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Cephalopods have acquired numerous novelties and expanded their habitats to various marine environments as highly agile predators. Among cephalopod novelties, multiple arms are used for complex behaviors, including prey capture. Suckers on arms are innovative features for realizing these arm functions. In addition, tentacles in Decapodiformes (squids and cuttlefishes) are arms specialized in prey capture and tentacular suckers show unique morphologies. However, little is known about the developmental process of sucker formation that should differ between tentacles and other arms. In this study, therefore, sucker formation processes on second arms and tentacles were observed and compared in a bigfin reef squid, Sepioteuthis lessoniana, to reveal the developmental processes forming the unique sucker morphologies, especially in tentacles. Morphological and histological observations of suckers during embryogenesis showed that, in second arms, the sucker-producing area appeared at the most distal part. At the most proximal side of the sucker-producing area, new sucker buds were isolated by invagination of the epithelial tissue. At the proximal arm parts, suckers with functional structures were observed. In tentacles, although the basic sucker formation pattern was similar to that in second arms, sucker formation started at earlier embryonic stages and the number of suckers was drastically increased compared to that in second arms. In addition, although 4 sucker rows were observed at the tentacular club, i.e., the thickest part of a tentacle, our observations suggested that 2 sets of 2 sucker rows are compressed to form the 4 rows. Therefore, the sucker-formation processes are temporally and spatially different between arms and tentacles. In addition, S. lessoniana shows conserved and unique patterns of sucker formation in comparison with previously described species, suggesting that sucker formation patterns were diversified among Decapodiformes lineages. This article is protected by copyright. All rights reserved.

The measured gross alpha and gross beta activities in the edible muscle tissues of eleven selected marine species along the coast of North Vietnam varied from 10.2 ± 1.5 to 73.2 ± 8.1 Bq/kg (wwt) and from 10.6 ± 0.4 to 68.8 ± 2.8 Bq/kg (wwt), respectively. The lowest gross alpha activity was recorded for bigfin reef squid (Sepioteuthis lessoniana) as a result of its carnivorous diet, and the highest alpha activity was noted for blood cockle (Anadara granosa) as a result of its omnivorous diet. However, the gross beta activities in both carnivorous and omnivorous species were similar. The highest and lowest gross beta activities were observed for narrow-barred Spanish mackerel (Scomberomorus commerson) and for bigfin reef squid and squid (Teuthida), respectively. All three aforementioned species have carnivorous diets. The calculated annual committed effective dose resulting from the consumption of 25 kg of muscle tissue per year varied from 192 to 1375 μS with an average of 689 μS.

Oval squids are polyandrous, with one female mating with multiple males during the spawning season. There are two alternative male mating tactics used by Sepioteuthis lessoniana. Larger males place spermatophores at the opening of the oviduct using a male-parallel mating posture, whereas smaller males attach spermatophores around the female buccal membrane using a male-upturned mating posture. If the route of egg transportation is taken into consideration, male-parallel mating would be expected to result in higher fertilization success than male-upturned mating. Although these male mating tactics are largely dependent on the body size of the male relative to that of the female, it is unclear how female choice affects the male's mating tactics and his mating success. Squids are highly visual animals, and they communicate through dynamic body patterning. In the present study, we observed that smaller male squids in captivity would attempt to mate with a larger female using the male-parallel tactic repeatedly, but they failed to be successful most of the time because of a rejection signal by the female. In contrast, when the males switched to the male-upturned tactic, the mating success rate was increased significantly, with much less female rejection signal. This finding suggests that female squids signal their mating receptivity visually and that male squids alter their mating tactics accordingly. This is the evidence to support the hypothesis that the switch in male mating tactics depends on female choice in oval squids and that this is transmitted via visual communication.

Cephalopods in nature undergo highly dynamic skin coloration changes that allow rapid camouflage and intraspecies communication. The optic lobe is thought to play a key role in controlling the expansion of the chromatophores that generate these diverse body patterns. However, the functional organization of the optic lobe and neural control of the various body patterns by the optic lobe are largely unknown. We applied electrical stimulation within the optic lobe to investigate the neural basis of body patterning in the oval squid,Sepioteuthis lessoniana. Most areas in the optic lobe mediated predominately ipsilateral expansion of chromatophores present on the mantle, but not on the head and arms; furthermore, the expanded areas after electrical stimulation were positively correlated with an increase in stimulating voltage and stimulation depth. These results suggest a unilaterally dominant and vertically converged organization of the optic lobe. Furthermore, analyzing 14 of the elicited body pattern components and their corresponding stimulation sites revealed that the same components can be elicited by stimulating different parts of the optic lobe and that various subsets of these components can be coactivated by stimulating the same area. These findings suggest that many body pattern components may have multiple motor units in the optic lobe and that these are organized in a mosaic manner. The multiplicity associated with the nature of the neural controls of these components in the cephalopod brain thus reflects the versatility of the individual components during the generation of diverse body patterns.SIGNIFICANCE STATEMENTNeural control of the dynamic body patterning of cephalopods for camouflage and intraspecies communication is a fascinating research topic. Previous studies have shown that the optic lobe is the motor command center for dynamic body patterning. However, little is known about its neural organization and the mechanisms underlying its control of body pattern generation. By electrically stimulating the optic lobe of the oval squids and observing their body pattern changes, surprisingly, we found that there is no somatotopic organization of motor units. Instead, many of these components have multiple motor units within the optic lobe and are organized in a mosaic manner. The present work reveals a novel neural control of dynamic body patterning for communication in cephalopods.

Coleoid cephalopods (squid, cuttlefish, and octopus) have a well-developed and complex central nervous system. Its absolute size is the largest among invertebrates, and the brain-to-body mass ratio is larger than that of fish and reptiles and equivalent to that of birds and mammals. Although a number of histological studies have been conducted on the brains of cephalopods, most of them used a light microscope or an electron microscope, which show the microstructure of the brain, but often cannot image the whole brain instantaneously. Of late, micro computed tomography (CT) has gained popularity for imaging animal brains because it allows for noninvasive three-dimensional (3D) reconstruction and preprocessing that are not cumbersome. To perform micro-CT on cephalopod brains, we first tested conditions suitable for preprocessing, paying special attention to staining conditions that would provide high contrast images. Four agents, iodine in 99.5% ethanol, iodine potassium iodide in water (IKI), phosphotungstic acid in 70% ethanol, and nonionic iodinated contrast agent in water, were tested at various concentrations and durations on brain of juvenile oval squid. To evaluate the quality of staining, we calculated the contrast ratio of the two-dimensional (2D) images and compared 3D segmentation of the best and worst 2D images. We concluded that 3% IKI staining for 7 days was the best combination to enhance the images contrast of the oval squid brain, in which each brain lobe was clearly detected and 3D segmentation of the whole brain was possible. The wider applicability of this preprocessing method for micro-CT of the brains of other cephalopods is discussed.

The wide disparity in adult body size observed both within and among animal taxa has long attracted widespread interest, with several general rules having been proposed to explain trends in body size evolution. Adult body size disparity among the cephalopod mollusks is remarkable, with adult body sizes ranging from a few centimeters to several meters. Some of the smallest cephalopods are found within Pickfordiateuthis, a group comprising three described species of squid found in the western Atlantic and tropical eastern Pacific. Pickfordiateuthis pulchella, the type species of the genus, was initially proposed to be closely related to the loliginid squids (Loliginidae), with subsequent descriptions of additional species supporting a placement within Loliginidae. Pickfordiateuthis is remarkable in that all species reach sexual maturity at about one-fifth to one-tenth the size seen in most loliginid species. To date, no phylogenetic analyses have included representatives of Pickfordiateuthis. To infer the phylogenetic position of Pickfordiateuthis and explore its implications for body size evolution, we collected specimens of Pickfordiateuthis pulchella from Brazilian waters and sequenced regions of two loci—the mitochondrial large ribosomal subunit (rrnL a.k.a. 16S) gene and the nuclear gene rhodopsin. Maximum likelihood and Bayesian analyses of these sequences support a placement of Pickfordiateuthis pulchella as sister to a clade comprising the Western Hemisphere loliginid genera Doryteuthis and Lolliguncula. Analyses of body size evolution within Loliginidae suggest that a shift to a smaller body size optimum occurred along the lineage leading to P. pulchella, with some evidence of shifts toward larger sizes in the ancestors of Loligo and Sepioteuthis; these inferences seem to be robust to phylogenetic uncertainty and incomplete taxon sampling. The small size and juvenile-like morphological traits seen in adult Pickfordiateuthis (e.g., sepiolid-like fins and biserial sucker arrangement in the tentacles) may be due to paedomorphosis.

Unlike vitellogenin, which is the sole major precursor of yolk protein in all oviparous vertebrates, a variety of major precursor of yolk proteins are found among oviparous invertebrates. Sea urchins have a transferrin-like yolk protein, while all other major precursors of yolk proteins in oviparous invertebrates belong to the superfamily of large lipid transfer proteins (LLTPs). However, a comprehensive understanding of vitellogenesis is absent in cephalopods. To understand control of vitellogenesis by the LLTPs gene, two vitellogenins (VTG1 and VTG2), two apolipophorins (APOLP2A and APOLP2B), and a cytosolic large subunit of microsomal triglyceride transfer protein (MTTP) found in the bigfin reef squid. Only the two VTGs showed high levels of expression in mature females compared to males.We further analyzed the expression profile and localization of both VTGs/VTGs during ovarian development. Our data showed that VTGs/VTGs expressions were correlated to the female reproductive cycle. Ovarian VTG1 and VTG2 were localized in the follicle cells but not in oocytes. In addition, VTG1 and VTG2 were represented in follicle cells and oocytes. Thus, our results showed that both VTGs were synthesized by follicle cells and are then delivered to oocytes. In addition, we demonstrated that VTGs were the major precursor of yolk protein in bigfin reef squid. We also found differential proteolytic cleavage processes of VTG1 and VTG2 during VTGs accumulation in oocytes. Therefore, our data shed light on the molecular mechanism of the yolk accumulation pathway in cephalopods.Summary SentenceTwo distinct vitellogenins, which are synthesized by the follicle cells, are the major precursor of yolk protein of oocytes in bigfin reef squid.

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.

β-Chitin and its chitosan from the gladius of Sepioteuthis lessoniana have been isolated, purified, characterised and compared with the commercial chitosan. Ash, moisture, mineral, metal and elemental content were analyzed using standard techniques. The optical activity of chitin was found to be levorotatory. The degree of deacetylation was calculated by potentiometric titration and (1)H NMR. Viscosity average molecular weight of β-chitosan was calculated by viscometry and size average molecular weight by GPC. The structure of β-chitosan was elucidated with FT-IR and NMR. Thermal nature, crystalline structure and morphology of β-chitosan were characterised through DSC, XRD and SEM, respectively. The water and fat binding capacity of β-chitosan presently studied was significantly higher than that of the commercial chitosan. The result of the present study adds that S. lessoniana gladius is also an additional source of β-chitin and chitosan of higher yield, lower molecular weight and higher degree of deacetylation.

The oval squid Sepioteuthis lessoniana is one of the most economically important squid species in Japan; however, its population structure is poorly understood due to the lack of hypervariable markers. Such information is critical for managing sustainable fisheries, as well as for ensuring the existence of wild S. lessoniana stocks. Eleven candidate microsatellite loci were isolated from a small insert genomic DNA library. Polymorphisms in these 11 loci were screened in 24 wild individuals. The number of alleles per locus was found to range from 5 to 19 alleles, and the observed heterozygosity ranged from 0.292 to 0.958. No evidence for linkage disequilibrium was detected among all the loci. The genotypic proportions conformed to Hardy-Weinberg equilibrium, except at one locus. In conclusion, these polymorphic microsatellite loci may be used to develop a genetic framework to manage S. lessoniana in the future.

The family Loliginidae Lesueur, 1821, is currently considered to include seven genera and approximately 50 species of neritic and coastal squids. These commercially important species occur in tropical and temperate coastal waters around the world. The taxonomy of the family has been revised a number of times in recent years, focusing in particular on genera such as Doryteuthis, Sepioteuthis, Alloteuthis, and Uroteuthis, which are represented by populations in the New World, Oceania, Europe/Africa, and Asia. However, no detailed phylogenetic analysis is available for the loliginids of the southern Atlantic, in particular the genus Doryteuthis. The present molecular study analyzed 81 loliginid taxa from around the world. The partial sequencing of the mitochondrial 16S and Cytochrome Oxidase I genes, and the nuclear rhodopsin gene revealed a number of important patterns, recovering the monophyletic status of the majority of the genera and revealing possible cryptic species in Doryteuthis plei D. pealei, Uroteuthis duvauceli and Sepioteuthis lessoniana.

Cephalopods have the largest and most complex nervous system of all invertebrates, and the brain-to-body weight ratio exceeds those of most fish and reptiles. The brain is composed of lobe units, the functions of which have been studied through surgical manipulation and electrical stimulation. However, how information is processed in each lobe for the animal to make a behavioral decision has rarely been investigated. To perform such functional analyses, it is necessary to precisely describe how brain lobes are spatially organized and mutually interconnected as a whole. We thus made three-dimensional digital brain atlases of both hatchling and juvenile pygmy squid, Idiosepius paradoxus. I. paradoxus is the smallest squid and has a brain small enough to scan as a whole region in the field-of-view of a low-magnification laser scan microscope objective. Precise analyses of the confocal images of the brains revealed one newly identified lobe and also that the relative volume of the vertical lobe system, the higher association center, in the pygmy squid represents the largest portion compared with the cephalopod species reported previously. In addition, principal component analyses of relative volumes of lobe complexes revealed that the organization of I. paradoxus brain is comparable to those of Decapodiformes species commonly used to analyze complex behaviors such as Sepia officinalis and Sepioteuthis sepioidea. These results suggest that the pygmy squid can be a good model to investigate the brain functions of coleoids utilizing physiological methods. J. Comp. Neurol. 524:2142-2157, 2016. © 2016 Wiley Periodicals, Inc.

We examined the relationship between mantle muscle structure and mantle kinematics in an ontogenetic series (5-85 mm dorsal mantle length) of oval squid, Sepioteuthis lessoniana. Thick filament length increased during growth in the mantle muscle fibres that power jet locomotion (i.e. the circular muscles). The thick filament length of both the superficial mitochondria-rich (SMR; analogous to vertebrate red muscle fibres) and central mitochondria-poor (CMP; analogous to vertebrate white muscle fibres) circular muscles increased significantly during ontogeny. Thick filaments in the SMR circular muscle fibres of newly hatched squid (N=5) ranged from 0.7 to 1.4 microm and averaged 1.0 microm, while the thick filaments of the SMR fibres of the largest squids (N=4) studied ranged from 1.2 to 3.4 microm and averaged 1.9 microm. The ontogeny of thick filament length in the CMP circular muscle fibres showed a similar trend. The range for hatchling CMP circular muscles was 0.7-1.4 microm, with an average of 1.0 microm, whereas the range and average for the largest squids studied were 0.9-2.2 microm and 1.5 microm, respectively. Within an individual hatchling, we noted no significant differences between the thick filament lengths of the SMR and CMP fibres. Within an individual juvenile, the thick filaments of the SMR fibres were approximately 25% longer than the CMP fibres. The change in thick filament length may alter the contractile properties of the circular muscles and may also result in a decrease in the rate of mantle contraction during jetting. In escape-jet locomotion, the maximum rate of mantle contraction was highest in newly hatched squid and declined during ontogeny. The maximum rate of mantle contraction varied from 7-13 muscle lengths per second in newly hatched squid (N=14) and from 3-5 muscle lengths per second in the largest squids (N=35) studied.

In Sepioteuthis lessoniana, the oval squid, on- togenetic changes in the kinematics of the mantle during escape-jet locomotion imply a decline in the relative mass flux of the escape jet and may affect the peak weight- specific thrust of the escape jet. To examine the relationship between ontogenetic changes in the kinematics of the man- tle and the thrust generated during the escape jet, we simul- taneously measured the peak thrust and the kinematics of the mantle of squid tethered to a force transducer. We tested an ontogenetic series of S. lessoniana that ranged in size from 5 to 40 mm dorsal mantle length (DML). In newly hatched squids, thrust peaked 40 ms after the start of the escape jet and reached a maximum of between 0.10 mN and 0.80 mN. In the largest animals, thrust peaked 70 ms after the start of the escape jet and reached a maximum of between 18 mN and 110 mN. Peak thrust was normalized by the wet weight of the squid and also by the cross-sectional area of the circumferential muscle that provides power for the escape jet. The weight-specific peak thrust of the escape jet averaged 0.36 in newly hatched squid and increased significantly to an average of 1.5 in the largest squids measured (P 0.01). The thrust per unit area of circum- ferential muscle averaged 0.25 mN/mm 2 in hatchlings and increased significantly to an average of 1.4 mN/mm 2 in the largest animals tested ( P 0.01). The impulse of the escape jet was also lowest in newly hatched individuals (1.3 mN s) and increased significantly to 1000 mN si n the largest squids measured (P 0.01). These ontogenetic changes in the mechanics of the escape jet suggest (1) that propulsion efficiency of the exhalant phase of the jet is highest in hatchlings, and (2) that the mechanics of the circumferential muscles of the mantle change during growth.

Mantle meat from the Japanese common squid (Todarodes pacificus) browns more than other squid meats during air-drying. The factors contributing to the browning of Japanese common squid, long-finned squid (Photololigo edulis) and bigfin reef squid (Sepioteuthis lessoniana) were studied in boiled and raw meat both before and after air-drying. Dried raw meat from the Japanese common squid browned more than dried boiled meat (b(∗) value, from 4.7 to 28.5). The results from SDS-PAGE showed significant degradation of myosin heavy chain (MHC) suggesting that protease activity in raw Japanese common squid meat was higher than in the other two species. The concentration of arginine (1932.0mg/100g) and ribose (28.8μmol/g) in Japanese common squid meat was higher than in the other two species. These results suggest that high protease activity and high concentrations of arginine and ribose increase the browning discoloration of Japanese common squid during air-drying.

The embryonic development of the central nervous system (CNS) in the oval squid Sepioteuthis lessoniana is described. It has three distinct phases: (1) The ganglionic accumulation phase: Ganglionic cell clusters develop by ingression, migration, and accumulation of neuroblasts. (2) The lobe differentiation phase: Ganglia differentiate into lobes. The phase is identified by the beginning of an axogenesis. During this phase, neuropils are first formed in the suboesophageal mass, then in the basal lobe system, and finally in the inferior frontal lobes and the superior frontal-vertical lobe systems. (3) The neuropil increment phase: After the shape of the lobes reached its typical form, neuropil growth occurs, specifically in the vertical lobe. The paralarval central nervous system (CNS) is characterized by neuronal gigantism of the giant fibers and some suboesophageal commissures and connectives. The neuropil formation in the CNS of S. lessoniana occurs somewhat earlier than in Octopus vulgaris, although the principal developmental plan is quite conservative among the other coleoids investigated. Some phylogenetic aspects are discussed based on the similarities in the morphologic organization of their brains. J. Comp. Neurol. 437:449–475, 2001. © 2001 Wiley-Liss, Inc.

In this study, the complete mitogenome sequence of the cryptic “lineage A” big-fin reef squid, Sepioteuthis lessoniana (Cephalopoda: Loliginidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome consists of 16,605 bp, which includes 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs genes. The overall base composition of “lineage A” S. lessoniana is 37.5% for A, 17.4% for C, 9.1% for G, and 35.9% for T and shows 87% identities to “lineage C” S. lessoniana. It is also noticed by its high T + A content (73.4%), two non-coding regions with TA tandem repeats. The complete mitogenome of the cryptic “lineage A” S. lessoniana provides essential and important DNA molecular data for further phylogeography and evolutionary analysis for big-fin reef squid species complex.

Animals adopt various forms of hunting according to their ecological, morphological, and cognitive features, and their specific hunting skills are acquired ontogenetically in relation to these features. It is noted that cuttlefish and squid hunt prey through the elongation of tentacles used specifically to capture prey. However, these two cephalopods have different lifestyles, leading to questions such as whether hunting skill is acquired similarly after birth and whether tentacle elongation is behaviorally identical. To address these questions, we observed and compared how captive pharaoh cuttlefish (Sepia pharaonis) and oval squid (Sepioteuthis lessoniana) attack prey during their early life stages. Like the adults, S. pharaonis hatchlings used the tentacular lunge attack, whereas S. lessoniana hatchlings used the arm-opening attack. S. lessoniana began to exhibit the tentacular strike attack after 30 days of age. In addition to timing of the emergence of a specific hunting mode, some differences were observed in the physical aspect of hunting behavior. For cuttlefish, maximum tentacle length and maximum speed of tentacle elongation increased from hatching to 30 days of age and then decreased. In contrast, for squid, maximum tentacle length increased from hatching to 30 days of age and then became constant. The distance to prey was positively correlated with maximum length and speed of tentacle elongation in S. pharaonis and with maximum swimming speed in S. lessoniana. These results show that cuttlefish mainly use an ambush strategy and that squid use a pursuit strategy. Possible causes for the ontogenetic differences in hunting behavior are discussed.

Owing to their codominant, multiallelic, and highly polymorphic nature, microsatellite markers have been used widely in population genetics and biological resource conservation studies. To investigate the genetic structure of Sepioteuthis lessoniana, we developed 24 microsatellite DNA markers and assessed the polymorphism of each locus in a wild S. lessoniana population. The number of alleles per locus ranged from 4 to 26, and the observed and expected heterozygosities varied from 0.188 to 1.000 and 0.392 to 0.959 with an average of 0.675 and 0.852, respectively. These microsatellite loci will be useful tools in future studies of population genetic structure in this species.

Squid can rapidly change the chromatic patterns on their body. The patterns are created by the expansion and retraction of chromatophores. The chromatophore consists of a central pigment-containing cell surrounded by radial muscles that are controlled by motor neurons located in the central nervous system (CNS). In this study we used semi-intact squid (Sepioteuthis lessoniana) displaying centrally controlled natural patterns to analyze spatial and temporal activities of chromatophores located on the dorsal mantle skin. We found that chromatophores oscillated with miniature expansions/retractions at various frequencies, even when the chromatic patterns appear macroscopically stable. The frequencies of this miniature oscillation differed between "feature" and "background" areas of chromatic patterns. Higher frequencies occurred in feature areas, whereas lower frequencies were detected in background areas. We also observed synchronization of the oscillation during chromatic pattern expression. The expansion size of chromatophores oscillating at high frequency correlated with the number of synchronized chromatophores but not the oscillation frequency. Miniature oscillations were not observed in denervated chromatophores. These results suggest that miniature oscillations of chromatophores are driven by motor neuronal activities in the CNS and that frequency and synchrony of this oscillation determine the chromatic pattern and the expansion size, respectively.

SummarySquid and other cephalopods catch prey with remarkable speed and precision [1]. Before the strike occurs, they encounter the difficult task of judging an object’s distance and size in the contrast-poor world of the mid-water environment [1–4]. Here we describe a solution to this common problem underwater, where a large portion of a squid’s dorso-temporal retina is intentionally blurred. This apparently counter-adaptive ‘retinal bump’ is combined with a vertical bobbing behavior that scans objects of interest from focused to defocused retinal regions. The image focus differential changes sharply at precisely the distance equivalent to tentacle length and enables the squid, Sepioteuthis lessoniana, to capture prey. This unique range-finding mechanism is an adaptation to hunting, defense, and object size identification in an environment where the depth cues found on land are less reliable.

The cylindrical shape of soft-bodied invertebrates is well suited to functions in skeletal support and locomotion, but may result in a previously unrecognized cost—large non-uniformities in muscle strain and strain rate among the circular muscle fibres of the body wall. We investigated such gradients of strain and strain rate in the mantle of eight long-finned squidDoryteuthis pealeiiand two oval squidSepioteuthis lessoniana. Transmural gradients of circumferential strain were present during all jets (n= 312); i.e. for a given change in the circumference of the outer surface of the mantle, the inner surface experienced a greater proportional change. The magnitude of the difference increased with the amplitude of the mantle movement, with circular muscle fibres at the inner surface of the mantle experiencing a total range of strains up to 1.45 times greater than fibres at the outer surface during vigorous jets. Differences in strain rate between the circular fibres near the inner versus the outer surface of the mantle were also present in all jets, with the greatest differences occurring during vigorous jetting. The transmural gradients of circumferential strain and strain rate we describe probably apply not only to squids and other coleoid cephalopods, but also to diverse soft-bodied invertebrates with hollow cylindrical or conical bodies and muscular organs.

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development.

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

We observed the reproductive behavior of the oval squid Sepioteuthis lessoniana in captivity. The male used three different mating behaviors: male-parallel (MP), male-upturned (MU) and sneaking. Male competition over females frequently occurred before and during the female egg-laying period, and the outcome of most fights depended on male body size. Larger males guarded their partners from other males and performed MP mating during the egg-laying period of the paired females. In contrast, there was no pairing and mate guarding in MU mating and sneaking, which were adopted by smaller subordinate males as alternative tactics outside female egg-laying period and during the period, respectively. MP matings were 95% successful, but more than half of MU matings were unsuccessful. Higher mating success in MP mating was achieved through pairing, whereas males in MU mating were less successful because mating attempts without pair formation were often foiled by escape of the female. Sneaking was successful in all cases but occurred less frequently. Spermatophores were attached at the opening of the oviduct in MP mating, whereas they were attached around the female buccal membrane in MU mating and sneaking. Considering the route of egg transportation, higher fertilization success can be expected in MP mating because of the advantageous location of the attached spermatophores. Our results suggest that MP mating is used by larger, paired males during the female egg-laying period, and that MU mating and sneaking are alternative tactics adopted by smaller, subordinate males. These alternative mating behaviors would be conditional strategy dependent on relative body size, because some individual males displayed both MP and MU mating behaviors., Zoological Science, 22(6), pp.645-651; 2005

The renal organs of 32 species of cephalopods (renal appendage of all cephalopods, and renal and pancreatic appendages in decapods) were examined for parasite fauna and for histological comparison. Two phylogenetically distant organisms, dicyemid mesozoans and chromidinid ciliates, were found in 20 cephalopod species. Most benthic cephalopods (octopus and cuttlefish) were infected with dicyemids. Two pelagic cephalopod species, Sepioteuthis lessoniana and Todarodes pacificus, also harbored dicyemids. Chromidinid ciliates were found only in decapods (squid and cuttlefish). One dicyemid species was found in branchial heart appendages of Rossia pacifica. Dicyemids and chromidinids occasionally occurred simultaneously in Euprymna morsei, Sepia kobiensis, S. peterseni, and T. pacificus. The small-sized cephalopod species, Idiosepius paradoxus and Octopus parvus, harbored no parasites. Comparative histology revealed that the external surface of renal organs varies morphologically in various cephalopod species. The small-sized cephalopod species have a simple external surface. In contrast, the medium- to large-sized cephalopod species have a complex external surface. In the medium- to large-sized cephalopod species, their juveniles have a simple external surface of the renal organs. The external surface subsequently becomes complicated as they grow. Dicyemids and chromidinids attach their heads to epithelia or insert their heads into folds of renal appendages, pancreatic appendages, and branchial heart appendages. The rugged and convoluted external surface provides a foothold for dicyemids and chromidinids with a conical head. They apparently do not harm these tissues of their host cephalopods.

SUMMARYTo determine which physiological properties contribute to temperature adaptation in the squid giant axon, action potentials were recorded from four species of squid whose habitats span a temperature range of 20°C. The environments of these species can be ranked from coldest to warmest as follows: Loligo opalescens>Loligo pealei>Loligo plei>Sepioteuthis sepioidea. Action potential conduction velocities and rise times,recorded at many temperatures, were equivalent for all Loligospecies, but significantly slower in S. sepioidea. By contrast, the action potential's fall time differed among species and correlated well with the thermal environment of the species (`warmer' species had slower decay times). The biophysical underpinnings of these differences were examined in voltage-clamped axons. Surprisingly, no differences were found between the activation kinetics or voltage-dependence of Na+ and K+currents. Conductance levels, however, did vary. Maximum Na+conductance (gNa) in S. sepiodea was significantly less than in the Loligo species. K+ conductance (gK) was highest in L. pealei, intermediate in L. plei and smallest in S. sepiodea. The time course and magnitude of gK and gNa were measured directly during membrane action potentials. These data reveal clear species-dependent differences in the amount of gK and gNa recruited during an action potential.

We investigated the kinematics of mantle movement during escape jet behavior in an ontogenetic series of Sepioteuthis lessoniana, the oval squid. Changes in mantle diameter during the jet were measured from digitized S-VHS video fields of tethered animals that ranged in age from hatchlings to 9 weeks. The amplitude of both mantle contraction and mantle hyperinflation (expressed as percent change from the resting mantle diameter) during an escape jet was significantly greater in hatchlings than in older, larger squid (P < 0.05). The maximum amplitude of mantle contraction during the escape jet decreased from an average of -40% in hatchlings to -30% in the largest animals studied. The maximum amplitude of mantle hyper-inflation decreased from an average of 18% in hatchlings to 9% in the largest squid examined. In addition, the maximum rate of mantle contraction decreased significantly during ontogeny (P < 0.05), from a maximum of 8.6 mantle circumference lengths per second (L/s) in hatchlings to 3.8 L/s in the largest animals studied. The ontogenetic changes in the mantle kinematics of the escape jet occurred concomitantly with changes in the organization of collagenous connective tissue fiber networks in the mantle. The alteration in mantle kinematics during growth may result in proportionately greater mass flux during the escape jet in newly hatched squid than in larger animals.

Ontogenetic changes in the organization and volume fraction of collagenous connective tissues were examined in the mantle of Sepioteuthis lessoniana, the oval squid. Outer tunic fiber angle (the angle of a tunic collagen fiber relative to the long axis of the squid) decreased from 33.5 degrees in newly hatched animals to 17.7 degrees in the largest animals studied. The arrangement of intramuscular collagen fiber systems 1 (IM-1) and 2 (IM-2) also changed significantly during ontogeny. Because of the oblique trajectory of the IM-1 collagen fibers, two fiber angles were needed to describe their organization: (1) IM-1(SAG), the angle of an IM-1 collagen fiber relative to the squid's long axis when viewed from a sagittal plane and (2) IM-1(TAN), the angle of an IM-1 collagen fiber relative to the squid's long axis when viewed from a plane tangential to the outer curvature of the mantle. The sagittal component (IM-1(SAG)) of the IM-1 collagen fiber angle was lowest in hatchling squid (32.7 degrees ) and increased exponentially during growth to 43 degrees in squid with a dorsal mantle length (DML) of 15 mm. In squid larger than 15 mm DML, IM-1(SAG) fiber angle did not change. The tangential component (IM-1(TAN)) of IM-1 collagen fiber angle was highest in hatchling squid (39 degrees ) and decreased to 32 degrees in the largest squid examined. IM-2 collagen fiber angle (the angle of an IM-2 collagen fiber relative to the outer surface of the mantle) was lowest in hatchling squid (34.6 degrees ) and increased exponentially to about 50 degrees in 15-mm DML animals. In squid larger than 15 mm DML, IM-2 fiber angle increased slightly with size. The volume fraction of collagen in IM-1 and IM-2 increased 68 and 36 times, respectively, during growth. The ontogenetic changes in the organization of collagen fibers in the outer tunic, IM-1, and IM-2 may lead to ontogenetic differences in the kinematics of mantle movement and in elastic energy storage during jet locomotion.

The cephalopod taxon Loliginidae (Cephalopoda: Myopsida) is a species-rich group of tropical and temperate shallow-water squids, many of which are commercial fisheries objects and neurophysiological research organisms. The worldwide distribution of these squids could make Loliginidae a useful case study in shallow-water marine biogeography, but the phylogeny of the group is unknown. To clarify loliginid phylogeny, regions of two mitochondrial genes (the 16S rRNA and the cytochrome c oxidase subunit I genes) were sequenced for members of 19 loliginid species and several outgroups. Maximum-parsimony and maximum-likelihood analyses were performed on a combined data set, as well as on each data set individually. Analyses of the combined data support loliginid monophyly and reveal four clades-one consisting primarily of species in American waters from two genera, one composed of 3 east Atlantic species, one consisting of the bioluminescent loliginids (Uroteuthis sensu Vecchione et al., 1998) plus Loliolus japonica, and one represented by a Loligo (Alloteuthis) subulata-Lolliguncula mercatoris pair. The likelihood of the unconstrained maximum-likelihood tree is not significantly better than the likelihoods of the best trees constrained to Sepioteuthis monophyly or Uroteuthis monophyly, but there is significant support for Lolliguncula polyphyly. Tests of alternative hypotheses of loliginid cladogenesis suggest that cladogenesis within Loliginidae is correlated with the widening of the Atlantic and the closure of the Tethys Sea, although dispersal from the Indo-West Pacific is a reasonable explanation for the origin of the clade of American loliginines.

Lens focal properties (spherical aberration), diameter and relative anterior/posterior proportions were measured photographically for Japanese squid (Sepioteuthis lessoniana) at ages 4-6 weeks, 7-9 weeks and 7-8 months. The measures involved photographing the refractive effects of lenses in a physiological solution, with and without an index matching fluid (polyvinylpyrroloidone solution), on a parallel array of fine helium-neon laser beams. Spherical aberration was determined from measurement of back vertex distance. Similar measurements were made on lenses from the eyes of cuttlefish (Sepia officinalis). The cephalopod lens develops as hemispheric halves from separate ectodermal sources. The posterior component contributes, on average, about 60% of axial lens diameter in S. lessoniana of all ages and 55% in S. officinalis. However, these proportions vary widely in both species. All lenses of both species show that spherical aberration is neutralized, although small variations in back vertex distance (positive and negative spherical aberration) were measured. Preliminary measures indicate that the refractive index distribution within the cephalopod lens varies in a manner reminiscent of teleost lenses. Squid lenses from animals 7-9 weeks of age were optimally corrected for spherical aberration. Some squid of this age, from a separate tank, showed a high incidence of cataract development. In each case, lens opacification was caused by deterioration of the posterior lens component.

Cytoplasmic pH (pHi) of haemocytes of bigfin reef squid (Sepioteuthis lesso-niana Lesson) was determined with the fluorescent probe, 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The pHi of haemocytes suspended in nominally HCO3−-free medium (extracellular pH 7.4) averaged (±S.E.) 7.32±0.02. Intracellular pH was independent of external Na+ concentration and varied only slightly with changes in extracellular pH (pHe) (ΔpHi/ΔpHe=0.16 over the pHe range 6.8–7.8). Addition of weak acids (sodium propionate, potassium acetate) to haemocyte suspensions resulted in a rapid decrease in pHi. Haemocyte pHi then recovered with an average half-time of 3–4 min. Recovery of pHi was independent of external Na+ concentration and insensitive to amiloride, but was abolished by N-ethylmaleimide (NEM). These results argue against the involvement of plasma membrane Na+/H+ exchange or other Na+-dependent transport mechanisms in the pHi recovery of acid-loaded haemocytes. The results suggest that there is an NEM-sensitive proton extrusion mechanism in the plasma membrane of squid haemocytes.

Light microscopical observation of serial sections of four kinds of decapoda which living in seas around Japan (Ommastrephes sloanei pacificus, Sepia esculenta, Doryteuthis bleekeri, Sepioteuthis lessoniana) was carried out. These specimens were fixed alive with 10% formaldehyde solution diluted with sea water and then dehydrated without decalcification with ethylalcohol diluted with sea water. After embedding in 6% celloidin, the specimens were cut into 30 microns thick and sections were stained with hematoxilineosin. Under light microscopic observation, the maculae, statoconial layers, cristae, cupulae, statoliths, central nervous system and static nerves were well preserved. The statocysts of all four decapod species were similar in shape. Color photographs of horizontal sections of the head, statocysts, processes, gravity recepter systems, angular acceleration receptor systems, statoliths and nerve fibers with the central nervous system of these four decapod species are shown.

Acetyltransferase enzymatic activity was detected and measured in homogenates obtained from intact nerve fibers and their separate cellular components, in the tropical squid Sepioteuthis sepioidea. The levels of acetylcholine synthesis were determined in pooled samples of whole stellar nerve, intact giant nerve fiber, extruded axoplasm, axoplasm-free giant nerve fiber sheaths, and small nerve fibers. The values found per mg of protein for the axoplasm-free sheaths are about 3–9 times those of the extruded axoplasm, and comparable to those found for the intact giant nerve fiber. These experimental findings settle the question of whether the Schwann cells of the giant nerve fiber of S. sepioidea, under physiological conditions, contain acetyltransferase activity and are able to synthesize acetylcholine.

The effect of tetrodotoxin on the sodium currents of the squid (Doryteuthis plei and Sepioteuthis sepiodea) giant axons was studied under potential control conditions. The axons were immersed in artificial seawater at 21 degrees C and pH 7.5. When the effect of the toxin is studied in concentrations ranging from 0.1 to 50 nM the Eadie-Haldane plot is not a straight line and indicates that there are two populations of sodium channels open during activity. 19.0 +/- 4.7% of the channels are accociated to receptors with an apparent dissociation constant of 0.11 +/- 0.05 nM and 84.0 +/- 4.1% of the channels are related to receptors having an affinity constant of 4.90 +/- 0.49 nM (nine nerves).

Anion conductances of giant axons of squid, Sepioteuthis, were measured. The axons were internally perfused with a 100-mM tetraethylammonium-phosphate solution and immersed in a 100-mM Ca-salt solution (or Mg-salt solution) containing 0.3 microns tetrodotoxin. The external anion composition was changed. The membrane currents had a large amount of outward rectification due to anion influx across Cl- channels of the membrane (Inoue, 1985). The amount of outward rectification depended on the species of anion used and was strongly influenced by temperature and internal pH. In contrast to the anion conductances themselves, the conductance relative to Cl- (gA/gCl) was found to be quite stable against changes in the membrane potential, temperature, and pH. It is therefore suggested that each gA/gCl is an intrinsic quantity of the Cl- channel of the squid axon membrane. The sequence and values of gA/gCl obtained in this study were NO3- (1.80) greater than I- (1.40) greater than Br- (1.07) greater than Cl- (1.00) greater than MeSO3- (0.46) greater than H2PO2- (0.33) greater than CH3COO- (0.29) greater than SO4(2-) (0.06).

We report the formation of purple hemocyanin upon addition of ethyleneglycol to a solution of Sepioteuthis lessoniana oxyhemocyanin in 0.05 M Tris-HCl buffer (pH 7.5). Spectroscopic studies on the purple hemocyanin indicate that this transformation between the normal blue state and the purple state of oxyhemocyanin is essentially a reversible one. The purple hemocyanin exhibits esr signals that correspond to about one-fifth of the total copper obtained. The results suggest that ethyleneglycol causes a moderate and reversible deformation of the active site of the oxyhemocyanin.