At the end of their life cycles, loliginid squids migrate to shallow coastal areas to mate and spawn. 1 Their distribution is affected by environmental factors such as salinity, turbidity and water temperature (Loligo forbesi, 2,3 L. vulgaris vulgaris 2 and L. vulgaris reynaudii 4), and during the spawning season, they avoid low temperature more than other periods of the life cycle. 2 The family Loliginidae generally occur in tropical and subtropical coastal areas worldwide, but

Male animals are not given equal mating opportunities under competitive circumstances. Small males often exhibit alternative mating behaviours and produce spermatozoa of higher quality to compensate for their lower chances of winning physical contests against larger competitors [1]. Because the reproductive benefits of these phenotypes depend on social status/agonistic ranks that can change during growth or aging [2], sperm traits should be developed/switched into fitness optima according to their prospects. However, reproductive success largely relies upon social contexts arising instantaneously from intra- and inter-sexual interactions, which deter males from developing extreme traits and instead favour behavioural plasticity. Nevertheless, the extent to which such plasticity influences developmentally regulated alternative sperm traits remains unexplored. Squids of the family Loliginidae are excellent models to investigate this, because they show sophisticated alternative reprodu...

Loliginid squids provide a unique model system to explore male alternative reproductive tactics (ARTs) and their linkage to size, behavioral decision making, and possibly age. Large individuals fight one another and the winners form temporary consortships with females, while smaller individuals do not engage in male-male agonistic bouts but use various sneaker tactics to obtain matings, each with varying mating and fertilization success. There is substantial behavioral flexibility in most species, as smaller males can facultatively switch to the alternative consort behaviors as the behavioral context changes. These forms of ARTs can involve different: mating posture; site of spermatophore deposition; fertilization success; and sperm traits. Most of the traits of male dimorphism (both anatomical and behavioral) are consistent with traditional sexual selection theory, while others have unique features that may have evolved in response to the fertilization environment faced by each temporary or permanent male morph.

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.

Background: Sperm 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. Results: Large 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. Conclusions: Here 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.

We have developed and characterized 13 novel polymorphic microsatellite markers for 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 Notoro Lagoon 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.

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.

Animal species usually have a single sperm-storage site in the female body, but females of the squid Heterololigo bleekeri possess two distinct sperm-storage sites simultaneously. Use of two sperm-storage sites correlates with alternative male mating behaviours: large consorts guard females and place spermatophores inside the oviduct just before spawning, whereas small 'sneaker' males place spermatophores on the membrane around the female's mouth within the arm crown, where a seminal receptacle is present. Previous work showed that spermatophore and sperm morphology diverge between consort and sneaker males. Here we show novel dichotomous adaptations in the sperm-transfer strategy of males of this squid, associated with the use of two distinct sperm-storage sites on females. The spermatangia ejaculated from spermatophores were clearly dimorphic: all spermatangia ejaculated from spermatophores smaller than 12 mm from small sneaker males were drop-shaped, whereas all spermatangia from spermatophores longer than 12 mm from larger consort males were rope-shaped. In addition, the dropshaped spermatangia were distinct in having a spine on their base, which might reduce the risk of it being shed from the female's body surface. Our findings suggest that existence of alternative spermstorage sites, and related biological and environmental factors, lead to the evolution of divergent spermtransfer strategies.

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 eithe...

Size variations in eggs and larvae and their relationship with female body size in Paciˆc herring Clupea pallasii were examined. Spawning adults were captured in Ishikari Bay, Lake Furen, Lake Akkeshi and Miyako Bay in 2013 and 2014. Compositions of body size and age of adults were diŠerent among spawning areas. Fish older than age 3 (larger than 25 cm in body length) dominated in Ishikari Bay and Miyako Bay, while age 2ˆsh (20 23 cm in body length) dominated in Lake Furen and Lake Akkeshi. Artiˆcially fertilized eggs and hatched larvae were obtained from adults caught from those four areas. Positive relationships were found between fork length of parental females and egg diameter, dry weight, larval notochord length, dry body weight, and yolk sac volume.

Squid are characterized by flexible life-history traits (LHTs) that change in response to changing oceanic parameters. Male alternative reproductive tactics (ARTs), characterized by large-sized ‘consorts’ versus small-sized ‘sneakers’, are commonly observed in loliginid species. This study reports on LHTs flexibility in male squids displaying ARTs. LHTs of consorts and sneakers in Uroteuthis edulis, including body size, age, growth rate and gonado-somatic energy allocation, were compared among seasonal and geographical groups from Japan and Taiwan. The ratio of consorts to sneakers was highest in the group spawning in the ‘Japan-warm’ season (June-November), followed by that of the ‘Japan-cold’ season (December-May), and lowest in Taiwan (spring and autumn). LHTs were compared among cohorts separated by hatching season and catch location (Jwarm, Jcold and Taiwan cohorts). Mean body size of consorts showed no difference among cohorts, although Taiwan consorts were relatively younger ...

Recent studies have highlighted the intriguing reproductive behaviors in some coastal squid species. Here we review briefly pre-copulative male-male and male-female interactions, alternative mating tactics and their associating sperm traits in squids. In one scenario, males compete with one another for mating opportunities by conventional means such as body pattern displays. After the male-male contests being settled, the winners are allowed to mate with the female more predominantly over others by transferring their spermatophores to the particular location of the female body. In another scenario, males pursue alternative reproductive tactics by which male individuals display either mate guarding or sneaking behavior, resulting in insemination dimorphism with different fertilization success, i.e., sperm transfer occurs near the oviduct or around the mouth adjacent to the seminal receptacle, in favor of leaching to eggs (immediate fertilization) or the seminal receptacle (storage), respectively. These characteristics of male mating behavior can lead to diverged evolution in sperm morphology, physiology, and behavior. Hence, the reproductive systems of squids would provide rare opportunities to analyze sperm evolution mechanisms.

As marine species that are harvested during spawning aggregation tend to be overfished, sufficient "reproductive escapement" is required to manage such overexploited populations. Because the reproductive value of short-lived organisms changes towards the end of their life span, information is required not only on the total amount harvested but also on the characteristics of the individuals caught. As spawning aggregations of the squid Loligo bleekeri are targeted by the commercial fishing industry, we analysed biological data of harvested squid to identify indicators of reproductive status. Females of this species had several sperm resources associated with alternative mating behaviours: in seminal receptacle near the mouth, around the seminal receptacle, and inside the opening of the oviduct, associated with alternative mating behaviours. However, only the mass of sperm on the oviduct was significantly related to oviduct weight (including ovulated eggs): oviduct weight was less when sperm mass was attached to the oviduct than when it was not, indicating that the female had paired with a consort male and had spawned before she was caught. Using this indicator to confirm a recent spawning event, we determined that only 22.9% of females spawned before they were harvested. Our results show that the commercial fishing industry mainly harvests squid that have not spawned and have high reproductive values. Further information on the movement and reproductive behaviour of squid is required to develop a management strategy for the coastal fishing industry that ensure sufficient reproductive escapement.