Thiotepa, a reliable marker for sperm precedence measurement in a polyandrous moth

The determination of paternity of offspring produced by polyandrous females is essential for the understanding of sperm competition mechanisms. The sterile male technique using radiation is one of the most commonly employed methods for this purpose. However, sterilization using radiation is likely to be restricted by the equipment availability and cost. Chemosterilization may thus be a cheaper and easier alternative for sterilizing male insects in sperm competition studies. Here we report a reliable chemomarker, thiotepa (N,N′,N″-triethylenethiophosphoramide), for the study of sperm competition and precedence in a polyandrous moth, Ephestia kuehniella (Lepidoptera: Pyralidae). Dipping heads of male moths in 1% thiotepa aqueous solution for 10 s resulted in complete sterilization, i.e. their sperm still fertilized eggs but those eggs did not hatch. The sterilization treatment did not significantly affect male copulation ability, female fecundity, and sperm transfer, motility and fertilization. Our results indicate that 86% offspring of the twice-copulated females were fathered by the second males and 14% by the first males. Males treated with 5% thiotepa aqueous solution died within 24 h while those treated with 0.5% thiotepa were not fully sterilized.