Purification and characterization of a matrix shell protein from the shell of scallop <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   A new protein named MSP-SC (matrix shell protein from scallop) with a molecular weight of 14 kDa was isolated from the shell of a scallop, Patinopecten yessoensis , using gel filtration column chromatography, ion exchange column chromatography, and reverse phase C4 column chromatography. A comparison of the known protein sequences with the N-terminal sequence of MSP-SC showed that the protein sequence of MSP-SC was novel. Immunohistochemistry using a polyclonal antibody against MSP-SC showed that MSP-SC is expressed in the mantle pallial cell layer but not in the muscle tissue, and showed a punctate distribution along the horizontal calcified layer in the shell. The isolated MSP-SC inhibited the formation of calcium carbonate (CaCO₃) crystals in a dose-dependent manner. The CaCO₃ crystals grown in the presence of a lower concentration of MSP-SC were much larger and aggregated when compared with those formed in the absence of MSP-SC. In addition, the crystal had a radial and not cubical morphology. These results suggest that MSP-SC regulates the formation and the morphology of CaCO₃ crystals in the shell. Moreover, its ability to aggregate CaCO₃ crystals and its localization along the horizontal calcified layer in the shell suggest that MSP-SC may serve to connect the CaCO₃ layers in the scallop shell.     

Evaluation of genetic diversity of eight grouper species <Emphasis Type="Italic">Epinephelus</Emphasis> spp. based on microsatellite variations

ABSTRACT:   Genetic diversity among eight species of grouper, Epinephelus bleekeri , E. coioides , E. malabaricus , E. ongus , E. akaara , E. maculatus , E. merra and E. fuscoguttatus , was studied using six microsatellite loci, Em-01 *, Em-03 *, Em-07 *, Em-08 *, Em-10 *, and CA-07 *, with the aim of exploring the feasibility of using microsatellite data for species identification. The results showed high levels of genetic differentiation among species ( F _ST  = 0.4403 and R _ST  = 0.4954). Species identification based on fixed allelic differences was possible between E. coioides , E. ongus , and E. fuscoguttatus at Em-01 * and between E. fuscoguttatus and E. ongus at Em-08 *. Private alleles were found in all species, except for E. ongus . Pairwise F _ST ranged 0.238–0.578 ( P  < 0.008 Bonferroni correction), and Nei's genetic distance ranged 0.433–2.710. Size homoplasy was observed at Em-03 * 157 allele, which was characterized by a T–C transition at the 119th nucleotide site of PCR products. The genetic assignment test unambiguously assigned each individual to the correct species. Thus, this test can be used for species identification of unknown individuals when the multilocus genotypes of the six microsatellite loci are available. The phylogenetic (neighbor-joining) tree, which was constructed based on the genetic distance matrix, separated the eight grouper species into two main groups.     

Effects of irradiance of various wavelengths from light-emitting diodes on the growth of the harmful dinoflagellate <Emphasis Type="Italic">Heterocapsa circularisquama</Emphasis> and the diatom <Emphasis Type="Italic">Skeletonema costatum</Emphasis>

ABSTRACT:   We investigated the effects of specific light wavelengths from light-emitting diodes (LEDs) on the growth of the dinoflagellate Heterocapsa circularisquama , which kills bivalves, and the diatom Skeletonema costatum , which is an important food source for bivalves. Growth of H. circularisquama was obviously inhibited at 590 nm and a photon flux density less than 75 μmol quanta/m²/s. However, growth of S. costatum was not suppressed by irradiance from any LEDs tested from near-ultraviolet to near-infrared wavelengths at 75 μmol quanta/m²/s. The growth rate of H. circularisquama in an experimental treatment group with irradiance provided by both cool-white fluorescent lamps (12:12 h L : D cycle) and a 590-nm LED (continuous irradiance) was 0.43/day. In the control group with irradiance provided only by cool-white fluorescent lamps (12:12 h L : D cycle), the growth rate was 0.63/day, indicating that growth of H. circularisquama was suppressed by 590 nm (less than 75 μmol quanta/m²/s) irradiance from the LED and the continuous irradiance. The use of 590-nm LEDs in bivalve culture at irradiance levels less than 75 μmol quanta/m²/s might encourage the growth of the useful diatom S. costatum without stimulating growth of the harmful dinoflagellate H. circularisquama .     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.     

Chemical composition of dried eyeballs from <Emphasis Type="Italic">Euphausia superba</Emphasis> and <Emphasis Type="Italic">Euphausia pacifica</Emphasis>

ABSTRACT:   Dried krill eyeballs were obtained from Euphausia superba and Euphausia pacifica by using a patented industry method and their chemical compositions were examined. Crude protein content was 77.7% and 80.8% of the dry matter of E. superba and E. pacifica , respectively. The dominating amino acids in both krill were glutamic acid, aspartic acid, glycine, arginine, leucine and tyrosine. Crude fat content was 10.9% and 5.4% of dry matter of E. superba and E. pacifica , respectively. The main lipid class of the extracted lipids was phospholipids at 88.5% in E. superba and 96.4% in E. pacifica . The dominating fatty acids in both krill were 22:6 ( n- 3), 20:5 ( n- 3), 16:0 and 18:1 ( n- 9). Astaxanthin (3, 3'-dihydroxy-β, β-carotene-4, 4'-dione) content of E. superba and E. pacifica was 566 mg/100 g and 252 mg/100 g of dry matter, respectively. Retinol of E. superba and E. pacifica was 153.0 mg/100 g (510 000 IU/100 g) and 57.6 mg/100 g (192 000 IU/100 g) of dry matter, respectively. The lipophilic extract of E. superba by using n -hexane contained 1923 mg/100 g of astaxanthin at approximately four times higher than the dried eyeballs.     

Characteristics of burnt meat in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis>

ABSTRACT:   In order to understand the characteristics of burnt meat in cultured yellowtail Seriola quinqueradiata , fish were kept at two different temperatures (13 and 30°C) and slaughtered by either spinal cord destruction (SCD) or suffocation in air (SA). Early postmortem changes during storage at 32°C were analyzed by rheological, biochemical, and histological methods. The burnt meat (with lightness parameter, L* ≥ 55) was observed at 1-h storage in the SA 30°C group, at 2 h in SCD 30°C, and at 4 h in SA 13°C; meat was normal for the SCD 13°C group until 6 h of storage. Breaking strength scores were higher for the normal meat (200 g/cm²) than burnt meat (70 g/cm²) at 4 h of storage. Expressible water content was higher for the burnt meat than for the normal meat. Adenosine triphosphate concentrations for the SCD groups were higher than for the SA counterparts. Moreover, pH decrease was much faster in the 30°C groups, showing pH 5.6 at 2 h of storage. A negative correlation between the pH and lactic acid contents in muscle ( P  < 0.001) was found. Histological analysis evidenced a larger pericellular area (40%) in the burnt samples than in the normal samples (16%). It was confirmed that a higher fish-keeping water temperature and a stressful slaughter method (faster glycolytic process) were determinative factors that influence the occurrence of burnt muscle in yellowtail, and that the effect of the former is larger than the latter.     

Conditions for the induction of some selective enzymes from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and their hydrolysis ability against mackerel and gracilar (asparagus, <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>)

ABSTRACT:   This study was conducted to optimize the cultivation conditions for Bacillus subtilis to produce proteases, amylases and cellulase, and to further investigate the hydrolysis ability against mackerel and asparagus. The extracellular enzymes from B. subtilis after 2 and 4 days incubation in a modified medium, containing 1% skim milk, 1% soya meal, 0.25% starch, 0.25% K₂HPO₄, 0.5% NaCl and 0.05% MgCl₂ were collected for the hydrolysis of asparagus and minced mackerel, respectively. Except for the α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging ability of the hydrolyzed asparagus, the trolox equivalent antioxidation capacity and DPPH scavenging ability of both samples increased significantly ( P  < 0.05) after 1 h hydrolysis and further increased during elongated hydrolysis at 50°C. Sodium dodecylsulfate–polyacrylamide gel electrophoresis indicated severe degradation of muscle proteins during hydrolysis. Changes in reducing sugar, soluble proteins and peptides before/after hydrolysis suggested the extracellular enzymes from B. subtilis could effectively hydrolyze the mackerel or asparagus, and subsequently improve their antioxidation ability.     

Visual acuity of Pacific Saury <Emphasis Type="Italic">Cololabis saira</Emphasis> for understanding capture process

ABSTRACT:   To understand the mechanism of the behavioral response in the capture process of how fish recognize fishing gear and then how they can avoid the gear, the visual acuity of Pacific saury Cololabis saira was investigated by histological examination of the retina of individuals in the size range of 75–365 mm fork length (FL). The contour map of cone density distribution shows that the highest cone density is located in the temporal area of the retina, which indicated the visual axis as the forward direction. The visual acuity (VA) depends both on the focal length of the lens and the number of cones in the retina. The lens diameter increased linearly from 1.40 to 4.73 mm with fish growth, while the cone density decreased gradually from 765 to 378 cells/0.01 mm². Our results show that the visual acuity increases proportionately from 0.057 to 0.140 for individuals ranging in FL from 75 to 365 mm as expressed by the equation VA  = 0.0065 ×  FL ^0.5271 ( r ² = 0.9624).     

Effects of fish size and water temperature on the acute toxicity of boron to Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:     The acute toxicities of boron were examined for Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major in terms of fish size and water temperature. Japanese flounder of 0.1–70.0 g and red sea bream of 0.6–20.3 g were exposed to different concentrations of boron for 96 h at 20°C under semi-static conditions. In both fish species, the median lethal concentration (LC₅₀) for 96 h of boron increased linearly with increasing fish size, ranging from 108 to 252 mg B/L for the flounder, and from 97 to 172 mg B/L for the sea bream. The effect of water temperature on acute toxicity was examined for Japanese flounder of 0.6 and 1.5 g at 10, 15, 20 and 25°C, and for red sea bream of 0.6 and 2.4 g at 12, 15, 20 and 25°C. The toxicity of boron for the flounder increased linearly with increasing water temperature. The 96 h LC₅₀ values ranged from 299 to 108 mg B/L for the 0.6 g flounder and from 350 to 113 mg B/L for the 1.5 g flounder. A similar trend was shown for the 2.4 g red sea bream; however, the relationship for the 0.6 g red sea bream was not significant.     

Identification and expression analysis of ghrelin gene in common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

ABSTRACT:   A ghrelin gene has been cloned and sequenced in common carp Cyprinus carpio . Ghrelin cDNA is composed of 461 bp [with a 36-bp 5'-untranslated region (UTR) and a 113-bp 3'-UTR], which translates into a protein of 103 amino acid residues. Carp ghrelin (preproghrelin) contained a predicted signal peptide of 26 amino acid residues, the ghrelin domain ( Gly ²⁷– Val ⁴⁵) and C-terminal peptide ( Gly ⁴⁶– Phe ¹⁰³). Homology analysis of the ghrelin domain of carp with that of other known ghrelin in vertebrates showed good similarity to teleost ghrelin (50–81.8%). Hydropathy analysis based on the deduced amino acid sequence of ghrelin domains in teleosts showed a similar profile. Carp ghrelin clustered with ghrelin of goldfish Carassius auratus and other teleosts, away from mammalian, reptilian, avian, amphibian and chondrichthian ghrelin, by phylogenetic analysis. Genomic organization of carp ghrelin gene was composed of four exons and three introns, which was the same as that of other teleosts and human ghrelin genes. The carp ghrelin gene was expressed in unstimulated tissues such as foregut, hindgut, spleen and brain. In spleen cells, expression of the ghrelin gene increased upon stimulation with lipopolysaccharide (LPS), phytohemagglutinin (PHA) or imiquimod. The identification of carp ghrelin gene and the analysis of the modulation of its expression in immune-activated conditions will allow a more complete analysis of the roles of ghrelin in teleosts.     

Multigenerational effects of 17β-estradiol and nonylphenol on euryhaline cladoceran <Emphasis Type="Italic">Diaphanosoma celebensis</Emphasis>

ABSTRACT:     The effects of parental exposure to 17β-estradiol (E2) and 4-nonylphenol (4-NP) on life history parameters of three successive generations of the euryhaline cladoceran Diaphanosoma celebensis were assessed under laboratory conditions. Less than 24-h-old neonates (P) were exposed to five sublethal concentrations of E2 (0.1, 1, 10, 100 and 1000 μg/L) and 4-NP (0.01, 0.1, 1, 10 and 50 μg/L) at 25°C. Age at first reproduction, reproductive period, fecundity and lifespan were investigated. Successive generations (F₁–F₃) were monitored further in the absence of toxicants. Results showed that cladocerans exposed to 10–1000 μg/L E2 produced more neonates at a younger age compared to the control. The same effects were also observed in the F₁ and F₂ but ceased in F₃. Cladocerans exposed to 1 μg/L 4-NP also produced significantly more neonates compared to the control, but this effect was not found in successive generations. The results suggest that vertebrate hormone can modify the reproduction of D. celebensis , and the effects are multigenerational.     

Effect of combination feeding of <Emphasis Type="Italic">Nannochloropsis</Emphasis> and freshwater <Emphasis Type="Italic">Chlorella</Emphasis> on the fatty acid composition of rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in a continuous culture

ABSTRACT:   A continuous culture of rotifer was conducted to investigate the effect of combination feeding of both a high density of Nannochloropsis oculata (N) and condensed freshwater Chlorella (FC) on the fatty acid composition of L-type rotifer Brachionus plicatilis in a continuous culture system. The algal feeding of the rotifers was carried out in three successive steps: N-feeding → N+FC-feeding → FC-feeding. The culture was conducted at 24°C and 25–27 psu in a 2000 mL bottle with 50% of water exchanged daily. The combination N+FC-feeding was effective in increasing rotifer density. The rotifers fed on N+FC (N+FC-R) had more non-polar lipids than polar ones, similar to those on N (N-R), opposite to the rotifers fed on FC (FC-R). N+FC-R contained higher levels of 16:2, 18:2n-6 (linoleic acid [LA]) and 20:2n-6, but lower levels of 18:1, 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid [EPA]) and 22:5n-3 (docosapentaenoic acid [DPA]) compared with N-R. Whereas N+FC-R contained higher levels of 16:1n-7, EPA and DPA, but lower levels of 16:2 and LA compared with FC-R. N+FC-R had more DPA in polar lipids than in non-polar ones. The Σn-6/Σn-3 ratio in N+FC-R was 0.9–1.0, significantly different from those in N-R (0.4) and FC-R (6.6–8.4). Therefore, it is inferred that the fatty acid profile of the N+FC-R cultured in a continuous culture system was affected by both N and FC. Also, the combination N+FC-feeding may be effective in manipulating the Σn-6/Σn-3 ratio in continuously cultured rotifers.     

Proposal for stock-recruitment relationship for Japanese sardine <Emphasis Type="Italic">Sardinops melanostictus</Emphasis> in North-Western Pacific

ABSTRACT:   This study assessed the stock-recruitment relationship (SRR) for the Japanese sardine Sardinops melanostictus in the North-western Pacific. Of the 20 SRR models investigated, the Akaike information criterion (AIC) was the minimum (AIC = 551.2) when the data were separated into two groups (A and B) and the log-normal distribution was applied as the error term. Group A was constructed with SRR data from 1976–1987 and 1992–2004. Group B consisted of data from 1988–1991. The AIC minimum model was R  = 22.8 S  ×  e ^ε for Group A, where R , S , and ε denote the recruitment of sardine (individual number of 0-year old fish), spawning stock biomass (SSB), and error term, respectively. This model indicated that recruitment was proportional to the SSB and that no density-dependent effect operated over the range of SSB investigated (51 000–11.3 million t). Recruitment was markedly higher (lower) when the sea surface temperature (SST) of the Kuroshio Extension area in February was low (high). The essential SRR can simply be expressed as R  = 22.8 S  ×  e ^ε with the level of recruitment deviating from the model to a greater or lesser degree depending on the environmental conditions.     

Community and trophic structures of abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis> habitat in Sagami Bay,Japan

Abstract:   The community and trophic structures of the subtidal habitat of the abalone Haliotis diversicolor were examined in Nagai, Sagami Bay, Japan. Conventional community indices showed no significant differences among three sampling stations. The overall continuum of stable isotope ratios was structured into three different trophic linkages: (i) brown algae-dependent benthic food chain; (ii) red algae-dependent benthic food chain; and (iii) planktonic food chain. Brown algae and red algae likely play different roles with respect to carbon sources in the habitat. Conventional fractionation values indicated that the abalone H. diversicolor (δ¹³C = −12.4 ± 1.0‰, δ¹⁵N = 9.3 ± 0.5‰) feeds on the lamina of Undaria pinnatifida during juvenile and adult stages (8.0–65.6 mm shell length). Stable isotope signatures suggested that the juveniles of other abalone species as well as some amphipods and a sea cucumber Holothuria decorata are competitors of H. diversicolor , whereas some Muricidae gastropods such as Thais bronni and Ergalatax contractus are predators. The isotopic differences among macroalgal species and the subsequent transfer to consumers indicate that stable isotopic analysis is an effective means of studying food webs in an open rocky shore community with little influence from external primary production such as terrestrial vegetation.     

Genetic comparison between torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and its closely related species karasu <Emphasis Type="Italic">Takifugu chinensis</Emphasis>

Abstract:   Sequence analyses of mitochondrial (mt) and nuclear genes were performed for genetic comparison between two Takifugu pufferfish species: torafugu T. rubripes and karasu T.  chinensis . With a sequence coverage of 20% in mtDNA, 640, 308, 344, 522 and 697 bp encoding mt 16S ribosomal RNA (rRNA), adenosine triphosphatase 6 ( ATPase 6 ), nicotinamide adenine dinucleotide dehydrogenase subunit 4 ( ND4 ), ND5 and cytochrome b (cyt b ), respectively, among 24 wild torafugu, 24 wild karasu and six hybrid-like samples, 15% of the torafugu identified by external color patterns showed nucleotide sequences consistent with karasu. Meanwhile, sequences of 60% karasu were consistent with those registered for torafugu (AJ421455). As for the hybrid-like samples, two possessed karasu-specific sequences in some base positions while torafugu-specific sequences in others. The remaining hybrid-like samples possessed torafugu-specific sequences. On the other hand, the mt control region did not show such type of consistency. Analysis of nuclear melanocortin receptor genes ( MC1R , MC4R ) among 54 samples showed 99–100% inter- and intraspecific sequence identity. Partial nuclear 18S  rRNA, complete internal transcribed spacer 1 ( ITS1 ), partial 5.8S  rRNA and ITS2 genes showed similar levels of identity, indicating a very low level of variation in their respective gene fragments between the two Takifugu species.     

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.     

Analysis of bacterial communities in <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp. cultures used for larval fish production

ABSTRACT:   Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72 × 10⁵−2.39 × 10⁶ cells/mL. High proportions of the total bacteria (31.6–53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that α-proteobacteria and the Cytophaga – Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks.     

Effects of organic acids on growth and phosphorus utilization in red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   A feeding experiment was conducted to investigate the effects of various organic acids and supplements on the growth, phosphorus (P) utilization, and environmental loading of nitrogen (N) and P in red sea bream Pagrus major. Seven diets designated as PA (0.5% inorganic P as Ca[H₂PO₄]₂), PO (no inorganic P), CA (1% citric acid), MA (1% malic acid), LA (1% lactic acid), MHA (1% methionine hydroxy analog) and LTE (1% liquid trace elements) were formulated. Duplicate groups of 25 fish were fed four times daily to near satiation for 75 days. Fish fed CA and LTE diets showed significantly better weight gain and feed conversion ratio compared to fish fed diet PO. Absorption of P was significantly improved in all organic acid-supplemented groups compared to the PA and PO groups. The P retention in CA and LTE groups were significantly higher compared to PA and PO diets. The P excretion was also significantly decreased in all organic acid-supplemented groups. Thus, use of organic acids, in particular CA and LTE, in red sea bream diets can reduce the use of inorganic phosphorus and contribute to the development of much-desired eco-friendly diets.