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The harmful dinoflagellate, Heterocapsa circularisquama, has been causing mass mortalities of bivalve molluscs in Japan, at relatively low cell densities. Although several studies have been conducted to determine the toxicity mechanisms, the specific cause of death is still unclear. In a previous study, in our laboratory, it was shown that H. circularisquama (10(3) cells ml(-1)) caused extensive cytotoxicity in the gills of short-neck clams, Ruditapes philippinarum. In the present study, Mediterranean mussels, Mytilus galloprovincialis, were exposed to H. circularisquama at four cell densities (5, 50, 500, 10(3) cells ml(-1)), three temperatures (15, 20, and 25°C), and three exposure durations (3, 24, and 48 h), and the pathologies in nine organs (gills, labial palps, mantle, hepatopancreas, stomach, intestines, exhalant siphon, adductor muscles, and foot) were assessed. Foot, adductor muscles, and exhalent siphons of mussels were not affected; however, 16 inflammatory (hemocytic infiltration and aggregation, diapedesis, hyperplasia, hypertrophy, edema, melanization, and firbrosis) and degenerative (thrombus, thrombosed edema, cilia matting and exfoliation, epithelial desquamation, atrophy, and necrosis) pathologies were identified in the gills, labial palps, mantle, hepatopancreas, stomach, and intestines. The total prevalence and total intensity of pathology in each individual mussel, and the prevalence and intensity of pathology in each organ increased significantly with increased cell density, exposure duration, and temperature. The prevalence of pathology was the highest in gills, followed by the prevalence in labial palps, mantle, stomach, and intestines. Pathology was least prevalent in the hepatopancreas. The intensity of pathology was the highest in the gills, followed by the labial palps and mantle, the stomach and intestines, and the hepatopancreas. This detailed quantitative histopathological study demonstrates that exposure to H. circularisquama induces a broad cytotoxic effect in six vital organs, even at low density (5 cells ml(-1)) and low temperature (15°C), but not in muscular organs. Combining cell density, time, and duration of exposure, the organ most affected by the harmful alga was the gill, followed by the labial palps and mantle, the stomach and intestines, and the hepatopancreas. The results of this pathological analysis show that exposure to H. ciruclarisquama severely affects the gills, the labial palps, and mantle thereby interfering with particle clearance and sorting, cleansing, and respiration, but also affects the stomach, intestines, and hepatopancreas, altering the digestive processes and possibly detoxification pathways, if mussels are able to detoxify the toxins of H. circularisquama. In the most severe cases, bivalves would most likely have died as a result of combined severe alterations of the vital functions, failure of tissue repair, and moderate to heavy hemorrhaging in both the external organs and the digestive organs concomitantly with light to moderate alterations in the detoxifying processes.

Blooms of the toxic dinoflagellate Heterocapsa circularisquama cause massive bivalve kills in Japan. Mariculture of the Japanese pearl oyster, Pinctada fucata martensii, is the industry most affected by these blooms, especially in Ago Bay, Mie Prefecture, where they are frequent, cause mass mortality of oysters, and overlap with their spawning season. The goal of this August 2009 study was to assess the effects of a toxic strain of H. circularisquama isolated from Ago Bay on gametes, fertilization, and embryo development of pearl oysters. Spermatozoa, eggs, spermatozoa and eggs, and fertilized eggs of pearl oysters from Ago Bay were exposed to H. circularisquama at cell densities reported during the bloom (10–104 cells mL−1) for different periods of time. The concentration of H. circularisquama, exposure duration, and their interactions all had significant effects on gamete quality, fertilization, and embryo development. The motility and swimming velocity of spermatozoa, egg viability, fertilization, and embryo development rate were significantly reduced in all concentrations, with a cell density of 10 cells mL−1 determined to be the critical density of H. circularisquama for deleterious effects. This is the first evidence of inimical effects of an HAB species on bivalve spermatozoa upon direct exposure. Further field and laboratory studies are required to investigate the potential effects of H. circularisquama blooms on the reproduction and recruitment of Japanese pearl oysters and other bivalves.

In a laboratory study, we investigated the clearance rates (CRs), respiration rates (RRs), total hemocyte count (THC), pathological alterations, and mucocyte densities in the gills of the short-neck clam Ruditapes philippinarum when exposed to the toxic dinoflagellate Heterocapsa circularisquama. Within 2 h of exposure to H. circularisquama at a concentration of 5–103 cells/mL, both the CRs and the RRs were significantly decreased by 43–52% compared with the control CRs and 43–93% compared with the control RRs, respectively (ANOVA, Newman-Keuls, P < 0.05). Clams exposed to H. circularisquama at 103 cells/mL for 96 h showed a set of defensive and degenerative pathologies that were absent in control clams. Within 3 h, the gills exhibited cilia matting, followed within 24 h by heavy hemocytic infiltration in the connective tissue of plicae, distortion of filaments, hyperplasia, and fusion of adjacent filaments with matted cilia. Within 48 h, the gill plicae became contracted and showed multifocal ep...

We examined the feeding behavior and energy consumption of Octopus ocellatus preying on short-neck clams under experimental conditions. Observed feeding patterns of individual octopuses of 7.0–52.1 g in wet weight were classified as “pulling”, “pulling–drilling–pulling” or variations of pulling and drilling. Octopuses used pulling for clams with shell lengths (SL) of 0.9–2.9 cm and a combination of pulling and drilling for clams with SL of 0.8–3.6 cm. Pulling alone resulted in a mean time for opening a clam (handling time) of 3.1 min and energy consumption of 8.0 μgO 2 g − 1 . Pulling plus drilling involved longer handling times. Prior to drilling, octopuses showed one of two pulling strategies, “low-energy pulling” or “high-energy pulling”. The latter was more similar to the type of pulling behavior seen when drilling was not used. The high-energy strategy resulted in a mean handling time of 55.4 min and an energy cost of 78.9 μgO 2 g − 1 . Low-energy pulling plus drilling resulted in a shorter handling time (29.5 min) and a lower energy cost (26.4 μgO 2 g − 1 ). We calculated the profitability (yield of energy gain per unit handling time) and energy efficiency (yield of energy gain per unit energy cost) of these feeding strategies. Pulling alone was the most efficient method in terms of profitability, whereas low-energy pulling plus drilling was the most efficient in terms of energy expenditure for young octopuses eating clams.

The effects of the toxic dinoflagellate Heterocapsa circularisquama on the activity rate, development rate, prevalence of damage, and survival rate of trochophore and D-shaped larvae of the pearl oyster Pinctada fucata martensii were studied in relation to H. circularisquama cell densities and exposure duration. In addition, larvae were regularly processed via scanning electron microscopy to investigate morphological damage. The activity rate of both larval stages was significantly decreased after 3–6 h of exposure to H. circularisquama at densities ranging from 100 to 2 × 104 cells/mL. The prevalence of damage was significantly high after 3–6 h of exposure to H. circularisquama at densities of 100 to 2 × 104 cells/mL and 5 × 103 to 2 × 104 cells/mL for trochophores and D-shaped larvae, respectively. Cytoplasmic discharge, mass mucus production, irregular shape, delayed or inhibited mineralization of the shell, mantle protrusion, the appearance of abnormal masses in the velum, and the exfoliation...

Mortality of the short-neck clam Ruditapes philippinarum exposed to the toxic dinoflagellate Heterocapsa circularisquama was studied under controlled conditions to clarify the mechanisms of recurrent mass deaths of clams occurring in western Japanese coastal areas. One-week mortality tests, involving three water temperatures, six H. circularisquama concentrations, and two clam body sizes, showed a significant increase in mortality with increasing temperature, H. circularisquama concentration, exposure duration, and body size (ANOVA, P

The effects of the toxic dinoflagellate Heterocapsa circularisquama on the valve movements of adult Ruditapes philippinarum (Bivalvia, Veneridae) were investigated. The valve movement behaviour was continuously measured using a non invasive Hall element sensor-automated data acquisition system. Individual clams acclimated to the experimental conditions were subjected to different food regimes over a 96 h period: (1) unfed in filtered seawater for the first 24 h, (2) batch-fed1, in which clams were given one ration of non-toxic Isochrysis galbana at the beginning of the second 24 h, (3) batch-fed2, in which clams were given a mixture of I. galbana and H. circularisquama (5 × 10 4 cells ml − 1 ) for the third 24 h, and (4) starved for 24 h in filtered seawater after exposure to the toxic alga. For each food regime, the valve activity was analyzed by quantifying: (1) the duration of valve opening (DVO, %), (2) the amplitude of valve opening (AVO, mm), and (3) the frequency of valve adductions (FVA, adductions h − 1 ). The real-time monitoring of the clams' valve movements proved that R. philippinarum detects H. circularisquama in the mixture of food at as low as 0.5 cells ml − 1 . Typically, the addition of the toxic alga induced a short and incomplete valve closure reaction. A strong correlation between the closure reaction time and H. circularisquama concentration was found ( R 2 = 0.91), the closure reaction occurring faster with increasing concentrations. Reopening of the valves was characterized by a significant increase in the FVA starting from 5 cells ml − 1 (ANOVA, P − 1 (Wilcoxon, P − 1 (ANOVA, LSD, P P Our results indicate that the Manila clam is highly sensitive to the toxic H. circularisquama and detects efficiently its presence at extremely low concentrations, in mixture of food, and in a time- and concentration-dependent manner. The Manila clam and the valve movement monitor could thus be used as an early biological warning system to monitor the outbreaks of this toxic alga along marine coastal areas and in bivalve farms. However, field experiments are required to provide further evidence for the use of this monitor in natural environment where several factors may interfere with the valve movement behaviour.

In octopus, growth trajectories have implications for survivorship, adult size and fecun- dity. Many species exhibit a 2-phase growth pattern starting with rapid exponential growth before shifting to a slower (commonly power) growth rate. Based on the concept that energy conservation enforces this threshold, we developed a temperature-dependent model which incorporates the energy balance between food intake and expenditure in growth and metabolism. We employed the model to investigate growth patterns occurring at different temperatures for 2 octopus species, Octo- pus ocellatus and O. pallidus. Model projections were consistent with laboratory data and suggest that increases in temperature as small as 1°C could have a significant influence on cephalopod growth, affecting the threshold body mass by up to 15.5% and the body mass at 100 d by up to 62.6%. Sensitivity analyses suggest that threshold size is more sensitive than threshold age to any given change in parameter values, and that metabolic rate has the greatest influence on the growth thresh- old. This model provides a basis for predicting individual growth trajectories and consequential pop- ulation structure of natural octopus populations. This type of analysis also has the potential to predict optimum conditions for a species and could be a powerful tool for predicting how climate change might affect species distribution as well as population structure and abundance.

The cephalopod receptor of particle motion was identified. In a previous study, it was suggested that statocysts served this function, but there was no direct supporting evidence, and epidermal hair cells had not been conclusively ruled out. Experiments on Octopus ocellatus were conducted using respiratory activity as an indicator of sound perception. Intact animals clearly responded to 141-Hz particle motion at particle accelerations below 1.3 ¥ 10 -3 m/s 2 , and the mean perception threshold at this frequency was approximately 6.0 ¥ 10 -4 m/s 2 . Specimens in which the statoliths had been surgically removed did not show any response for accelerations up to 3.9 ¥ 10 -3 m/s 2 at 141 Hz, which was approximately 16 dB greater than the mean perception threshold at this frequency. Specimens that had undergone a control operation in which the statoliths remained intact showed positive responses at 2.8 ¥ 10 -3 m/s 2 for the same frequency stimulus. This indicates that the statocyst, which is morphologically similar to the inner ear system in fish, is responsible for the observed responses to particle motion in O. ocellatus. This is the first direct evidence that cephalo- pods detect kinetic sound components using statocysts.

Oxygen consumption and ammonia excretion rates of three abalone species, Haliotis discus discus, H. gigantea, H. madaka and their hybrids were measured at 20 °C by incubating for 3 h under dark and light (N = 9–13 for each species and hybrid). Animals were fasted before and during the experiment and measurements were made first under dark followed by light on the next day. The rates increased with the increase in body weight and were higher under light than dark. On average, H. discus discus had higher oxygen consumption (DD; dark = 0.039, light = 0.04 ml/g/h) than H. gigantea (G; D = 0.033, L = 0.036) and H. madaka (M; D = 0.034, L = 0.035); the hybrids had varied patterns with respect to their parental species [DD × M (D = 0.032, L = 0.038); M × DD (D = 0.03, L = 0.038); G × DD (D = 0.035, L = 0.04) and DD × G (D = 0.03, L = 0.034), mother first]. M (0.261, 0.298 μmol/g/h) had the highest ammonia excretion rate while G (0.162; 0.264) and DD (0.229; 0.232) had the lowest under dark and light, respectively. The hybrids had varied patterns in comparison with their parents (DD × M = 0.247, 0.32;M × DD= 0.177, 0.28; DD × G = 0.249, 0.364 and G × DD = 0.116, 0.155). The O/N ratios under both conditions in all species and hybrids indicated that they had carbohydrate dominated metabolism. Results demonstrated physiological variability among the species and hybrids indicating necessity of different strategies for their management and aquaculture.

In this study, we prove that the mass die-offs occurring in conjunction with red adductor disease in pearl oysters are caused by an infection. Our research also reveals the influence of water temperature on the onset of the disease and its progress, and we propose measures that are effective in mitigating harm from the disease. To analyze the effect of water temperature on the onset and progress of the disease, laboratory infection experiments were performed over a range of water temperatures (13 °C–31 °C). The experiments showed that disease onset and progress were highly dependent on water temperature. No outbreak was observed at temperatures below 16 °C, while the adductors of experimentally infected oysters turned red in a short time at temperatures between 19 °C and 31 °C. Our research showed that the cumulative water temperature value for days when water temperature was above 19 °C (AT 19 = Σ( T i ), T i > 19) is an effective numerical indicator for the onset of red adductor disease. We also found that when pearl oysters already infected with red adductor disease at a high rate are kept at water temperatures below 16 °C in the winter for a certain minimum period of time, clinical signs regress, and that this treatment is also effective in delaying relapses of the disease with rising water temperatures in the spring. We determined a numerical indicator for finding the low-temperature index that is most effective in delaying disease onset. This is the low-temperature burden based on the number of days with water temperatures of 16 °C or below and those days' temperatures (LTI 16 = Σ(16 − T i ), T i 16 value, the greater the effectiveness in suppressing the onset of red adductor disease. Hence, appropriate low-water-temperature management in the winter helps prevent the relapse and progress of the disease in the spring and beyond when water temperature rises. Results of this research strongly suggest that the LTI 16 value in wintering fisheries and the cumulative water temperature of the days in spring and beyond when the water temperature is above 19 °C (AT 19 ) make it possible to predict when red adductor disease will break out in pearl oysters, and therefore help efforts to mitigate damage from relapses.

Estimated age, hatching date and sexual maturation were investigated for the loliginid squids Loligo duvauceli and L. chinensis, the commercially important neritic species in the Andaman Sea of Thailand. The specimens caught by commercial bottom-trawl in Phang-nga Bay and southern area off Phuket Island were obtained monthly from Phuket fish landing between April and August 2005. Age was estimated based on the counting of statolith increments from a total of 329 individuals of L. duvauceli (ML ranges from 41 to 224 mm) and 116 individuals of L. chinensis (ML ranges from 42 to 186 mm). The estimated age ranged from 41 to 161 and 67 to 158 days old for L. duvauceli and L. chinensis, respectively. The length–weight relationships differed among sexes and the sexual dimorphism was expressed as mature females having a greater body weight than males of the same length. Males showed a wide range of maturity size possibly because of the seasonal change in size at maturity. The results of the back calculation for the hatching date of individuals fell between November 2004 and June 2005 which suggests that L. duvauceli and L. chinensis hatch continuously and recruit to the fishing ground for a relatively short period with a shorter lifespan than expected.

The adoption of an upright posture in abalones is essential to enable them to use their foot, and hence to be able to move and seek shelter, as well as to avoid exposure of soft parts and possible predation. In mass restocking programs for abalone, juveniles are released by divers near the seabed, but without control over their posture when they reach the bottom. Thus, the time to recover the upright posture is an important consideration in abalone restocking programs as the quicker they assume this posture the higher the likelihood of survival. This study reports significant differences in the speed of recovering the upright posture between juveniles of the abalones Haliotis discus discus, H. gigantea and H. madaka and between tests conducted under stagnant and flowing water conditions. Longer times were required for recovery in all species in stagnant than flowing water. On average, juveniles of H. discus discus (17.16 and 10.43 s) and H. gigantea (22.54 and 11.89 s) recovered faster than those of H. madaka (161.13 and 49.02 s) under stagnant and flowing water conditions respectively. These results suggest that different species require different levels of care and that water flow or current at the time of release may affect post-release survival.

Bioaccumulation of cadmium (Cd) by oval squid, Sepioteuthis lessoniana, the relative contributions of Cd in water and food to bioaccumulation, and the distribution of cadmium among organs were studied. In a bioconcentration test, oval squid was exposed to 0.2 mg Cd/l seawater for 14 days, then was reared in Cd-free seawater for 14 days. After the exposure and elimination periods, the Cd concentrations in the whole body were 3.10 ± 0.18 and 1.90 ± 0.05 μg/g wet weight (ww), respectively. The bioconcentration factor (BCF) was 15.5 after exposure for 14 days. The liver exhibited the highest Cd concentration ( 49.3 μg / g ww), and had the highest proportion of organ Cd content (42.8%) to whole-body burden at the end of the exposure period. In a biomagnification test, oval squid was exposed to 0.12 mg Cd/l seawater for 15 days and was simultaneously fed mummichog, Fundulus heteroclitus, that were simultaneously exposed to the same Cd concentration. For a further eight days the squid was kept in Cd-free seawater and was fed mummichog that were also kept in Cd-free seawater. After the exposure and elimination periods, the Cd concentrations in the whole body were 4.18 ± 0.69 and 2.80 ± 0.44 μg/g ww, respectively. The concentration of whole-body Cd in the oval squid that was derived from water was estimated at 1.86 μg / g ww after calculation of a BCF from the bioconcentration test. The contribution of Cd derived from water to the whole-body Cd burden appeared similar to that of the Cd derived from food. In the biomagnification test, the liver Cd concentration ( 58.8 μg / g ww) and its proportion of the whole-body Cd concentration (40.6%) were the highest among those of all the organs after exposure for 15 days, regardless of the uptake route. Because wild squid seem to be exposed to much lower Cd concentrations in water and are subjected to similar Cd concentrations in their prey, compared with squid of the present study, the main Cd source for squid would appear to be dietary.

Oxygen consumption rates of the young oval squid Sepioteuthis lessoniana (0.033-27.54g BW) reared from eggs and those captured from wild population were investigated on an individual basis by both water bottle and flow-through methods. Oxygen consumption rate (R in ml oxygen/ind./h) increased linearly with increase in BW (W in g) expressed as R=aWb. For squid smaller than about 2g BW, there was not a significant difference in the values of the exponent b and the coef-ficient a between R's measured respectively by water bottle and flow-through methods. The R was not significantly different from each other between squid reared from eggs and captured from the sea. The exponent b and coefficient a for young squids ranged from 0.741 to 0.944 and from 0.697 to 0.966, respectively. There was a tendency for the exponent to change with growth, such as b for the squids less than 0.5g BW being 0.944, while for the squids over 0.5g was 0.813, respec-tively. This suggests that R in very early life stage tends to increase more rapidly with growth than in older stages.

Oxygen consumption and ammonia excretion were investigated during a long term starvation in an abalone Sulculus diversicolor aquatilis. Abalone used in the present study were 14 specimens ranged from 0.14 to 17.10g in wet weight of soft body (W, g). They were kept starved in a closed aquarium at 24°C. Oxygen consumption and ammonia excretion rates were measured by individual in a closed bottle with several days interval until death by starvation. Oxygen consumption rate (R, μl O2/ind./h) of the abalone increased linearly with an increase in W and decreased with the period of starvation (T, days); R=92.019 W0.914 0.987T. Ammonia excretion rate (E, μmol/ind./h) increased linearly with an increase in W. Until the 17th day from commencement of the experiment E increased with an extention in T, however, it decreased with extention of T after the 17th day and they are expressed as E=0.369 W0.78 T0.413 and E=2.151 W1.012 T-0.454, respectively. The O/N ratios for abalones just after feeding were relatively high and indicate an apparent dependence upon carbohydrates in the feeding period. After about 2 weeks starvation, the O/N ratios showed relatively low and constant, followed by standard protein energy utilization thereafter.

Food consumption, food conversion and growth rates of the oval squid Sepioteuthis lessoniana were measured on an individaul basis fed on three kinds of live organisms, namely, fish, mysid and squid hatchling. Squid used in the present study were all reared from egg to the experimental size, ranged from 0.06 to 42.1g in wet body weight in an indoor aquarium. The daily food con-sumption rate ranged from 0 to 72% of body weight and depended upon the growth stage of the squid. Food conversion rate on a wet weight basis ranged from 15.4 to 43.8% and the rate was largely independent of the size of squid. Growth rate of the oval squid decreased with increasing size. The daily growth rate in body weight of hatchling reached 12.9% and that of the squid grown up to 30-50g in body weight ranged from 1.8 to 4.1%. The growth was dependent upon quality of food. Squid fed on squid hatchlings showed the highest growth rate followed by those fed on fish. The daily growth rate increased with each increment of the daily food consumption rate.