Masculinization of Convict Cichlid (<Emphasis Type="Italic">Cichlasoma nigrofasciatum</Emphasis>) by immersion in <Emphasis Type="Italic">Tribulus terrestris</Emphasis> extract

We have examined the effects of Tribulus terrestris (TT), a non-toxic herb, on sex reversal in the Convict Cichlid Cichlasoma nigrofasciatum with the aim of introducing a new environmentally friendly method for masculinization in C. nigrofasciatum. TT is a natural plant product that elevates the testosterone levels in humans and animals. Different concentrations (0.0, 0.10, 0.20 and 0.30 g/l) of TT extract were tested for their effect on sex reversal in C. nigrofasciatum by immersing newly hatched offspring once weekly for 2 months in TT extract. Of the dosages used in the present study, 0.30 g/l TT was the most effective in terms of masculinization, resulting in a maximum male ratio of 87.23% (P < 0.001). Sex ratios of 79 and 85% at 0.10 and 0.20 g/l TT, respectively, were also significantly different from the expected 1:1 ratio (P < 0.001). Histological examinations revealed that the testes of fish treated with TT extract contained all stages of spermatogenesis, clearly demonstrating that the administration of TT extract to C. nigrofasciatum stimulated spermatogenesis. Total survival rates in all treatments and the control were uniformly high, ranging from 88.57 to 90% (P > 0.05). We concluded that TT had no negative effect on the survival rate of C. nigrofasciatum. In addition, all groups of TT-treated fish exhibited successful growth acceleration compared to the control group, but only the 0.30 g/l TT treatment significantly improved the growth rate of C. nigrofasciatum. (P < 0.01). Sex reversal in C. nigrofasciatum demonstrated that TT-treated 0-day-old larvae showed successful sex reversal, spermatogenesis and a better growth rate than untreated progenies.     

Sterols from bivalves <Emphasis Type="Italic">Calyptogena soyoae</Emphasis> and <Emphasis Type="Italic">Bathymodiolus septemdierum</Emphasis> living in deep sea

From the two species of bivalves, Calyptogena soyoae around a cold seep and Bathymodiolus septemdierum near hydrothermal vents in the sea, sterols were isolated using high-pressure liquid chromatography analysis of the lipid fraction guided by characteristic ¹H NMR signals of the sterol skeleton. The minor sterol composition of C. soyoae included 24-methylenecycloartanal, cycloeucalenol, and obutusifoliol, which are known phytosterols. From B. Septemdierum, lathosterol and cholesterol as main sterols together with more diverse sterols were obtained. The difference between these species and their sterol contents is most likely because of feeding modes and metabolism of nutrients from their habitat.     

Growth of wild pearl oysters <Emphasis Type="Italic">Pinctada fucata,Pinctada margaritifera</Emphasis> and <Emphasis Type="Italic">Pinctada sugillata</Emphasis> (Bivalvia: Pteriidae) in Taiwan

In order to understand growth features of pearl oysters in the genus Pinctada, i.e. Pinctada fucata, Pinctada margaritifera, and Pinctada sugillata in Taiwan, a total of 3062 wild individuals of these species from juvenile to adult were collected monthyly from March 2001 to April 2002 in Jukeng, Pingtung County, south-west Taiwan. Quantitative measurements of live oysters were conducted for shell height (SH), shell length (SL), shell width (SW), hinge length (HL), and wet weight (WW). Different cohorts were identified through multiple length frequency analysis on SH of P. fucata and P. margaritifera, and growth curves with seasonal variation were estimated for these species. Pinctada fucata in Taiwan had a different seasonal growth pattern from the Japanese population, but had similar growth rates during the high growth period. The growth rate of P. margaritifera in Taiwan was slower than in French Polynesia, the Solomon Islands, and the Red Sea. Comparisons of morphological growth features among the three species show large differences in the SW-related features. Pinctada fucata in Taiwan had larger SW than in Japan and Korea. The differences in growth rates and morphological features suggested that the wild Taiwanese oysters may retain genetically pristine characteristics, thus genetic conservation might be urgently needed.     

Suspension feeding and growth of ark shell <Emphasis Type="Italic">Anadara granosa</Emphasis>: comparison with ubiquitous species <Emphasis Type="Italic">Scapharca subcrenata</Emphasis>

The ark shell Anadara granosa is a species peculiar to the Ariake Sound. To determine why distribution of A. granosa in Japan is largely confined to this area, we examined feeding and growth of A. granosa as functions of environmental and biological variables. The results were compared with those of another ark shell Scapharca subcrenata, which is ubiquitous in Japan. Feeding experiments indicated that A. granosa is eurythermal and euryhaline, as is S. subcrenata, but is adapted to temperature slightly higher than S. subcrenata. Weight-specific clearance rate (CR) of A. granosa as a function of soft-body dry-weight (w) followed the power function of w (CR=2.7×w ^−0.37), with coefficient and exponent very close to those for S. subcrenata. Growth rate of A. granosa increased linearly with daily ration, similar to S. subcrenata. Thus, feeding and growth characteristics of A. granosa were comparable to those of S. subcrenata and no ‘peculiarities’ of the former were detected. Therefore, the factors that make A. granosa a species restricted to the sound are probably not directly related to feeding or growth characteristics.     

Morphological studies of peripheral blood cells of the Chinese sturgeon, <Emphasis Type="Italic">Acipenser sinensis</Emphasis>

The peripheral blood cells of one-year-old Chinese sturgeon (Acipenser sinensis) have been studied by light microscopy and transmission electron microscopy. The erythrocyte count was 84.86 × 10⁴ cell mm⁻³ in the peripheral blood of the fish and that of leukocytes was 2.24 × 10⁴ cell mm⁻³. The erythrocytes and four main types of leucocyte—thrombocytes, lymphocytes, granulocytes (including neutrophils and eosinophils), and monocytes, were identified in the peripheral blood. In addition to normal erythrocytes, reticulocytes and division of erythrocytes were observed. Thrombocytes were the most numerous among the leukocytes, and the number of neutrophils with lobated nuclei was larger than for other fish. The structures of the erythrocytes, lymphocytes, monocytes, granulocytes, and thrombocytes of the fish were studied. The erythrocytes were almost completely devoid of organelles, except for some mitochondria and granules. A large number of vacuoles and a few organelles were observed in cytoplasm of the monocytes. There were many microvilli on the membrane and pseudopodia-like cytoplasm bulge in the lymphocytes. The neutrophils were round or oval in shape with bilobed, trilobed, or multilobed nuclei whereas the eosinophils had big special granules, dark stained. There were many vesicles in some thrombocytes, which were related to its phagocytosis; some thrombocytes had almost no cytoplasm or organelles.     

Annual variation of potential predation impacts on larval and juvenile marbled sole <Emphasis Type="Italic">Pseudopleuronectes yokohamae</Emphasis> by sand shrimp <Emphasis Type="Italic">Crangon uritai</Emphasis> in Hakodate Bay,Hokkaido

An investigation was conducted to evaluate the annual variation in potential predation impact (PPI) on larval and juvenile Pseudopleuronectes yokohamae by Crangon uritai in Hakodate Bay using predator-prey size relationships. Laboratory experiments were designed to estimate the favorable prey size of C. uritai through back-calculation of body length (BL) of P. yokohamae from sagittal otolith diameter observed in the stomachs of C. uritai. The most favorable prey-predator size ratio (BL of P. yokohamae-total length (TL) of C. uritai) class was 0.15–0.19, and ranged from 0.12–0.31. There was a significant positive correlation between the BL increase of P. yokohamae and the bottom water temperature in the field, such that BLs stagnated during the cold year of 1999 from April to June, and increased during the warm year of 2002. In contrast, no significant correlation was found between the TL increase of C. uritai and the bottom water temperature. Moreover, there were no significant differences in the mean TL of C. uritai between months (April–June) or years (1998–2002). Variation of PPI depended on the relationships between the growth rates of P. yokohamae and bottom water temperature. Therefore, the warm year of 2002 resulted in higher potential predation impact (PPI≥20), and it was at least 20 days shorter than that of the cold year of 1999. These results suggest that increased bottom water temperature in the nursery area was one of the most important factors for cumulative predation loss.     

Tench (<Emphasis Type="Italic">Tinca tinca</Emphasis> L.) larvae rearing under controlled conditions: density and basic supply of <Emphasis Type="Italic">Artemia</Emphasis> nauplii as the sole food

After artificial reproduction of tench, larvae must be maintained indoors, and studies on rearing conditions are needed, focussing on the reduction of labour and costs. Three experiments on larvae (5th day post-hatch) were conducted for 25 days using Artemia nauplii as the sole food in order to determine basic feeding and density conditions during the first rearing period. Tench were maintained in 25 l fibreglass tanks, supplied with an artesian water flow throughout of 0.2 l min⁻¹. Water temperature was 22.5 ± 1°C, and the photoperiod was natural. Larvae fed on a restricted amount of nauplii reached high survival rates, even with the minimum of 50 nauplii larva⁻¹ day⁻¹. This amount of food may be sufficient at least for the first 25 days of exogenous feeding if fast growth is not the priority, and high densities can be maintained with good survival rates (over 90% up to 160 larvae l⁻¹ and 77% with 320 larvae l⁻¹). When food was supplied in excess once a day, high survival rates were achieved (91–97%), without differences among the densities tested. Animals at a density of 100 l⁻¹ reached the highest length (15.57 mm) and individual weight (46.8 mg). This growth is greater than those reported in studies feeding several times a day. It could be deduced that, while live food remains available for tench, it is not necessary to feed so frequently. Considering the relationship among the initial number of animals, final survival and growth and ration supplied, the new data reported here are useful to establish suitable stocking densities under both culture and experimental conditions.     

Effects of dietary thiamin on the physiological status of the grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

This study was conducted to evaluate the effects of dietary thiamin on the physiological status of the juvenile grouper, Epinephelus coioides. Graded levels of thiamin (0.08, 0.50, 2.12, 3.15, 4.63, 12.37 mg thiamin kg⁻¹ diet) were fed to grouper juveniles (mean weight: 16.97 ± 0.14 g) for 10 weeks. Although fish fed the thiamin-deficient (TD) diet showed no obvious symptoms of thiamin deficiency or increased mortality, those fed the lowest doses of thiamin (0.08 and 0.50 mg thiamin kg⁻¹ diet) had significantly decreased transketolase activity in the liver. In addition, the level of liver thiobarbituric acid reactive substances in fish fed the TD diet was 33–67% higher than that in fish with the thiamin-supplemented diet. There were no significant differences in superoxide dismutase activity between the different groups of fish.     

Genetic variation in mitochondrial genes and intergenic spacer region in harmful algae <Emphasis Type="Italic">Chattonella</Emphasis> species

In this study, nuclear ribosomal RNA gene internal transcribed spacer regions and the cox2-cox1 fragment of the mitochondrial (mt) genome were sequenced in 24 strains of Chattonella spp. Variability in both regions showed that the mt genome sequences of Chattonella spp. have a higher evolutionary rate than the nuclear rRNA gene sequences. A maximum likelihood tree based on the mt sequence grouped the Japanese Chattonella strains into two groups (Groups A and B), although no correlation was observed amongst the phylogenetic groups, their morphologies, or the isolated areas. Groups A and B were clearly identified by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using Fokl, without the need for a sequencing experiment. The PCR-RFLP assay revealed that Chattonella cells obtained from sea water in Oita, Japan, in 2004 and 2005 belonged to Group B. This is the first report showing the genetic variation in Chattonella spp. using a PCR-RFLP identification protocol.     

Effects of reactive radicals and heat on <Emphasis Type="Italic">trans</Emphasis>-isomerization of eicosapentaenoic acid

Trans geometric isomers of eicosapentaenoic acid (TEPA) have been found as minor components in human platelets. However, there is little information on the mechanism of trans-isomerization of eicosapentaenoic acid (EPA) in vitro and in vivo. The effects of reactive radicals and heat on trans-isomerization of EPA were examined. Trans-isomerization occurred when EPA ethyl ester reacted with nitrogen dioxide radical (NO₂) but not with 2,2′-azobis(2,4-dimethylvaleronitrile). TEPA was also produced from EPA ethyl ester heated at 200°C for 60 h. No TEPA, however, was detected in sardines Sardinops melanostictus after boiling, roasting, or microwave heating. These results suggest that EPA is trans-isomerized by NO₂ in vivo while trans-isomerization of EPA does not occur during conventional cooking.     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

Dietary amino acid <Emphasis Type="SmallCaps">l</Emphasis>-threonine requirement of fingerling Indian catfish, <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis> (Bloch) estimated by growth and biochemical parameters

An eight-week feeding experiment was conducted to quantify the dietary threonine requirement of young catfish, Heteropneustes fossilis (9.20 ± 0.85 cm, 3.60 ± 0.45 g) using isonitrogenous and isoenergetic diets [40% crude protein (CP); 4.28 kcal g/100 g, gross energy (GE)] containing casein, gelatin and l-crystalline amino acids. Six dietary treatments supplemented with graded levels of l-threonine (0.50, 0.75, 1.00, 1.25, 1.50 and 1.75 g per 100 g, dry diet), in gradations of 0.25 g per 100 g dry diet were formulated. Fish were randomly stocked, in triplicate groups, in 55-l indoor polyvinyl flow-through circular tanks and fed experimental diets at 4% of their body weight divided over two equal feedings at 08:00 and 16:00 hours. Feeding schedule and ration size were worked out prior to the start of the feeding trial. Live weight gain (263%), feed conversion ratio (FCR) (1.35) and protein efficiency ratio (PER) (1.85) were significantly higher (P < 0.05) in fish fed a diet containing 1.25% dietary threonine. However, second-degree polynomial regression analysis of live weight gain, FCR, PER and body protein deposition data indicated the dietary threonine requirement to be 1.37, 1.26, 1.23 and 1.24 g per 100 g of dry diet, respectively. Whole-body moisture decreased significantly (P < 0.05) with the increase of dietary concentration up to 1.25%. A significantly (P < 0.05) higher protein content was evident in fish fed a diet containing 1.25% threonine. Body fat increased significantly (P < 0.05) with the increase of dietary concentration and was found to be highest at a 1.75% threonine concentration. A significantly (P < 0.05) higher ash content was reported at the 0.50 and 0.75% threonine levels. Body protein deposition was also found to be significantly (P < 0.05) higher at the 1.25% threonine level, followed by the 1.50% threonine level. Based on these results, it is recommended that the diet for fingerling H. fossilis should contain threonine at a level of 1.27 g per 100 g of dry diet, corresponding to 3.17 g per 100 g of dietary protein for optimum growth and efficient feed utilization. No mortality was observed during the experiment.     

Scale regeneration and calcification in goldfish <Emphasis Type="Italic">Carassius auratus</Emphasis>: quantitative and morphological processes

The time-course changes in calcium and phosphorus contents, dry weight, and area during scale regeneration in the goldfish, Carassius auratus L. were quantified. Histological observations were then conducted to understand the mutual relationship between the quantitative and morphological processes of scale regeneration. The quantitative study revealed that regenerating scales grow most rapidly in area during the first 5 days of regeneration. The gradual decrease in the area growth rate coupled with the continuous linear weight growth over the period of 5–28 days suggests a shift in growth priority from area growth to the apposition, of the basal plate. Calcium and phosphorus deposition proceeded almost linearly during scale regeneration. Calcification of the bony layer preceded that of the basal plate and, after 14 days of regeneration, calcification of the basal plate started and gradually progressed. On day 28, recovery of calcium and phosphorus contents in the regenerating scales were approximatley 72% of ontogenetic scales, which is lower than the rates of area and weight regeneration (104 and 85%, respectively). Late initiation and slow progress of calcification in the basal plate is suggested to be responsible for the slow regeneration in calcium and phosphorus contents.     

Feeding habits of stone flounder <Emphasis Type="Italic">Platichthys bicoloratus</Emphasis> larvae in Mutsu Bay,Japan

To clarify the feeding strategy of pelagic larvae of stone flounder in Mutsu Bay, the dietary composition and pray size was investigated from February to April during 1989–1999. Diets were compared with the numerical and volumetric compositions and frequency of occurrence of each prey species. Mensuration formulae were applied to estimate individual prey volume in the diet, while the length of planktonic species was measured from net and water samples. Prey shapes were assumed as sphere, cylinder, ellipsoid, pyramid, two elliptical cones, or a combination of ellipsoid and cylinder. Prey-size range increased as the larvae grew. Preflexion larvae fed mainly on copepod nauplii. Flexion and postflexion larvae ingested primarily appendicularians, with a suggestion that these larvae might depend on some parts of the microbial food web. Low frequencies of flexion and postflexion larvae with empty guts (1.7 and 1.4%, respectively) might be derived from feeding on slow-swimming appendicularians. From a size comparison between ‘house’-like organ length and trunk length of the appendicularian Oikopleura sp., almost all house-like organs with trunks in the larval diet were nonexpanded ‘house rudiments’, not expanded ‘houses’. Thus, stone flounder larvae may not chew the houses, but swallow the house rudiments with trunks.     

<Emphasis Type="Italic"> Xenostrobus securis </Emphasis>(Lamarck, 1819) (Mollusca: Bivalvia): first report of an introduced species in Galician waters

The presence of the non-indigenous species, the black-pygmy mussel Xenostrobus securis, is reported here for the first time in an intense shellfish farming area off Galicia (NW Spain). Very high concentrations of this mytilid bivalve have colonized estuarine waters located at the inner part of the Ria de Vigo. The invasive role of X. securis is discussed in the context of the wide ecological tolerance of the species and the recent finding of settlements of this species on numerous colonies of the economically-important blue mussel Mytilus galloprovincialis. The mode of introduction of the black-pygmy mussel is also discussed in relation to human management activities.     

Fortification of <Emphasis Type="Italic">niboshi</Emphasis> with histidine prepared from <Emphasis Type="Italic">katsuobushi</Emphasis> protein digest

ABSTRACT:   Free and total histidine contents in the muscle of various fishes and invertebrates caught in the Seto Inland Sea were estimated. These contents were higher in the muscles from skipjack, yellowfin tuna, yellowtail and mackerel than in those from lizard fish, sea bream and hairtail. Invertebrate muscles, except squid, contained low free and total histidine. Boiled and dried anchovy ( niboshi ) contained lower free histidine than that of dried anchovy (suboshi). To prepare free histidine-rich extracts, the waste residue of soup stock from smoke-dried and shaved skipjack ( katsuobushi ) was digested with proteases. The extract of the enzyme digest contained high free histidine and was used to fortify niboshi with free histidine. If taken, the fortified niboshi may suppress food intake, and prevent obesity.     

Improvement of larval rearing technique for mass seed production of snow crab <Emphasis Type="Italic">Chionoecetes opilio</Emphasis>

Larval rearing experiments were conducted to examine the potential for mass seed production of the snow crab Chionoecetes opilio using a total of eight 20-kL tanks. Tanks were equipped with agitators, which move the water using a rectangular blade to prevent the zoeas sinking to the bottom of the tank. Larval rearing water was treated with sodium nifurstyrenate once a week to reduce the chance of larval infection by pathogenic bacteria. Zo eas were fed with rotifers and Artemia nauplii. A total of 122 830 megalops and 16 660 first-stage crabs were produced. Thus, the potential for mass seed production of snow crab was determined. Survival rates up to the megalopal stage were high in tanks with a feeding regime that fed rotifers to larvae through an entire zoeal stage. This study also describes the fatty acid composition of snow crab larvae. It revealed that the first zoeas had a high DHA content and DHA/EPA ratio, but these values significantly decreased in the second stage zoeas and megalops. Improving the DHA content and/or DHA/EPA ratio of larvae should be important in studies on mass seed production technology of the snow crab.     

Partial characterization of the digestive enzymes of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis> under culture conditions

The digestive enzyme activities of Pacific bluefin tuna Thunnus orientalis were evaluated for specific activity and characterized for pH and temperature optima in crude extracts of stomach, caecal mass, and proximal, middle and distal intestine. A higher level of alkaline proteolytic activity was detected in the caecal mass than in the proximal intestine. Total alkaline proteases, trypsin, chymotrypsin and leucine aminopeptidase (LAP) were tested. The temperature and pH analyses showed that proteolytic activity as well as lipase were maximal in the alkaline range, with a maximum at pH 9.0 and at temperatures between 35 and 60°C, except for the pepsin, which showed maximum activity at the same temperatures but in the acid range (pH 3.0). The α-amylase activity showed a broader range in activity, both for pH and temperature, with higher activity over the alkaline pH values and higher temperature. The lipase activity seems to be nondependent on bile salts under our assay conditions, resulting in a significant activity reduction in the presence of bile salts. This knowledge will allow the development of a gastrointestinal model (everted intestine) where food or feed will be hydrolysed with the fish’s own enzymes, a project that is being undertaken in our laboratory as a contribution to the development of novel diets for tuna fish.     

Research on the jellyfish (<Emphasis Type="Italic">Rhopilema esculentum</Emphasis> Kishinouye) and associated aquaculture techniques in China: current status

The jellyfish is a popular seafood in eastern and southern Asian nations where there is a high market demand that stimulates large-scale jellyfish production. Due to its economic importance in China, many biological studies have focused on the jellyfish Rhopilema esculentum Kishinouye in terms of the environmental impact of aquaculture activities and culture techniques. In recent years, the commercial aquaculture of R. esculentum has expanded greatly in China. This paper summarizes the current status of research in China on R. esculentum and associated aquacultural practices, focusing on a number of related technological approaches.