The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster
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eLife assessment
This important study describes an atypical role of the odorant binding protein Obp56g in mating plug formation in Drosophila melanogaster suggesting that Obps may play roles in reproduction in addition to their originally described roles in olfaction. Mutant males lacking Obp56g fail to induce the formation of a mating plug in the female reproductive tract-leading to ejaculate loss and reduced sperm storage. The evidence supporting the claims of the authors is solid and compelling. The work will be of interest to biologists studying Obps and seminal fluid protein function and their evolution.
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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. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). 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, though functional evidence in any species is lacking. Here, we used functional genetics 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 are dispensable. 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, likely due to its expression in the male ejaculatory bulb. 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 in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.
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eLife assessment
This important study describes an atypical role of the odorant binding protein Obp56g in mating plug formation in Drosophila melanogaster suggesting that Obps may play roles in reproduction in addition to their originally described roles in olfaction. Mutant males lacking Obp56g fail to induce the formation of a mating plug in the female reproductive tract-leading to ejaculate loss and reduced sperm storage. The evidence supporting the claims of the authors is solid and compelling. The work will be of interest to biologists studying Obps and seminal fluid protein function and their evolution.
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Reviewer #1 (Public Review):
This study characterized the role of the Drosophila odorant-binding protein Obp56g in mediating post-mating responses in females. The authors show that this Obp56g is expressed in the male ejaculatory bulb, use genetic approaches to disrupt Obp56g, and show that males with disrupted Obp56g fail to form mating plugs in their mates.
Despite the fact that Obp56g deficient males generate and transfer sperm normally, the lack of formation of a functional mating plug leads to sperm loss in females mated to these males and thus a failure to induce normal post-mating responses.
This study identifies an unexpected role for an Obp and raises new questions about the function of this class of protein and the variety of roles that they may play in sensory function and reproduction.
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Reviewer #2 (Public Review):
Here, Brown and colleagues report a valuable finding on the function and evolution of the seminal odorant-binding protein Obp56g in Drosophila melanogaster. Previous studies have shown that this family of proteins is highly expressed in olfactory tissues like the antennae and maxillary palps. Some of these proteins have been shown to mediate behavioural responses to specific odorants-hence the general moniker odorant binding proteins. This slightly misleading historical naming convention implies an exclusive role in olfaction-however, many of these proteins are expressed in other tissues of the animal, including the male reproductive system. In addition, seminal fluid proteins exhibit a fascinating evolutionary history, with rapid evolution and turnover across taxa.
The authors suggest that the Obp56g …
Reviewer #2 (Public Review):
Here, Brown and colleagues report a valuable finding on the function and evolution of the seminal odorant-binding protein Obp56g in Drosophila melanogaster. Previous studies have shown that this family of proteins is highly expressed in olfactory tissues like the antennae and maxillary palps. Some of these proteins have been shown to mediate behavioural responses to specific odorants-hence the general moniker odorant binding proteins. This slightly misleading historical naming convention implies an exclusive role in olfaction-however, many of these proteins are expressed in other tissues of the animal, including the male reproductive system. In addition, seminal fluid proteins exhibit a fascinating evolutionary history, with rapid evolution and turnover across taxa.
The authors suggest that the Obp56g protein may have been co-opted for a reproductive role in Drosophila melanogaster during evolution. The authors show that Obp56g is required for male fertility and the induction of the post-mating response in females. Mutant males lacking Obp56g fail to form a mating plug in the female reproductive tract-leading to ejaculate loss and reduced sperm storage. The experimental evidence supporting the claims of the authors is solid and compelling. The data were collected and analyzed using solid and validated methodologies. The author's findings can be used as a starting point for understanding the mechanistic roles of this family of proteins in mating plug coagulation. The work will interest biologists studying non-sperm seminal fluid protein function and evolution.
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Reviewer #3 (Public Review):
Male seminal fluid proteins play a crucial role in fertility and influence female physiology and behavior after mating. Brown et al. have discovered a new reproductive function for odorant-binding proteins (Obps) in Drosophila. The study shows that Obp56g is expressed in male reproductive tissues that produce seminal fluid proteins and is required for the formation of the mating plug in the mated female. The study demonstrates that RNAi knockdown and CRISPR/Cas9-generated mutations in Obp56g result in a defective mating plug, reduced sperm storage, and subsequent effects on female post-mating responses. The research also suggests that Obp56g has been co-opted for a reproductive function over evolutionary time, as supported by functional and comparative RNAseq data across Drosophila species. Finally, the …
Reviewer #3 (Public Review):
Male seminal fluid proteins play a crucial role in fertility and influence female physiology and behavior after mating. Brown et al. have discovered a new reproductive function for odorant-binding proteins (Obps) in Drosophila. The study shows that Obp56g is expressed in male reproductive tissues that produce seminal fluid proteins and is required for the formation of the mating plug in the mated female. The study demonstrates that RNAi knockdown and CRISPR/Cas9-generated mutations in Obp56g result in a defective mating plug, reduced sperm storage, and subsequent effects on female post-mating responses. The research also suggests that Obp56g has been co-opted for a reproductive function over evolutionary time, as supported by functional and comparative RNAseq data across Drosophila species. Finally, the study reports expression shifts, duplication, and divergence in the evolution of these seminal protein genes.
Overall, the study represents a significant contribution to our understanding of seminal proteins and their reproductive function. The creation of novel Obp mutants using CRISPR/Cas9 technology is a valuable resource for future research in the Drosophila community. The manuscript successfully conveys the key findings and their potential implications for the field. However, to reinforce the study's conclusions, more quantitative data is necessary. Furthermore, improving the statistical analysis and incorporating additional genetic controls would enhance the quality of the study and provide a stronger foundation for its conclusions.
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