Figure 1.

Assessing sperm competitive ability in D. melanogaster. (A) During copulation, sperm, as part of the ejaculate, are transferred to the uterus or bursa. Only one third of the ∼1,500 sperm transferred travel to the storage organs—a pair of spermatheca and a seminal receptacle (Miller and Pitnick, 2002; Manier et al., 2010). Sperm in storage can remain functional for long time periods, thus increasing the probability of sperm competition (Parker, 1970). The paired spermathecae are mushroom-shaped organs surrounded by spermathecal secretory cells (SSCs) while the seminal receptacle is a long tubular organ. Seminal receptacle and spermatheca function independently but there is communication between them (Schnakenberg et al., 2011). The two storage organs are differentially related to the sperm dynamics. The seminal receptacle is the first to get filled up by sperm, stores the highest sperm numbers, and is also the organ to first deplete sperm (Nonidez, 1920). The seminal receptacle is the most relevant organ in determining the fertilization set (Manier et al., 2010), i.e., the set of sperm from different males that actually engages in sperm competition to fertilize the eggs (Manier et al., 2010). Fertilization occurs when the sperm released from the storage organs penetrate the anterior pole of the eggs through a particular structure named the micropyle, which happens upon the eggs have been pushed through the oviduct into the uterus (Nonidez, 1920). Sperm recruitment by the storage organs is influenced by the proper functioning of the female central nervous system (Arthur et al., 1998). Additionally, sperm recruitment rate by storage organs, modification, stability, and usage in the female reproductive tract are influenced by a diverse compendium of molecules present in the ejaculate and others secreted by the female spermatheca and accessory glands, the latter also known as parovaria (Schnakenberg et al., 2011; Sun and Spradling, 2013; Sirot et al., 2014). Displaced resident sperm and the excess of subsequent male sperm are ejected along with the mating plug—a seminal component-coagulated structure that forms in the female reproductive tract upon mating (Manier et al., 2010). Drawing by J.L. Sitnik from (Wolfner, 2011). (B) Typical double-mating assay to test for significant variation in sperm competitive ability between different experimental males. Females of known genotype mate consecutively with two males (1st, reference male; 2nd, experimental male), which carry alleles associated with particular markers (eye color in this case). Paternity identity for each offspring can be tracked based on the markers used and the relative contribution of each father to the total progeny number summarized through a score –P–. In this way, different experimental males can be compared against a common reference male (e.g., knockouts against wildtype) to test if their corresponding P scores are statistically significantly different. The design shown, called offense, evaluates the ability of the sperm from the second male to outcompete first-male sperm from the female sperm storage organs, increasing the probability to fertilize the ova; P is denoted as P2. In a different version of this experimental design called defense, the experimental male is the first to mate while the reference male is second. In this case, the sperm ability to avoid being outcompeted by the sperm from the second male is evaluated and P denoted as P1. Alternatively, female contribution to sperm competition outcomes can be assessed by keeping both first and second male’s genotypes constant and varying the genotype of the females being tested. Further, this experimental design can be adapted to analyze sperm behavior in the female reproductive tract instead of differential paternity contribution. For this, both the experimental and reference males, or at least one of them, must carry transgenes for sperm monitoring purposes as they glow under the fluorescent microscope.