Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2

Abstract

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the female's body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

Sneaker Male Squid Produce Long-lived Spermatozoa by Modulating Their Energy Metabolism

“…Because they are inseminated externally and yet moved to the seminal receptacle, self-clustering could be associated with postcopulatory event(s) specific to sneaker spermatozoa, such as sustained longevity, migration to the seminal receptacle, or switching their respiratory mode. This study, together with previous reports (19,20), suggests that there has been coordinated evolution of complex adaptive traits constrained by postcopulatory reproductive contexts. Future studies should address the molecular and cellular mechanisms of how squid spermatozoa are capable of storing glycogen at different levels.…”

“…Consort Spermatozoa-We demonstrated previously that, in H. bleekeri, both sneaker and consort male individuals produce euspermatozoa that are distinguishable from each other in morphological (flagellar length) and behavioral (self-swarming) properties (19,20). Although they are both fertilization-competent, we noted that sneaker spermatozoa exhibited greater fertilization success than consort spermatozoa when inseminated in vitro (19).…”

“…Our previous observations revealed that, even after ejaculation, sneaker spermatozoa do not freely diffuse into seawater; instead, they preferentially stay in the vicinity of the ejaculatory duct of the spermatangium by forming a cluster (25). This phenomenon is absent in consort spermatozoa and can be explained by sneaker-specific sperm chemotaxis in response to self-emitted CO 2 (20). The physiological role of this clustering trait in sneaker spermatozoa remains to be determined; however, a sharp contrast in swimming behavior between sneaker and consort spermatozoa led us to examine the relationship between sperm concentration and sperm viability.…”

“…The chemoreceptor for CO 2 is a membrane-anchored carbonic anhydrase (CA) that catalyzes the following reaction: CO 2 ϩ HO % HCO 3 Ϫ ϩ H ϩ . Protons produced through this reaction are the actual chemoattractants, so a proper proton gradient, created naturally or experimentally in the microenvironment, can lead to the formation of sperm clusters (20). Considering the facilitated lactate efflux from sneaker spermatozoa and rapid conversion into protons, we hypothesized that lactic acidosis might enhance sperm clustering.…”

“…Sperm Clusters Are Stabilized in a Nutrient-rich Environment-Previously, we found that spermatozoa from sneaker males exhibit self-clustering behavior caused by chemotaxis to respiratory CO 2 (20,25). The chemoreceptor for CO 2 is a membrane-anchored carbonic anhydrase (CA) that catalyzes the following reaction: CO 2 ϩ HO % HCO 3 Ϫ ϩ H ϩ .…”