Squid Loligo Bleekeri Research Articles

Structural and Phylogenetic Profiles of Muscle Actins from Cephalopods

Structural properties of cephalopod muscle actins have not been characterized in detail to date. Sequences of these actins have been determined by cDNA cloning from the arm muscles of four cephalopod species, namely, Japanese common squid Todarodes pacificus, spear squid Loligo bleekeri, common cuttlefish Sepia esculenta, and common octopus Octopus vulgaris. The sequence identities of the deduced amino acid sequences among the cephalopod actins were around 98%, and the substitutions were mostly limited to Subdomains 1 and 3. Phylogenetic analyses revealed that cephalopod actins formed one cluster and were distinguishable from the bivalve counterparts. The modeled tertiary structure of squid actin was very similar to that of rabbit counterpart, but the structures of Subdomains 1 and 3 were slightly different.

Estimating the Strength of Single Chitin Nanofibrils via Sonication-Induced Fragmentation.

We report the mechanical strength of native chitin nanofibrils. Highly crystalline α-chitin nanofibrils were purified from filaments produced by a microalgae Phaeocystis globosa, and two types of β-chitin nanofibrils were purified from pens of a squid Loligo bleekeri and tubes of a tubeworm Lamellibrachia satsuma, with relatively low and high crystallinity, respectively. These chitin nanofibrils were fully dispersed in water. The strength of individualized nanofibrils was estimated using cavitation-induced tensile fracture of nanoscale filaments in a liquid medium. Both types of β-chitin nanofibrils exhibited similar strength values of approximately 3 GP; in contrast, the α-chitin nanofibrils exhibited a much lower strength value of 1.6 GPa. These strength estimates suggest that the tensile strength of chitin nanofibrils is governed by the molecular packing modes of chitin rather than their crystallinity.

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.

Synchrony in the abundance trend of spear squid Loligo bleekeri in the Japan Sea and the Pacific Ocean with special reference to the latitudinal differences in response to the climate regime shift

Abstract Tian, Y., Nashida, K., and Sakaji, H. 2013. Synchrony in the abundance trend of spear squid Loligo bleekeri in the Japan Sea and the Pacific Ocean with special reference to the latitudinal differences in response to the climate regime shift. – ICES Journal of Marine Science, 70: 968–979. Spear squid Loligo bleekeri is widely distributed in the Japanese coastal waters. The fisheries depend largely on four stocks: the southern and northern stocks both in the Japan Sea and the coastal regions of the Pacific. The catch per unit effort (cpue) for the northern stock in the Japan Sea decreased substantially during the 1980s but increased during the 1990s, while the abundance index for the southern stock showed the opposite trend. The cpue for the southern and northern stocks in the Pacific coast showed a similar pattern to that in the Japan Sea. The synchrony in the abundance trends between the Japan Sea and the Pacific Ocean, and latitudinal differences between the northern and southern stocks indicate the impact of the climate regime shift. Generalized additive model analysis identified significant effects of environmental factors. Increased water temperature had a positive effect on the northern stock but a negative effect on the southern stock in the Japan Sea and the Pacific, whereas El Niño–southern oscillation events and the Asian monsoon had additional significant effects on the Pacific stocks. These results suggest that the abundance trends of spear squid were largely forced by environmental factors with latitudinal differences in the response to the climate regime shift.

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.

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 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.

Identification of enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri by expressed sequence tag analysis

Expressed sequence tag (EST) analyses were performed with the aim of identifying enzyme genes in the liver of the Bleeker’s squid Loligo bleekeri. Of the 768 ESTs identified and sequenced, 669 were grouped into 324 clusters. Of these clusters, 123 comprising 245 ESTs were found to be homologous to genes reported to date. Among these, 43 clusters were annotated as enzymes according to the Enzyme Commission (EC) numbering system. Two EC groups, oxidoreductases and hydrolases, possessed a large number of ESTs. A cluster homologous to the glutathione peroxidase, an enzyme in the oxidoreductase group, contained 16 ESTs, which accounted for 2.4% of the total ESTs sequenced. There are three serine proteases, three cathepsins, two triacylgricerol lipases, and two chitinases among the clusters homologous to the enzymes in the hydrolase group. Since the squid liver functions in the digestive process, these enzymes would be involved in food digestion. Our data provide information on the various types of enzymes expressed in the squid liver and may provide a useful basis for further characterization of these enzymes.

Interannual–interdecadal variations of spear squid Loligo bleekeri abundance in the southwestern Japan Sea during 1975–2006: Impact of the trawl fishing and recommendations for management under the different climate regimes

A climatic regime shift, characterized by an abrupt change from cool to warm conditions, occurred in the Tsushima Warm Current (TWC) region in the Japan Sea in the late 1980s. The abundance of spear squid Loligo bleekeri in the southwestern part of the Japan Sea responded strongly to the changing thermal regime. Spear squid are widely distributed in Japanese coastal waters and form one of the most important target species of pair trawlers in the southwestern Japan Sea. Catch from the southwestern Japan Sea has fluctuated from a minimum of 16 tons in 2003 to a maximum of 13,700 tons in 1977. Catch has decreased to less than 100 tons in recent years, and consequently the stock is at the point of collapse. The abundance index of spear squid shows a decadal pattern of variation with a step change from positive to negative annual anomalies that occurred around 1990; this pattern corresponds closely with the changing regime in water temperature in the TWC, strongly indicating that the decadal variability in spear squid was largely affected by the climatic regime shift in the late 1980s. Significant negative correlations between water temperature and an abundance index of spear squid ( R 2 = 0.39, p < 0.01) indicate that increasing ambient water temperature reduced spear squid abundances during the 1990s. Monthly CPUE (catch per unit effort) decreases sharply during the fishing season from autumn through winter, a patterns that was well described by a DeLury model. DeLury estimates of apparent mortality showed that the fishing mortality coefficient was relatively stable during 1975–1987, but has increased greatly since the late 1980s. The average fishing mortality coefficient averaged during the warmer 1990s was about 2.1 times higher than during the colder late 1970s and 1980s, indicating that the fishing mortality has intensified during the 1990s and has accelerated the collapse of the stock. This suggests that the decadal pattern of variation of spear squid was largely forced by the late 1980s climatic regime shift of the TWC, while high fishing magnified interannual variation of spear squid and attributed to the subsequent collapse of the stock. Reducing fishing pressure on juvenile squid during the early part of the fishing season in autumn is considered to be particularly important for the recovery of this stock under the current unfavorable thermal regime.

Effect of low temperature on mating behavior of squidLoligo bleekeri

Effect of low temperature on mating behavior of squid<i>Loligo bleekeri</i>

Threshold dimorphism in ejaculate characteristics in the squid Loligo bleekeri

Male dimorphism has been thought to correlate with alternative reproductive behav- iors. Alternative reproductive behaviors promote asymmetry in sperm competition, and the differ- ences 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 pro- duced 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.

Dependance of paternity rates on alternative reproductive behaviors in the squid Loligo bleekeri

The mating behavior of captive Loligo bleekeri and the paternity of the resulting prog- eny 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, result- ing 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 different- sized 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 be- haviors would change these factors and lead to different paternity rates.

Skin Color Changes of Squids Todarodes pacificus and Loligo bleekeri During Chilled Storage

ABSTRACT: Squid skin color is one of many important factors to evaluate market values. The effects of chilling and hypoxia treatments on skin color of Japanese common squid Todarodes pacificus and spear squid Loligo bleekeri were investigated to prevent highly fresh squids from abnormal fading skin color with inappropriate commercial value losses. Skin color of the squids gradually faded away during ice‐cold storage. Measurements of chromato‐phores expansion showed that skin color can be maintained well after death by keeping the bodies well‐oxygenated and avoiding direct contact with ice.

Cloning and functional characterization of squid voltage-dependent Ca 2+ channel β subunits: involvement of N-terminal sequences in differential modulation of the current

cDNAs that encode β subunits of voltage-dependent Ca 2+ channel were cloned from the optic lobe of the squid Loligo bleekeri. The subunits, LoCa vβ 1a and LoCa vβ 1b are 96% identical in amino acid sequence. The sole sequence differences are in the N-terminal region and in a five amino acid insertion in the central region of LoCa vβ 1b. RT-PCR revealed that LoCa vβ 1a and LoCa vβ 1b transcripts were expressed mainly in the optic lobe and stellate ganglion, and more weakly in mantle muscle, systemic heart, gill, branchial heart, stomach and liver. Coexpression of LoCa vβ 1a or LoCa vβ 1b with mammalian Ca v2.3 and α 2/δ subunits in the Xenopus oocyte resulted in high-voltage-activated currents, and showed slow current inactivation and moderate steady-state inactivation. Comparison of the squid subunits with four mammalian β subunits, β 1b, β 2a, β 3 and β 4, demonstrated that the modulatory effects of the β subunits on steady-state inactivation kinetics were β 3<β 4≈β 1b< LoCa vβ 1a≈ LoCa vβ 1b<β 2a. LoCa vβ 1a-induced current amplitude was about two to four times higher than that of LoCa vβ 1b. Experiments with point mutants and chimeras suggest that potential PKC and CK2 phosphorylation sites in the N-terminal region of LoCa vβ 1b affect the current amplitude reciprocally, and may be responsible for regulating current amplitude.

イカの各組織由来のホスファチジルコリン及びホスファチジルエタノールアミンにおけるＤＨＡ及びＥＰＡの位置分布（原標題は英語）

The distribution of docosahexaenoic (22:6n-3; DHA) and icosapentaenoic (20:5n-3; EPA) acids in phosphatidylcholine (PC) and/or phosphatidylethanolamine (PE) from different tissues of two species of squids, spear squid Loligo bleekeri and Pacific flying squid Todarodes pacificus, was studied. Mantles, fins, arms, and integuments of squid and liver of the former species contained small amounts of lipid (TL: 1.8-4.3% of wet weight) comprised almost entirely of PC (47.9-67.0%), sterols (14.2-32.7%), and PE (12.2-15.7%). Liver of the latter species contained copious amounts of TL (36.2%) with higher triacylglycerols (97.0%). In PC and PE from squid tissue, DHA (24.7-37.6% for PC and 7.4-25.0% for PE), EPA (6.3-18.4% and 18.9-38.8%, respectively) and palmitic acid (28.6-44.0% and 11.0-22.5%, respectively) were characteristically present. In squid tissue, except the liver, component fatty acids at sn-position 2 of PC and PE were polyunsaturated for the most part with the predominant component of DHA (71.3-78.7% for PC and 17.1-45.3% for PE) and EPA (10.9-14.4% and 37.2-58.0%, respectively). These acids at sn-position 1 of PC and PE were more saturated with palmitic (66.4-72.9% for PC and 15.9-41.6% for PE) and stearic (4.2-6.9% and 9.8-30.0%, respectively) acids. LysoPC, enriched with DHA (66.1%) and EPA (11.1%), was obtained from squid tissue PC (DHA: 36.4%, EPA: 6.7%) by Lipase M10 hydrolysis.

Cultured Giant Fiber Lobe of Squid Expresses Three Distinct Potassium Channel Activities in Selective Combinations

Neurons from the giant fiber lobe (GFL) of squid Loligo bleekeri were dissociated and cultured. The ionic currents were recorded using whole-cell patch clamp methods. The sodium current and the noninactivating potassium current like those elicited by the giant axon were among the currents expressed in axonal bulbs and bulblike structures upon dissociation. Meanwhile axonless cell bodies did not elicit such currents. Axonless cell bodies and some bulblike structures elicited two kinds of inactivating potassium currents, the slow- and the fast-inactivating current, which differed in their inactivation kinetics and pharmacology. Within 24 hr of plating, the current composition remained the same. While the noninactivating current was not sensitive to 4-aminopyridine, the two inactivating currents were sensitive, the slow-inactivating current being more sensitive. Selective combinations of the sodium current and the three potassium currents expressed in different structures of the acutely dissociated GFL could have resulted from cellular control of synthesis and transportation of the channel proteins to the somatic and the axonal membrane. The sodium current and the noninactivating potassium current could be recorded from some axonless cell bodies maintained in culture for over three days, indicating that the separation of the giant axon from its somata could result in the transportation of the channels normally expressed on the giant axon membrane to the somatic membrane.

115 Four-wavelength ratiometric Ca 2+ imaging using btc and fura-2 simultaneously

On the basis of our recent results of the complete amino acid sequence of the squid Loligo bleekeri sodium channel deduced by cloning and sequence analysis of the complementary DNA (Sato, C. and Matsumoto, G. Biochem. Biophys. Res. Comm. 186, 1), we have proposed a tertiary structure model of the sodium channel where the transmembrane segments are octagonally aligned and the four linkers of S5–6 between segments S5 and S6 play a crucial role in the activation gate, voltage sensor and ion selective pore, which can slide, depending on membrane potentials, along inner walls consisting of segments S2 and S4 alternately. The proposed model is contrasted with that of Noda et al. (Nature 320; 188–192, 1986).

Myosin heavy chain-degrading proteinase from spear squid muscle

Trypsin-like proteinase responsible for textural deterioration of thermally induced gel was purified to apparent homogeneity from spear squid Loligo bleekeri mantle muscle by chromatography on Sephacryl S-400 and DEAE Sepharose. The enzyme gave a protein band with a molecular weight of 42 kDa on SDS-PAGE. The proteinase readily hydrolyzed casein, synthetic peptide butyloxycarbonyl-valine-leucine-lysine-4-methylcoumaryl-7-amide (Boc-Val-Leu-Lys-MCA) and myosin heavy chain. The proteolytic activity was effectively inhibited by serine protease inhibitors such as soybean trypsin inhibitor and N ga- p-tosyl- l-lysine-cnloromethyl ketone, but was not affected by the other types of protease inhibitors (e.g. EDTA, EGTA, iodoacetic acid, mercuric chloride and N-tosyl- l-phenylalanine-chloromethyl ketone). The optimal temperature was 40 °C for hydrolysis of both casein and Boc-Val-Leu-Lys-MCA. The optimal pH values were 6.8 and 7.9 for caseinolytic and peptide hydrolyzing activity, respectively. The proteolytic activity was increased 1.3-fold by addition of 0.25 m NaCl, but not by the addition of Ca 2+. Myosin heavy chain was shown to be cleaved into smaller fragments by incubation with the proteinase on SDS-PAGE. These results revealed that the enzyme was involved in degradation of myosin heavy chain from the squid mantle meat.

Neuronal Specificity of Subtype SQSC1 of Squid Putative Sodium Channel

The distribution of SQSC1 mRNA in tissues of squid Loligo bleekeri was studied by the blot hybridization method. The complete cDNA for the coding region of SQSC1, the invertebrate putative sodium channel, was prepared from squid optic lobe (Sate and Matsumoto, Biochem. Biophys. Res. Comm. 186, 61-68, 1992). Transcriptional products of the SQSC1 gene were found to consist of two main different lengths (12 and 9 kb). The transcriptional products were detected in all the nervous tissues examined: optic robes, cerebral ganglia and giant stellate ganglia. However, it was not detected in the muscle, suggesting the SQSC1 gene is specific for sodium channels of squid nerve cells. SQSC1 appears more widely distributed in the nervous system than GFLN1 which they reported as expressed specifically in stellate ganglion of the squid (Rosenthal and Gilly, Proc. Natl. Acad. Sci. USA90, 10026-10030, 1993).

Primary structure of squid sodium channel deduced from the complementary DNA sequence

The complete amino acid sequence of a sodium channel from squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The deduced sequence revealed an organization virtually identical to the vertebrate sodium channel proteins; four homologous domains containing all six membrane-spanning structures are repeated in tandem with connecting linkers of various sizes. A unique feature of the squid Na channel is the 1,522 residue sequence, approximately three fourths of those of the rat sodium channels I, II and III.