Microsatellite analysis of sperm-use patterns in the bushcricket Requena verticalis

The proportion of offspring sired by the second male to mate with a doubly mated female, P2, is a ubiquitously measured statistic in the study of insect sperm competition. Nevertheless, the underlying mechanisms of sperm transfer, storage, and use that determine P2 are poorly understood. Typically the second male to mate gains moderate to high paternity. More rarely, the first male to mate gains the majority of fertilizations. Here we examine the transfer, storage, and use of sperm in the bushcricket Requena verticalis, a species with male parental investment and almost complete first male paternity. Sperm drain from an externally attached spermatophore into the female's reproductive tract, where they are transported to the sperm store or spermatheca. We find that only sperm from the first male to mate are transported to the spermatheca. We provide some data that address a number of different mechanisms that might account for the lack of storage of second-male sperm. DNA microsatellite markers are developed to assign paternity. By manipulating the numbers of sperm transferred by first and second males, we show that the size of the ejaculate transferred by the first male has a major impact on paternity; increasing ejaculate size of the first male assures his paternity. Paternity assurance in R. verticalis holds significant implications for the evolution of paternal investment via the male's nuptial gift.