The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster

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Abstract

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Previous work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility in the ejaculate, though functional evidence in any species is lacking. Here, we used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the male's ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. We found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Data availability

All data generated or analyzed for this study are included in the manuscript, supporting files, or are available on Github. Source data files have been provided for Figure 1C, Figure 2B, Figure 4C, Figure 1-figure supplement 2B, Figure 1-figure supplement 3A & B, Figure 4-figure supplement 2, and Figure 6-figure supplement 1. All mating data, R code to analyze mating data, RNAseq data across species, and tree files/alignments for use in PAML are available on Github: https://github.com/WolfnerLab/Obps

The following previously published data sets were used

(2020) Reanalysis of FlyAtlas2.0 RNAseq data
Github, https://github.com/YazBraimah/FlyAtlas2.

https://github.com/YazBraimah/FlyAtlas2
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(2018) RNA-seq of sexed adult tissues/body parts from eight Drosophila species
NCBI Gene Expression Omnibus, GSE99574.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99574
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2. et al
(2022) 10x stringent male reproductive gland
Fly Cell Atlas, https://cloud.flycellatlas.org/index.php/s/rSgw2GGSqRkLHgM.

https://cloud.flycellatlas.org/index.php/s/rSgw2GGSqRkLHgM

Article and author information

Author details

Nora C Brown

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8567-1273
Benjamin Gordon

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3856-0500
Caitlin E McDonough-Goldstein

Department of Evolutionary Biology, University of Vienna, Vienna, Austria

Competing interests
The authors declare that no competing interests exist.
Snigdha Misra

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
Geoffrey D Findlay

Department of Biology, College of the Holy Cross, Worcester, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8052-2017
Andrew G Clark

Department of Computational Biology, Cornell University, Ithaca, United States

For correspondence
ac347@cornell.edu

Competing interests
The authors declare that no competing interests exist.
Mariana Federica Wolfner

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

For correspondence
mfw5@cornell.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2701-9505

Funding

National Institutes of Health (HD059060)

Andrew G Clark

National Institutes of Health (HD059060)

Mariana Federica Wolfner

National Institutes of Health (F32GM097789)

Geoffrey D Findlay

National Science Foundation (2212972)

Geoffrey D Findlay

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.