The mating behavior of captive Loligo bleekeri and the paternity of the resulting progeny were examined based on behavioral observations and genetic analyses. In this species, there are 3 mating behaviors (male-parallel, head-to-head, and extra-pair), and 2 sperm storage sites in females (seminal receptacle and the opening of the oviduct), which suggest that sperm competition occurs. All 3 mating behaviors were observed, and females mated often with different males, resulting in multiple paternity within 3 of the 4 broods examined. In each brood, the male to mate last and frequently before the female spawned fertilized the most eggs (87 to 100%). A sneaker male that mated by extra-pair copulation sired 8.5% of the eggs in a brood. Some eggs were fertilized by sperm received before the start of the study, indicating that sperm can be stored for at least several days before a spawning. In the broods with multiple paternity, the paternity patterns differed among egg capsules. Male competition was more intense between similar-sized males than between differentsized males, but body size did not affect the copulative success in the male-parallel position. We found multiple mating and multiple paternity in L. bleekeri. Paternity rates differed depending on the complex of factors, mating position, timing, frequency and duration. Alternative reproductive behaviors would change these factors and lead to different paternity rates.KEY WORDS: Multiple paternity · Reproductive strategy · Squid · Loligo · Microsatellite · Sperm competition Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 298: [219][220][221][222][223][224][225][226][227][228] 2005 just before she spawns . These consort males guard females before and after copulation and while the females spawn, but sometimes large intruders replace the consort males and copulate with the females . Furthermore, extra-pair copulations (EPCs) also occur, in which a small 'sneaker male' copulates with a female that is paired with another male by attaching spermatophores near the female's mouth (Hanlon 1996. The spermatozoa are stored until they are released at spawning, so loliginid females can use spermatozoa from several males when they spawn.When squids spawn, eggs are extruded from the oviduct and pass through the funnel to a position near the mouth. The eggs can be fertilized either as they leave the oviduct, by spermatozoa placed in the mantle cavity during a male-parallel mating, or near the mouth, by spermatozoa stored in the seminal receptacle after a head-to-head mating or EPC. This difference in the fertilization mechanism will presumably affect the reproductive success of each copulative behavior and play a role in sexual selection (Birkhead & Møller 1998).If the reproductive success of each copulative behavior differs, males will compete to mate in the favorable position. Agonistic behavior between males has been shown to escalate through several phases, from visual signaling to physical contact (DiMarco & Ha...

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 female's 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.

Male dimorphism has been thought to correlate with alternative reproductive behaviors. Alternative reproductive behaviors promote asymmetry in sperm competition, and the differences in fertilization success could promote adaptations in ejaculate characteristics in relation to each reproductive behavior. Using allometric analysis, we show that ejaculate dimorphism clearly exists in males of the squid Loligo bleekeri, a cephalopod species with body size-related alternative mating behaviors. A morphological switch point was detected for internal characters: larger individuals produced discontinuously longer spermatophores than did smaller individuals, although no switch point was detected for external characteristics (fin length, fin width, head width, mantle width, tentacle length and hectocotylus length) except for bimodal body size. This clear internal switch point could be an adaptation to the characteristic alternative mating behaviors of loliginid squid, in which males use different mating tactics to pass spermatophores to different sperm storage sites in and on the females. Our results indicated that alternative reproductive behaviors can result in morphological adjustment in internal characteristics. KEY WORDS: Male dimorphism · Sperm competition · Alternative reproductive behavior · Squid · Loligo bleekeri Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 345: [141][142][143][144][145][146] 2007 squid, however, have 2 distinct sperm storage sites (the seminal receptacle near the mouth and the opening of the oviduct within the mantle cavity), which corresponds to alternative mating behaviors (Hanlon & Messenger 1996). Loliginid squids form dense spawning aggregations on coastal spawning grounds, and males pair temporarily with females in order to mate (Hanlon & Messenger 1996). These spawning aggregations usually tend to have more males than females and the mating behaviors that males use correlate with their body size: large males pair with females and copulate in the male parallel position by attaching spermatophores to the female's oviduct in the mantle cavity, and small sneaker males mate in the head-to-head position by attaching spermatophores near the mouth of females that have already paired with other males (Hanlon et al. 1997(Hanlon et al. , 2002. Sneaker males fertilize fewer eggs than paired males do (Iwata et al. 2005). This suggests that sperm stored near the oviduct have an advantage, perhaps because they are stored closer to the eggs being spawned than are sperm stored in the seminal receptacle near the mouth. These size-dependent alternative mating behaviors and the differences in fertilization success could promote strategic ejaculation in males specializing on different sperm-passing sites. Furthermore, loliginid males pass sperm as spermatophores, enclosing the spermatozoa within a hard shell, and males store several hundred spermatophores when they mature. Therefore, the ejaculate characteristics associated with a male's status c...

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.

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 CO 2 -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.Sperm traits can evolve through postcopulatory contexts involving sperm competition, cryptic female choice, and insemination/fertilization environments. As consequences of sperm competition (1, 2) and cryptic female choice (3, 4), male individuals of certain species have evolved to produce giant spermatozoa to achieve better fertilization success. Besides morphological traits, the evolutionary forces driven by postcopulatory sexual selection should also favor more effective sperm motility and hence influence their morphological and energetic traits (5), thus facilitating fertilization in polyandrous mating systems. Sperm competition theory predicts that an increased risk of sperm competition (sometimes referred to as female promiscuity) should result in selection for increased sperm swimming velocity. A growing body of evidence has supported this prediction. Thus, a positive relationship between sperm swimming speed and sperm competitiveness was seen not only in external fertilizers such as fish (6 -8) but also in internal fertilizers such as birds (9, 10) and mammals (11). Commonly, polyandrous insemination can occur simultaneously (external fertilization) or sequentially (internal fertilization) toward the same set of eggs. However, in species that employ internal fertilization, female individuals often store spermatozoa in their reproductive tract and use them during multiple ovulation cycles (12-14). Thus, it is possible that spermatozoa with greater longevity might have greater reproductive fitness than those with increased velocity. This implicates that spermatozoa should be able to modulate their energy expenditure adequately depending on the postmating contexts.The coastal squid Heterololigo bleekeri exhibits alternative male mating tactics in which complex polyandrous inseminations occur at different places and...

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Chokka squid Loligo reynaudii have characteristic alternative mating tactics -large "consort" males form a temporal pair with a female and transfer spermatophores to within the female's mantle cavity. On the other hand, small "sneaker" males rush towards a pair and transfer spermatophores onto the female's buccal membrane. The differences in mating behaviour and the related sperm storage sites place clear constraints on the fertilization process and can drive divergent adaptation between consort and sneaker males. However, morphological adaptation associated with alternative mating tactics has not been examined in this species. We observed the morphology of spermatangium (sperm mass ejaculated from the spermatophore capsule) from each sperm storage site on the female body, and found dimorphism in spermatangium associated with the alternative sperm storage sites. We also observed spermtophores stored in the Needham's sac of mature males and confirmed that small males produced "sneaker-type" spermatangium.Therefore mating tactics adopted by individual males is distinguishable without a requirement for direct behavioural observation. The information helps for better understanding of the reproductive system and the population structure in this species.

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.