Squid male alternative reproductive tactics are determined by birth date.

Alternative reproductive tactics (ARTs) are discontinuous phenotypes associated with reproduction, observed in males of many species. Typically, large males adopt a tactic of competing with rivals for mating, while small males adopt a tactic of stealing fertilization opportunities from the large males. The 'birth date hypothesis', proposing that the date of birth influences the determination of each male's reproductive tactic, has been tested only in teleost fish to date. Here, the birth date hypothesis was tested in ARTs of Japanese spear squid Heterololigo bleekeri (consort/sneaker) by analysing statolith growth increments. The birth date significantly differed between consorts (early-hatched) and sneakers (late-hatched). However, no differences were detected in growth history up to 100 days from hatching. Most immature males caught during the reproductive season were larger than sneakers, and their hatch date was similar to that of consorts, suggesting that these immature males had already been following a life-history pathway as a consort. These results indicate that ARTs of H. bleekeri are determined based on their hatch date in early life. This study firstly suggests that the birth date hypothesis applies to aquatic invertebrates, suggesting that the mechanism by which birth date determines the individual phenotype is a phenomenon more common than previously believed.

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.

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.

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.

The involvement of spinal release of histamine on nociceptive behaviors induced by intrathecally administered spermine.

The involvement of spinal release of histamine on nociceptive behaviors induced by spermine was examined in mice. Intrathecal spermine produced dose-dependent nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by spermine at 0.02 amol and 10 pmol were markedly suppressed by i.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deficient mice. In histamine H1 receptor-deficient mice, the nociceptive behaviors induced by spermine were completely abolished after treatment with 0.02 amol of spermine and significantly suppressed after treatment with 10 pmol of spermine. The i.t. pretreatment with takykinin NK1 receptor antagonists eliminated the nociceptive behaviors induced by 0.02 amol of spermine, but did not affect the nociceptive behaviors induced by 10 pmol of spermine. On the other hand, the nociceptive behaviors induced by spermine at both 0.02 amol and 10 pmol were suppressed by i.t. pretreatment with antagonists for the NMDA receptor polyamine-binding site. The present results suggest that the nociceptive behaviors induced by i.t. administration of spermine are mediated through the spinal release of histamine and are elicited via activation of NMDA receptors.

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.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

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 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.

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.

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

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.

Partial involvement of NMDA receptors and glial cells in the nociceptive behaviors induced by intrathecally administered histamine.

The involvement of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine was determined in mice. Histamine at 800 pmol injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with tachykinin NK(1) receptor antagonist CP99,994 or competitive antagonist for N-methyl-d-aspartate (NMDA) receptor d-(-)-2-amino-5-phosphonovaleric acid (d-APV). The i.t. pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline failed to affect the nociceptive behaviors induced by histamine. However, in mice pretreated i.t. with dl-fluorocitric acid or minocycline, the nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with CP99,994 but not d-APV. In Western blot analysis using lumbar spinal cords, i.t. treatment with 800 pmol of histamine increased the phosphorylation of the NR1 subunit of NMDA receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with dl-fluorocitric acid or minocycline. The present results suggest that histamine at 800 pmol elicits nociceptive behaviors through activation of the neuronal NK(1) receptor and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord.

Involvement of glial cells in the nociceptive behaviors induced by a high-dose of histamine administered intrathecally.

The involvement of spinal glial cells in the nociceptive behaviors induced by 1600 pmol of histamine was determined in mice. Histamine injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. pretreatment with the glial cell inhibitor DL-fluorocitric acid or minocycline. In Western blot analysis using lumber spinal cords, i.t. treatment with histamine increased the phosphorylation of the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with DL-fluorocitric acid or minocycline. We have previously reported that the nociceptive behaviors induced by 1600 pmol of histamine were significantly suppressed by the i.t. co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine (MK-801), an ion channel blocker of NMDA receptors, or agmatine, an antagonist for the polyamine recognition site on the NR1 subunit of NMDA receptors. In the present study, the increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was also abolished by i.t. co-administration of agmatine or MK-801. The present results suggest that histamine at 1600 pmol elicits nociceptive behaviors by stimulating the polyamine recognition site on the NR1 subunit of NMDA receptors on spinal glial cells.

Telling children and adolescents about their cancer diagnosis: Cross-cultural comparisons between pediatric oncologists in the US and Japan.

PURPOSE: Over the last 50 years, direct communication about cancer with adults has shifted from an approach of not telling to one of telling. Less is known about communication practices with children. The purpose of this study is to (1) describe patterns of communication at diagnosis between pediatric oncologists and children with cancer and (2) compare cultural differences in these practices in the US and Japan. METHODS: This 2003 survey, developed in English and translated into Japanese was mailed to members of the American Society of Pediatric Hematology/Oncology and the two Japanese Societies of Pediatric Hematology and Oncology; there were 350 US and 362 Japanese respondents. Descriptive statistics and logistic regressions were performed. RESULTS: US physicians had a consistent pattern of telling children (65% always told the child; less than 1% rarely or never told). Japanese physicians had greater variability in their patterns of telling (with only 9.5% always telling, 34.5% rarely or never telling). Direct communication with the child was influenced by personal attitudes, patient factors, and work culture in both countries. Many more variables emerged as influencing Japanese physicians' communication practices than for US physicians. US physicians were influenced by their own sense of responsibility for telling, while Japanese physicians were more influenced by personal attitudes, patient factors, and work culture. CONCLUSIONS: US and Japanese physicians differed when communicating directly with the child about his or her cancer. The impact of these practices on children and their parents should be explored and the parent and child's perspectives elicited. This information will help facilitate culturally sensitive patient and family centered communication.