How female squid inseminate their eggs with stored sperm

How sperm reach ova after mating is one of the central questions in reproductive biology. Many species copulate and store sperm in female reproductive organs until spawning [1]. The way females use stored sperm is closely associated with sperm competition and cryptic female choice. However, it is difficult to observe the process of fertilization in natural spawning, as fertilization usually occurs in some 'hidden place' within the female's body. Here, we report the fertilization process of a squid using a glass plate as a spawning substratum, enabling observation within the female arm crown where the sperm storage organ is located and where fertilization may occur. Additionally, we detail the distribution of sperm around newly spawned eggs. Our observations reveal that: the female places her sperm-storage organ (seminal receptacle) over an egg held within her arm crown and inseminates the eggs one-by-one during attachment to the spawning substratum; sperm pass through a pathway within the jelly layers surrounding an egg; and such direct insemination behavior and the pathway through the egg jelly enables a female squid to externally fertilize her eggs using relatively few sperm. This study is the first to reveal the fertilization process using stored sperm, under female control.