Frequent occurrence of the tetrodotoxin-bearing horseshoe crab <Emphasis Type="Italic">Carcinoscorpius rotundicauda</Emphasis> in Vietnam

Tetrodotoxin (TTX) was detected by HPLC and LC-MS/MS in specimens of the horseshoe crab Carcinoscorpius rotundicauda collected from Tan Hai village, Vung Tau province, Vietnam, where cases of poisoning due to the consumption of horseshoe crab have frequently been reported. The soft tissue from 10 out of the 12 specimens analyzed showed considerable levels of TTX, making them unsafe for human consumption (81.2 ± 50.3 MU/g). Paralytic shellfish poisoning (PSP) toxins were also detected in all of the specimens, but the levels were low. These findings show that the frequency of occurrence of C. rotundicauda specimens with a high level of TTX is significantly high, and that the TTX is responsible for the food poisonings caused by the consumption of C. rotundicauda in this area.     

Accumulation of paralytic shellfish poisoning toxin by the swimming crab <Emphasis Type="Italic">Charybdis japonica</Emphasis> in Kure Bay,Hiroshima Prefecture

The accumulation of paralytic shellfish poisoning (PSP) toxin in the crab Charybdis japonica was investigated in Kure Bay, when a bivalve Mytilus galloprovincialis, in the same water, showed toxicity caused by PSP toxin. In 2005, 17 specimens of the crab had PSP toxin over 4 MU/g in the hepatopancreas, and the highest toxicity was at 37.4 MU/g. Since the regulation limit of PSP for crab hepatopancreas was set in 2004, this is the first observation of regulatory level of PSP toxin in C. japonica. Unlike in the hepatopancreas of the crab, the hazardous level of the toxin was not detected in the muscle of the cephalothorax and the appendage. The toxin accumulation in the crab was also investigated by feeding toxic mussels to the crab. The crab retained the toxin mainly in the hepatopancreas, and the ratio of retention in the crab was from 12.9 to 24.6%. The toxin profiles, shown in the feeding experiments, suggest that the transformation of PSP toxin occurs in the crab because dcGTX2 and dcGTX3 was detected in all crab specimens despite the lack of these analogs in the mussels used as feed.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Reproductive biology of blue marlin <Emphasis Type="Italic">Makaira nigricans</Emphasis> around Yonaguni Island,southwestern Japan

We studied the spawning seasonality and gonadal development of blue marlin Makaira nigricans using specimens captured around Yonaguni Island in southwestern Japan between February 2003 and February 2006. The mean (±SD) lower jaw–fork length of females (234 ± 24 cm) was greater than that of males (191 ± 12 cm). The smallest mature female and male were 183 and 160 cm, respectively. Most of the 717 females had immature ovaries. However, in March and from May to September, the ovaries of 26 females contained oocytes with yolk globules, hydrated oocytes, or postovulatory follicles. Most males had testes with a large amount of spermatozoa throughout the year. The occurrence of mature blue marlin at Yonaguni Island suggests that spawning occurs here. The mean condition factors (fatness of the fish) of both sexes decreased from March to June or July, presumably as they expended energy to reproduce. We discuss our results in the context of migration theory for blue marlin in the western North Pacific Ocean.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

Reproductive season and female maturity size of coconut crab <Emphasis Type="Italic">Birgus latro</Emphasis> on Hatoma Island,southern Japan

Information about reproductive season, size at sexual maturity and size-dependent reproductive potentials is important for efficient resource management. Such information is necessary to determine seasonal closures and minimum legal size limits. To conserve resources of coconut crab Birgus latro, which are in danger of extinction due to excessive exploitation, their reproductive season, female size at functional maturity and relationship between female size and reproductive potentials were investigated. The reproductive season started in early June and ended late August, and females with higher reproductive potentials showed a tendency to spawn early in the reproductive season. The prohibition of exploiting the resources during the reproductive season, especially early in the season, would be appropriate for sustainability of high reproductive outputs. Fifty percent of investigated females matured functionally at 24.5-mm thoracic length (TL). However, the size at which all females reached functional maturity was 32.3 mm TL, and there was a wide variation in the size of functional maturity among individuals. We must determine minimum legal size by taking the variation into account to allow all individuals to mate at least once before they are caught, which may prevent serious depression in reproductive rate and genetic diversity.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Effects of soy sauce <Emphasis Type="Italic">koji</Emphasis> and lactic acid bacteria on the fermentation of fish sauce from freshwater silver carp <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>

The effects of soy sauce koji and the lactic acid bacterium, Tetragenococcus halophilus, were studied on the fermentation of fish sauce prepared from Chinese silver carp. The fish sauce prepared without koji and the lactic acid bacterium contained low levels of organic materials, total nitrogen, and organic acids. The use of koji was effective in increasing these qualitative parameters and further improved the amino acid score of the fish sauce. Addition of T. halophilus had an effect on lowering the pH value during the initial period of fermentation when the soy sauce koji was also supplemented. In contrast, T. halophilus-like bacteria were found to be predominant for all tanks fermented under the different starting conditions. Although it was not examined whether the T. halophilus-like bacteria observed after fermentation were the same as the starter-bacterium or not, it was suggested that T. halophilus plays an important role in the successful fermentation of silver carp fish sauce. Sensory evaluation conducted with Japanese and Chinese panelists also suggested the superiority of the use of koji for fermentation of silver carp fish sauce.     

A simple method to distinguish two commercially valuable eel species in Japan <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">A. anguilla</Emphasis> using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Circadian melatonin release <Emphasis Type="Italic">in vitro</Emphasis> by European sea bass pineal

Melatonin production by the fish pineal is under the control of photoperiod. In addition, in some fish species pineal melatonin production is self-sustained by an intrapineal circadian pacemaker, not in salmonids, though. The aim of this research was to test melatonin release in vitro by the pineal of sea bass held under LD 12:12 and DD conditions to determine the endogenous nature of melatonin rhythm. Circadian rhythmicity ( = 28.3 h) was found since rhythmic melatonin release persisted on DD.     

Prediction of timing of mating and egg extrusion in the coconut crab <Emphasis Type="Italic">Birgus latro</Emphasis> judged from female pleonal expansion

A temporal pattern of reproductive activities of biological resources is essential information for the implementation of specific seasonal closures. First we investigated the relationship between the female pleonal expansion and female reproductive condition (i.e., non-ovigerous, ovigerous, or with spermatophores) to clarify the morphological characteristics of female coconut crabs that were able to mate. Second, we mated females with males in captivity to examine the relationship between the degree of female pleonal expansion and mating success. The index of pleonal expansion (ratio of inter-tergite distances to thoracic length) was significantly different among female reproductive conditions; females with spermatophores just before egg extrusion had the most expanded pleon. In the mating trials, all females showing index values higher than 0.35 mated with males. These results indicate that the female pleonal expansion has a strong relation to reproductive activities of females and can be applied as a morphological criterion for selecting females being able to mate. Next, we investigated whether reproductive activities of female coconut crabs are related to the lunar cycle. The reproductive activities were synchronized with the lunar cycle, and most females mated and extruded eggs around the new moon phase. These results have considerable implications for resource management and aquaculture for this species.     

Comparative fishing trials for <Emphasis Type="Italic">Charybdis japonica</Emphasis> using collapsible box-shaped and dome-shaped pots

ABSTRACT:   Comparative fishing trials were undertaken with two commercial collapsible pots for the crab Charybdis japonica . One pot was box-shaped with two slit entrances at the ends, while the other was dome-shaped with two open funnel entrances. Pots were placed at the bottom of a pond that is connected to Kagoshima Bay. Fishing trials were carried out using a 1-day soaking time; the bait used was mackerel. The box-shaped pot had higher catches of crabs and non-target organisms, but the number of large crabs (carapace width >8 cm) was almost half as many as that caught with the dome-shaped pot. The catch of non-target species was also greatly reduced in the dome-shaped pot, and fish caught were mostly enmeshed in the netting. The reduction in catches of small crabs and non-target organisms was due to the nature of the dome-shaped pot's larger mesh size and open entrances, which allowed for escape. The dome-shaped pot seemed more efficient for harvesting commercial size crabs, while the box-shaped pot seemed better for removing all crab sizes.     

Circadian locomotor activity in Japanese sea catfish <Emphasis Type="Italic">Plotosus lineatus</Emphasis>

To find out whether or not the locomotor activity of the Japanese sea catfish, Plotosus lineatus, shows a circadian rhythmicity, the locomotor activity of this catfish was recorded in a laboratory. All of the catfish recorded (n = 20) showed a nocturnal locomotor activity rhythm under a light–dark (LD) cycle (LD 12:12). The locomotor activity started after the light was turned off, and declined during the period of darkness. In five of the six catfish we examined, locomotor activity shifted gradually over the course of 3–4 days in order to synchronize to the LD cycle after it was advanced by 6 h. Locomotor activity persisted within a constant darkness (DD) in all eight of the catfish. The average free-running period of locomotor activity under DD lasted for 24.2 ± 0.4 h (mean ± SD). Thus, the present results demonstrate that the Japanese sea catfish has an endogenous circadian oscillator entrained by an LD cycle.     

Cryopreservation diluents for spermatozoa of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis>

To develop a suitable cryopreservation diluent for spermatozoa of the endangered Sakhalin taimen Hucho perryi, all possible combinations of cryoprotectants (glycerol, dimethyl sulfoxide [DMSO], methanol) and extenders (fetal bovine serum [FBS], 300 mM glucose solution [GS], artificial seminal plasma for masu salmon) were examined by observing sperm motility 10 s after thawing. Spermatozoa cryopreserved with diluents such as mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS showed the highest motility. The maximal post-thaw motility was observed at 10% among all concentrations (0, 5, 10, 15 and 20%) of these three cryoprotectants. No significant difference among three diluents was observed in motility at 10 s. Mixtures of 10% glycerol plus 90% FBS, 10% DMSO plus 90% FBS, and 10% methanol plus 90% GS are suitable cryopreservation diluents for Sakhalin taimen spermatozoa.