Genetic evidence for multiple mating and mixing of sperm from multiple males in the seminal receptacles of female Japanese flying squid (Todarodes pacificus)

The genetic diversity of sperm and spermatangia stored by mated Japanese flying squid (Todarodes pacificus) was examined to better understand how this ommastrephid squid mates and stores sperm. Analyses were conducted using spermatangia implanted in the lips of immature squid collected in Tsugaru Strait, northern Japan, in July–August 2019, spermatangia implanted in the lips of mature squid collected in the East China Sea in April 2019, and sperm stored in the seminal receptacles of squid collected off Tosashimizu, Kochi Prefecture, Japan, in February 2019. DNA was extracted, microsatellite loci were amplified using five PCR primers, and allele peaks were analysed. Analysis of the spermatangia confirmed that females in the field mate with multiple males, suggesting that males transfer from 1 to at least 14 spermatangia in each mating. Analysis of the seminal receptacles showed that sperm from multiple males are mixed within individual sperm chambers. Spermatangia were also found implanted in the lips of males, providing evidence of same-sex sexual behaviour. To date, studies of cephalopod mating systems have focused on coastal species, and the results of this study add to our growing knowledge of more oceanic species.