishment in the fruit fly, Drosophila melanogaster: an ELISA-based method for tracking individual ejaculates. Behav Ecol Sociobiol 63, 1505–1513 (2009). https://doi.org/10.1007/s00265-009-0806-6

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