Sperm competition between males with different reproductive tactics can be a strong selective force that shapes the evolution of tactic-specific reproductive traits. In fishes, sneak fertilization of the female's eggs is the most widespread male alternative reproductive tactic and sneaker males generally exhibit enlarged testes as a tactic-specific trait due to the higher risk of sperm competition they face. In addition, traits of the sperm themselves, such as size, density, velocity and longevity, vary with reproductive tactic in some species. Here, we investigated differences in the sperm traits between males of dusky frillgoby (Bathygobius fuscus) adopting nest-holding tactics and sneaking tactics. In this species, sneak nest intrusion lasts only a few seconds to a minute, whereas females lay eggs over several hours. A unique feature of this species is that both types of males deposit sperm-containing mucus on the nests, from which the sperm are gradually released into the water. We found that the sperm of sneaker males are present at a higher concentration in the testes, are longer lived and decrease in velocity more gradually than the sperm of nest-holding males, despite no differences in sperm size being detected. These tactic-specific sperm traits of sneaker males appear to contribute enhancing their fertilization success under the limited opportunity for sneaking in this species.

The removal of rival sperm from a female's sperm storage organ acts as a strong sperm competition avoidance mechanism, which has been reported only in internally fertilizing species and not at all in externally fertilizing species. This study demonstrated for the first time that nest-holding males of Bathygobius fuscus , an externally fertilizing marine fish, remove the sperm of rival sneaker males from the spawning nest by exhibiting tail-fanning behaviour within the nest. Males showed tail-fanning behaviour when semen was artificially injected into the nest but not when seawater was injected, and in open nests this behaviour resulted in higher paternity rates for the focal male. The sperm removal behaviour entails the risk of removing their own sperm; therefore, additional sperm release behaviour is likely necessary to benefit from the sperm removal effect. Consistent with this, males increased post-fanning sperm release behaviour more in the semen than in the seawater injection treatment. Moreover, males who had removed sperm for a longer time spent more time releasing sperm after the removal, suggesting that the additional sperm release behaviour compensated for the loss of their own sperm. These results suggest that sperm removal behaviour is not restricted to internally fertilizing organisms and deserves further investigation in this and other species.