Reproductive strategy is an important aspect of biological diversity. In tardigrades, several reproductive modes, including sexual reproduction, are known. However, tardigrade mating behavior has been observed only rarely in most species, and in some cases, especially in the freely ovipositing eutardigrades, remains entirely unknown. In the present study, we cultured two sexually reproducing tardigrade species that lay eggs freely, Paramacrobiotus sp. TYO strain and Macrobiotus shonaicus, to investigate and compare their courtship, mating, and chromosome morphology. Mating behavior was observed and recorded in both species. The entire mating sequence, including courtship, was categorized into five discrete steps common to two species, as follows:  Tracking: the male tracks and orientates toward the female;  Touching: the male makes contact with the cloaca of the female;  Standstill: the female ceases movement until male ejaculation is complete;  Ejaculation: the male curls its caudal end and ejaculates into the cloaca from close range;  Contraction: the female contracts its ventral side after ejaculation to capture spermatozoa deposited in the external environment in close proximity to the cloaca. Some notable differences between the two species were observed in the steps 3-4. First, oviposition was observed at 40 min in Paramacrobiotus sp. TYO strain, and a few days after mating in M. shonaicus, respectively. Comparisons of chromosome morphology before and after mating indicated that oocytes are arrested at metaphase I in both species. Spermatozoa attach to the interior of the chorion of laid eggs.
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