Expression of Mst89B and CG31287 is Needed for Effective Sperm Storage and Egg Fertilization in Drosophila

Gurman Grewal,Alberto Civetta,Bahar Patlar

doi:10.3390/cells10020289

Abstract

In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.

Highlights

Variation in fecundity and fertility influences an organism’s reproductive fitness
In Drosophila and other organisms, gene perturbation screens such as loss-of-function alleles or transgenic constructs that modulate gene expression have been used to identify sperm and seminal fluid protein genes that have an impact on different aspects of male fecundity
The KK lines from Vienna Drosophila Resource Center (VDRC) and the Harvard Transgenic RNAi Project (TRiP) lines from Bloomington Drosophila Stock Center (BDSC) were chosen because these stocks contain UAS-hairpin sequences inserted at specific locations in the second chromosome

Summary

Introduction

Variation in fecundity and fertility influences an organism’s reproductive fitness. The identification of genes contributing to variation in phenotypes, those that impact fitness, is a long-standing question among evolutionary geneticists [1]. Perturbation of specific genes can completely or partially block the production of mature sperm [2], drastically decrease the ability of males to store sperm [3,4,5], disturb the retention and release of sperm in/from storage [6,7,8], and even impair sperm viability once in female storage [9,10]. Both sperm and seminal fluid protein genes have been shown to influence male sperm competitive ability [11]. This drop in the number of sperm in storage caused a similar drop in egg fertilization over the same period of time

Gene Selection

Fly Stocks and Maintenance

Gene Knockdown

Fecundity and Sperm Competition

Statistical Analysis

Results