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

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.

. CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint immediately after the end of mating relative to females mated to control males (p<0.001, n=27-30). (C) Counts of eggs from mated CS females over 4 days. CS females mated to CrebA-GAL4>Obp56g RNAi lay significantly fewer numbers of eggs relative to CS females mated to control males (p<0.001, n=27-28). (D) Ejaculate loss (dotted line) from the bursa observed in females mated to CrebA-GAL4>Obp56g RNAi males, with the bursa dissected and imaged for GFP to visualize the autofluorescent speckles that comprise the uncoagulated mating plug. Error bars represent mean +/-SEM. Significance level: * p<0.05, ** p<0.01, *** p<0.001.  . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (Obp22a,Obp51a,Obp56e,Obp56f,Obp56i) and X-linked (Obp8a) Obp genes used in this study, with text boxes representing chromosomes X/Y, 2, and 3 (dot chromosome not shown).
The kinked line represents the Y chromosome. Obp and ebony CRISPR editing takes place in the germline of individuals in the P1 generation. ebony editing can happen on either the gRNA or Cas9 chromosomes (written out in the F1 generation as "ebony" for simplicity), which are removed from the genetic background before assaying males for reproductive phenotypes.
. CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023.  and merge + transillumination microscopy images. 9/9 females mated to these males had mating plugs, and 9/9 had sperm masses present in their bursas. B) Representative CS female mated to Obp56g 1 ;ProtB-eGFP mutant males. 0/10 females had mating plugs, though 10/10 had sperm masses present in their bursas.
. CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint  . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint     . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint

Supplemental methods:
To build our gRNA-expressing vectors, we used pAC-U63-tgRNA-Rev, a plasmid that expresses multiplexed gRNAs separated by rice Gly tRNA sequences, as well as the (F+E) . CC-BY 4.0 International license available under a ich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 7, 2023. ; https://doi.org/10.1101/2023.02.03.526941 doi: oRxiv preprint