Yoko Iwata

Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

BackgroundSperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri.ResultsLarge consort males transfer smaller (average total length = 73 μm) sperm to a females internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males.ConclusionsHere we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2

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 females 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.

The different types of sperm morphology and behavior within a single species: Why do sperm of squid sneaker males form a cluster?

Some coastal squids exhibit male dimorphism (large and small body size) that is linked to mating behaviors. Large “consort” males compete with other, rival males to copulate with a female, and thereby transfer their spermatophores to her internal site around the oviduct. Small “sneaker” males rush to a single female or copulating pair and transfer spermatophores to her external body surface around the seminal receptacle near the mouth. We previously found that in Loligo bleekeri, sneaker sperm are ~50% longer than consort sperm, and only the sneaker sperm, once ejaculated from the spermatophore (sperm mass), form a cluster because of chemoattraction toward their own respiratory CO2. Here, we report that sperm clusters are able to move en masse. Because a fraction of ejaculated sperm from a sneaker’s spermatophore are eventually located in the female’s seminal receptacle, we hypothesize that sperm clustering facilitates collective migration to the seminal receptacle or an egg micropyle. Sperm clustering is regarded as a cooperative behavior that may have evolved by sperm competition and/or physical and physiological constraints imposed by male mating tactics.

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

Spermatozoa released by males should remain viable until fertilization. Hence, sperm longevity is governed by intrinsic and environmental factors in accordance with the male mating strategy. However, whether intraspecific variation of insemination modes can impact sperm longevity remains to be elucidated. In the squid Heterololigo bleekeri, male dimorphism (consort and sneaker) is linked to two discontinuous insemination modes that differ in place and time. Notably, only sneaker male spermatozoa inseminated long before egg spawning can be stored in the seminal receptacle. We found that sneaker spermatozoa exhibited greater persistence in fertilization competence and flagellar motility than consort ones because of a larger amount of flagellar glycogen. Sneaker spermatozoa also showed higher capacities in glucose uptake and lactate efflux. Lactic acidosis was considered to stabilize CO2-triggered self-clustering of sneaker spermatozoa, thus establishing hypoxia-induced metabolic changes and sperm survival. These results, together with comparative omics analyses, suggest that postcopulatory reproductive contexts define sperm longevity by modulating the inherent energy levels and metabolic pathways.

Complex adaptive traits between mating behaviour and post-copulatory sperm behaviour in squids

Emergence of male dimorphism within a species is the evolutionary process of disruptive selection. In squids, two types of male mating behaviour, known as alternative reproductive tactics (ARTs), are causally associated with adult body size. Males inseminate promiscuously with the same females; large “consort” males internally, and small “sneaker” males externally. Previously we found that in Heterololigo bleekeri, sneaker (but not consort) spermatozoa are able to swarm by sensing self-emitted CO2. This suggests that a swarming trait might have arisen in sneakers as a “sperm cooperation” strategy among sibling sperm in order to compete with consort males, or as a consequence of adaptation to external fertilization. To address these possibilities, we examined six species where three patterns of insemination are present, namely, only internal, only external, or both ARTs. In three species that employ both ARTs (H. bleekeri, Loligo reynaudii and Uroteuthis edulis), sneaker spermatozoa always exhibited self-swarming capacity. In Idiosepius paradoxus and Todarodes pacificus, which use only external insemination, spermatozoa formed a swarm. However, in Euprymna morsei, which use only internal insemination, sperm were unable to swarm. These results suggest that the self-swarming trait is likely to be linked to the mode of insemination rather than the alternative strategy used by sneaker males. Thus we propose a new hypothesis in which cooperative sperm behaviour has evolved not only through kin selection against sperm competition risks, but also through adaptation to the insemination/fertilization environment.

A coordinated sequence of distinct flagellar waveforms enables a sharp flagellar turn mediated by squid sperm pH-taxis

Animal spermatozoa navigate by sensing ambient chemicals to reach the site of fertilization. Generally, such chemicals derive from the female reproductive organs or cells. Exceptionally, squid spermatozoa mutually release and perceive carbon dioxide to form clusters after ejaculation. We previously identified the pH-taxis by which each spermatozoon can execute a sharp turn, but how flagellar dynamics enable this movement remains unknown. Here, we show that initiation of the turn motion requires a swim down a steep proton gradient (a theoretical estimation of ≥0.025 pH/s), crossing a threshold pH value of ~5.5. Time-resolved kinematic analysis revealed that the turn sequence results from the rhythmic exercise of two flagellar motions: a stereotypical flagellar ‘bent-cane’ shape followed by asymmetric wave propagation, which enables a sharp turn in the realm of low Reynolds numbers. This turning episode is terminated by an ‘overshoot’ trajectory that differs from either straight-line motility or turning. As with bidirectional pH-taxes in some bacteria, squid spermatozoa also showed repulsion from strong acid conditions with similar flagellar kinematics as in positive pH-taxis. These findings indicate that squid spermatozoa might have a unique reorientation mechanism, which could be dissimilar to that of classical egg-guided sperm chemotaxis in other marine invertebrates.

Isolation and characterization of 13 polymorphic microsatellites for the Hokkai Shrimp, Pandalus latirostris

We have developed and characterized 13 novel polymorphic microsatellite markers for the Hokkai Shrimp, Pandalus latirostris, to provide an effective tool for conducting genetic studies on this species. No linkage disequilibria and no deviation from HWE were detected in these markers. In 32 individuals from Lake Notoro in Hokkaido, Japan, the number of alleles and expected heterozygosities ranged from 7 to 22 and from 0.84 to 0.95, respectively, suggesting the availability of these markers for ecological studies and conservation genetics in this species.

Postcopulatory Reproductive Strategies in Spermatozoa

To reproduce sexually, males and females produce very different gametes (sperm and eggs) in many animals. This difference gives rise to very different strategies in the two sexes and in gamete cells from the two sexes. Sperm meet eggs in harmony; however, the male and female do not always have common interests in reproduction. The battle of the sexes continues even after copulation. Female promiscuity is key to the understanding of reproductive behaviors not only in male individuals but also in sperm cells, because sexual selection continues after mating through sperm competition. Here, we highlight multiple sperm traits—the sperm acrosome reaction in sea urchins, sperm storage in birds, and sperm dimorphism in squid—that are tightly associated with postcopulatory reproductive strategies.

Spermatophore dimorphism in the chokka squid Loligo reynaudii associated with alternative mating tactics

Chokka squid (Loligo reynaudii) have characteristic alternative mating tactics: ‘consort’ males temporarily pair with and guard a female and transfer spermatophores onto her oviduct opening inside the mantle cavity, whereas ‘sneaker’ males rush towards a mating pair and transfer spermatophores onto the female’s buccal membrane near her sperm storage organ. Differences in mating behaviours and their related sperm-storage sites clearly constrain the fertilization process and can drive dimorphism between consort and sneaker males. The presence and character of male dimorphism has not yet been fully examined in this species, but consort males are commonly much larger than sneaker males. We observed clear dimorphism in spermatangia (the sperm mass ejaculated from the spermatophore), consistently associated with the two alternative sperm storage sites on the female’s body. Observations of spermatophores stored in the Needham’s sac of mature males confirmed that small males produce ‘sneaker-type’ spermatangia whereas larger males produce ‘consort-type’ spermatangia, and no individuals possessed both types. Therefore, by association, the mating tactic adopted (including the sperm deposition site used) by individual males can be determined from observation of their spermatangial type, without requiring direct behavioural observation of mating. This ability to infer information about mating tactic will improve our understanding of the reproductive system and mating dynamics in this species.

Respiratory CO2 Mediates Sperm Chemotaxis in Squids

The squid Loligo (Heterololigo) bleekeri uses two distinct insemination sites, inside or outside the female’s body, which links to the mating behavior of two distinct types of males, consort or sneaker, respectively. We found that sperm release a self-attracting molecule, which causes only sneaker sperm to swarm. We identified respiratory CO2 as the sperm chemoattractant and its sensor, membrane-bound flagellar carbonic anhydrase. Downstream signaling results from generation of an extracellular proton gradient, intracellular acidosis, and concomitant recovery from acidosis. This cycle in turn elicits Ca2+-dependent flagellar turning/tumbling, resulting in chemotactic swarming.