Male x Female Protein Interactions Mediating Reproductive Success in the Drosophila Mating Plug

PROJECT SUMMARY
In both mammals and invertebrates, initiation of post-mating responses in the female post-copulation is known
to contribute to reproductive success. Seminal fluid proteins (Sfps) have been shown to initiate many of these
responses. However, male x female interactions are underexplored as contributors to infertility. Investigating
the mechanisms and evolution of male x female interactions is critical for understanding the complexities of
reproduction. I will use D. melanogaster as a model system for investigating post-copulatory male x female
interactions via characterization of male and female contributions to the Drosophila mating plug’s (MP)
formation and ejection. In Drosophila, the MP forms in the uterus of the female via coagulation of male-
ejaculated Sfps and some female reproductive tract proteins. Genetic disruption of the MP impacts
reproductive outcomes by affecting sperm retention; however, little is known about the male and female
proteins (and their interactions) that modulate mating plug ejection (MPE) rates either directly by contributing to
MP composition/degradation or indirectly by regulating female ejection behavior. In Aims 1 & 2 I will
respectively use female or male phenotypic variation in the Drosophila Genomic Reference Panel to
perform a GWAS on MPE timing. I will then functionally investigate how top gene candidates mediate MPE
timing. I have concluded a GWAS on female MPE timing and have so far identified 4 neuronal genes that
regulate female MPE (Aim 1). Using highly tissue- and cell type- specific knockdown of gene candidate
expression via RNA interference, I will more deeply characterize neuronal regulation of female MPE timing.
After also identifying and characterizing male genes regulating MPE timing (Aim2) I will perform a 6x6 grid
cross of males and females with disrupted function of MPE genes to investigate the presence of
complex non-additive male x female MPE interactions. Finally, I will investigate the molecular evolution and
function of male and female genes contributing to MP composition (Aim 3). I discovered that many male and
female MP genes are closely paralogous to each other and evolving under positive selection,
potentially suggestive of evolution under sexual conflict. After fully characterizing the evolution and
coevolution of these male and female MP paralogs, I will investigate their sex-specific functions in MPE
and MP formation. Observation of opposing functions for paralogous male and female MP genes would point
to sexual conflict driven evolution caused by opposing sex-specific optimal mating strategies. Observation of
complementary functions would indicate similar evolutionary pressures acting on male and female paralogs to
cooperatively ensure optimal reproductive outcomes.

Grant Number: 5F32HD111231-02
NIH Institute/Center: NIH
Principal Investigator: Jolie Carlisle