The assessment of insemination success in yellow dung flies using competitive PCR

Abstract

In spite of considerable interest in postcopulatory sexual selection, separating the effects of sperm competition from cryptic female choice remains difficult because mechanisms underlying postcopulatory processes are poorly understood. One methodological challenge is to quantify insemination success for individual males within the sperm stores of multiply mated females to discover how insemination translates into eventual paternity. Any proposed method must be applicable in organisms without extensive DNA sequence information (which include the majority of model species for sexual selection). Here, we describe the development and application of microsatellite competitive-multiplex-PCR for quantifying relative contributions to a small number of sperm in storage. We studied how DNA template characteristics affect PCR amplification of known concentrations of mixed DNA and generated regressions for correcting observations of allelic signal strength based on such characteristics. We used these methods to examine patterns of sperm storage in twice-mated female yellow dung flies, Scathophaga stercoraria. We confirm previous findings supporting sperm displacement and demonstrate that average paternity for the last mate accords with the mean proportion of sperm stored. We further find consistent skew in storage across spermathecae, with more last male sperm stored in the singlet spermatheca on one side of the body than in the doublet on the opposite side. We also show that the time between copulations may be important for effectively sorting sperm. Finally, we demonstrate that male size may influence the opportunity for sperm choice, suggesting future work to disentangle the roles of male competition and cryptic female choice.

Wild yellow dung fly females may not select sperm based on dung pat microclimate but could nevertheless benefit from polyandry

“…Theoretical work and laboratory studies suggest that females could bias paternity toward certain males by differentially storing sperm from different males in each spermatheca and subsequently choosing sperm (or a sperm mix) from a particular spermatheca (Hellriegel and Ward 1998;Hellriegel and Bernasconi 2000;Bussière et al 2010). The present study revealed that sperm mixtures differ in wild yellow dung flies, as we found a significantly lower number of ejaculates in the singlet spermatheca compared with the doublet spermathecae.…”

“…After taking samples out of the thermocycler we again shook them carefully and spun them down, stored the plate at 4°C for 10-20 h and froze it at -20°C for at least 24 h before DNA extractions were used for subsequent processing. DNA template amount (1 ll), total PCR reaction volume (6 ll), and cycling parameters (number of cycles: 27) for the heads were the same as in Bussière et al (2010).…”

“…We photographed and measured the hind tibiae of all flies under a stereo microscope with the software ImageJ 1.37v (Wayne Rasband, National Institutes of Health, USA, http://rsb.info.nih.gov/ij/). Extraction, amplification and analysis of DNA DNA extraction was performed from sperm pellets according to Bussière et al (2010): we used a kit designed for small amounts of DNA (QIAamp Ò DNA Micro Kit, Qiagen AG, Switzerland) to extract the potentially very low number of DNA copies from sperm pellets, and added carrier RNA to buffer AL (1 ll dissolved carrier RNA in 200 ll buffer AL). Note that carrier RNA does not dissolve in buffer AL; it must first be dissolved in buffer These cycling conditions did not produce large stutter peaks for six of the applied markers.…”

Sexual Selection: The Logical Imperative

“…Although (as expected) females showed a greater preference to oviposit on north slopes as ambient temperature increased, they found no evidence that females biased paternity towards certain male genotypes depending on the offspring's microclimate. Bussière et al (2010) used molecular techniques to demonstrate that although the mean proportion of sperm stored in the spermathecae match the published mean average paternity for the last male (the P 2 value), sperm from different males are not stored randomly across the female's sperm stores (see also Otronen et al 1997;Hellriegel and Bernasconi 2000). The mean number of ejaculates stored also differs across spermathecae (Demont et al 2011.…”

Large Neurological Component to Genetic Differences Underlying Biased Sperm Use in Drosophila

“…Any of these processes could act to bias sperm use. In some insects, the female preferentially places a certain male's sperm in an advantageous position within the sperm storage organs (Bloch Qazi 2003;Bussiere et al 2010). These dynamic sperm movements might be influenced, in some part, by male seminal proteins or the morphology of sperm themselves, but it is also likely that muscle contractions and active movements within the female tract are important factors that facilitate such movement.…”