Ritualized ink use during visual courtship display by males of the sexually dimorphic cuttlefish Sepia andreana.

Visual display is a crucial aspect of courtship, and their success relies on both display quality and the surrounding environment, such as the visual background. Cephalopods may release ink when attacked by predators or during aggressive interactions with conspecifics. Here, we report that ink is used as a part of the courtship display by males of the cuttlefish species Sepia andreana. Males of this species engage in a highly ritualized multimodal courtship using a pair of markedly long sexually dimorphic arms. At the climax of the courtship, the male releases a diffuse backdrop of ink near himself and then performs the specific courtship display by extending his sexually dimorphic arms and altering his body pattern to pale in front of this ink backdrop, and then proceeds to mate. This novel use of cephalopod ink could make the surroundings darker and more homogeneous, potentially serving as a temporary modification of the visual environment for courtship display.

Context-dependent behavioural plasticity compromises disruptive selection of sperm traits in squid.

Sperm morphology is generally uniform within a species due to selective pressures that act to achieve better fertilization outcomes under postcopulatory competitive circumstances. Therefore, polyandry that intensifies post-mating sperm competition should constrain intraspecific sperm polymorphism. Contrary to this paradigm, we previously found that a polyandrous squid, Heterololigo bleekeri, produces dimorphic eusperm (flagellum length dimorphism; FLD), which is closely associated with alternative reproductive tactics (ARTs); large males (consorts) transfer their spermatophores inside the female's mantle cavity, while small males (sneakers) do so outside the mantle. Thus, FLD was considered as the consequence of different insemination strategies that arise from different modes of sperm competition, sperm storage and the fertilization environment. However, in other squid species showing ARTs, the choice of mating behaviour is rather conditional (i.e., switching mating tactic between consorts and sneakers), which poses the question of whether sperm FLD could have evolved. Here, we investigated five species in the family Loliginidae that exhibit ARTs and found that all species showed sneaker-biased FLD. However, in a species with conditional ARTs, we found FLD rather ambiguous and the testicular somatic index to be nearly continuous among individuals at transitional state, suggesting that plasticity in mating behaviour compromises the disruptive selection on a sperm morphological trait.

Sperm competition risk affects ejaculate strategy in terms of sperm number but not sperm size in squid.

In polygamous species, the mode of sperm storage in females influences evolution of sperm quantitative and qualitative traits because it provides the arena for sperm competition, cryptic female choice and fertilization processes. In this study, we compared ejaculate traits of two squid species, Heterololigo bleekeri and Loligo reynaudii. Both species show dimorphic sperm traits associated with alternative reproductive tactics where consort and sneaker males transfer sperm to different storage sites within a female (on the oviduct and near the mouth, respectively). Due to differences in reproductive behaviours and sperm placement, sperm competition risk is expected to be higher in sneakers than in consorts of both species and higher overall in L. reynaudii. Our results demonstrate that the instantaneous number of released sperm is adjusted to the expected sperm competition risk via an elaborate sperm package. Consort sperm are similar in size; however, sneaker sperm have a significantly longer flagellum in H. bleekeri than in L. reynaudii, most likely due to intra-tactic conflicts associated with sperm storage conditions. From consideration of the different mating tactics, we suggest that while levels of sperm competition determine quantitative traits, sperm quality traits are determined more by the mode of sperm storage and fertilization.

Evidence of sperm removal behaviour in an externally fertilizing species and compensatory behaviour for the risk of self-sperm removal.

The removal of rival sperm from a female's sperm storage organ acts as a strong sperm competition avoidance mechanism, which has been reported only in internally fertilizing species and not at all in externally fertilizing species. This study demonstrated for the first time that nest-holding males of Bathygobius fuscus, an externally fertilizing marine fish, remove the sperm of rival sneaker males from the spawning nest by exhibiting tail-fanning behaviour within the nest. Males showed tail-fanning behaviour when semen was artificially injected into the nest but not when seawater was injected, and in open nests this behaviour resulted in higher paternity rates for the focal male. The sperm removal behaviour entails the risk of removing their own sperm; therefore, additional sperm release behaviour is likely necessary to benefit from the sperm removal effect. Consistent with this, males increased post-fanning sperm release behaviour more in the semen than in the seawater injection treatment. Moreover, males who had removed sperm for a longer time spent more time releasing sperm after the removal, suggesting that the additional sperm release behaviour compensated for the loss of their own sperm. These results suggest that sperm removal behaviour is not restricted to internally fertilizing organisms and deserves further investigation in this and other species.

Rare polyandry and common monogamy in the firefly squid, Watasenia scintillans.

In cephalopods, all species are considered to be polyandrous because of their common life history and reproductive traits reflecting a polyandrous mating system. Contrary to this belief, here we show several lines of evidence for monogamy in the firefly squid, Watasenia scintillans. In this species, females are capable of long-term storage of spermatangia, and of egg spawning even after the complete disappearance of males following the breeding season. The stored spermatangia are distributed equally between bilateral pouches under the female's neck collar. Such a nonrandom pattern of sperm storage prompted us to hypothesize that females might engage in lifetime monandry. Hence, we genotyped female-stored spermatangia and offspring, and found that in 95% of females (18/19), all the spermatangia had been delivered from a single male and all the embryos in a clutch had been sired by spermatozoa from stored spermatangia. In males, throughout the reproductive season, relative testis mass was much smaller in W. scintillans than in all other cephalopods examined previously. The mean number of male-stored spermatophores was ~ 30, equivalent to only 2.5 matings. Our genetic, demographic and morphometrical data agree with a mathematical model predicting that monogyny is favored when potential mates are scarce. Together, these results suggest mutual monogamy in W. scintillans.

Male Alternative Reproductive Tactics and Associated Evolution of Anatomical Characteristics in Loliginid Squid.

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.

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.

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more, but females influence insemination success by removing spermatangia.

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.

Spermatangium formation and sperm discharge in the Japanese pygmy squid Idiosepius paradoxus.

In cephalopods, sperm discharge is an important event not only for sperm transfer but also influencing sperm storage capacity of attached spermatangia (everted spermatophores). To investigate sperm discharge from spermatangia and the condition of naturally attached spermatangia in Japanese pygmy squid (Idiosepius paradoxus) we (i) investigated the morphology of spermatophores and spermatangia, and the process of spermatophore evagination and sperm discharge from spermatangia obtained in vitro; (ii) observed spermatangia that were naturally attached to female squids at 6, 12, 18, 24 and 48 h after copulation to investigate alterations in naturally attached spermatangia with time. The spermatophore of I. paradoxus is slender and cylindrical and consists of a sperm mass, a cement body and an ejaculatory apparatus, which is similar to those of loliginid squids. The spermatangium is fishhook-shaped, its distal end being open and narrow. After the spermatangium is formed, the sperm mass gradually moves to the open end of the spermatangium, from where sperm are released. Sperm discharge is a rapid process immediately after the beginning of sperm release, but within 5 min changes to an intermittent release of sperm. Although the volume of residual spermatozoa differed among spermatangia that were naturally attached to a single individual, the probability that spermatangia would be empty increased with time. Most naturally attached spermatangia discharged almost all of their spermatozoa within 24h after copulation, and no spermatangia were attached to females 48 h after copulation. These results suggest that sperm transfer from the spermatangium to the seminal receptacle must occur within 24h, and that the spermatangium functions as a transient sperm storage organ in pygmy squids.

Evaluation of the effectiveness of light streamer tori-lines and characteristics of bait attacks by seabirds in the western North Pacific.

To improve the effectiveness of tori-lines it is necessary to evaluate the ability of tori-lines to mitigate seabird bycatch and determine what kind of seabird species gather during line settings, attack the bait and are incidentally caught. We conducted two experiments in the western North Pacific and examined the effectiveness for seabird mitigation of light streamer tori-lines which have no long streamers but many light (short) streamers and are mainly used in the North Pacific area. Firstly, the effectiveness of two different types of tori-line (light streamer (1 m) and long streamer (up to 7 m) tori-line) and of two different colors (yellow and red) of light streamers for seabird bycatch avoidance was evaluated using 567 sets based on data from 20 offshore surface commercial longliners. No significant difference in the bycatch number between the different tori-line types and streamer colors was found. Secondly, we investigated the characteristics of the seabird bycatch in the North Pacific and the effectiveness of three different types of streamers (light, hybrid and modified light types) by detailed observations of seabird attacks using a chartered longline vessel. Although the appearance rate of albatrosses and shearwaters were 40.9% and 27.7%, Laysan albatross was the main seabird species that followed the vessel but shearwaters seldom followed the vessel and did not aggregate during line setting. In all attacks on bait observed during line settings, 81% and 7% were by albatrosses and shearwaters, respectively. In the number of primary attacks by Laysan albatrosses which attacked most aggressively of all seabirds, there were no significant differences among the tori-line types. No individuals of shearwater were caught. The results of both experiments indicated that light streamer tori-lines were as effective as tori-lines with long streamers for mitigating seabird bycatch in the North Pacific.