Spermatozoa morphology changes during reproduction and first observation of acrosomal contact in two dioecious species of Macrobiotidae (Tardigrada: Eutardigrada).

Mating behaviours for two species of dioecious eutardigrades: a strain of Paramacrobiotus sp. and Macrobiotus shonaicus (Stec et al., 2018) have been recorded previously, and observations have indicated that spermatozoa of both species are first released into the environment, then swim through the cloaca of the females and into the spermatheca. The fusion of gamete nuclei has not yet occurred in a laid egg. Therefore, it has been suggested that fertilization is completed externally as the egg is released into the environment before the nuclei of the gametes fuse. In the present study, the spermatozoa of both Paramacrobiotus sp. and M. shonaicus spermatozoa underwent morphological changes during reproduction. In morphometrical analyses of testicular spermatozoa, the tail, mid-piece, nucleus, and acrosome were significantly longer in Paramacrobiotus sp. compared with M. shonaicus. The nuclei of both the testicular and spermathecal spermatozoa were equally coiled, but the latter had shorter tails in both species. These spermatozoa were present on the surface of the egg chorion after oviposition. The tip of the acrosomes lay buried in the chorion, suggesting that penetration had occurred. We also proposed that the reduced tail is a conserved trait, at least in Macrobiotidae.