Shiro Itoi

Rapid identification of eels Anguilla japonica and Anguilla anguilla by polymerase chain reaction with single nucleotide polymorphism-based specific probes

Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla. This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla. Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica and A. anguilla-specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla.

A simple method to distinguish two commercially valuable eel species in Japan Anguilla japonica and A. anguilla 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.

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish

Aims:  We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture.

Larval pufferfish protected by maternal tetrodotoxin

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae.

Difference in the localization of tetrodotoxin between the female and male pufferfish Takifugu niphobles, during spawning

In order to understand the sexual differences in TTX-usage in the pufferfish, Takifugu niphobles, localization of TTX and toxin amount in tissues of mature male and female specimens were investigated by immunohistochemical methods using anti-TTX antibody and LC/MS analysis. Subsequently, differences in the immunohistochemical signals were compared with the amount of TTX. The paraffin-embedded sections of the skin, muscle, liver, gonad and intestinal tract were subjected to anti-TTX monoclonal antibody based on the fluorescent immunohistochemical techniques. Immuno-positive reaction was observed in the skin and liver in males, and the skin and ovary in females. In the skin, TTX was localized at the epidermis, the basal cell layer, the mucous cells and the sacciform cells, and with intense immunoreaction at the flat epithelial cell layer and the sacciform cells. The signal from the liver cells was stronger in males than in females. The intensity of the signal from the tissues correlated with the toxin amounts therein. These results suggest that tissue distributions of TTX and toxin amount in the pufferfish were sex-dependent.

Chemical sensitivity of the male daphnid, Daphnia magna, induced by exposure to juvenile hormone and its analogs

It was reported that males daphnid Daphnia magna that have been induced by methyl farnesoate exposure exhibit higher tolerance to chemical compounds such as potassium dichromate and pentachlorophenol than females. Male neonates are also known to be induced by exposure to juvenile hormone analogs, such as fenoxycarb and pyriproxyfen. If these analogs can be used to produce male progeny, the biological and physiological studies of daphnid male would be progressed since the effects of these analogs were several hundred times higher than that of methyl farnesoate. Therefore, in the present study, it was investigated that the chemical sensitivity of male neonates induced by exposure to juvenile hormone (methyl farnesoate) and its analogs. The minimum concentrations of methyl farnesoate, fenoxycarb and pyriproxyfen to induce 100% male-reproduction were 200nM (50microg/l), 0.23nM (70ng/l) and 0.31nM (100ng/l), respectively. In addition, no reduction of relative reproduction was observed at the juvenoid concentrations in 24h exposure producing 100% male progeny. The median effective concentrations (EC50) of potassium dichromate for immobility of male neonates, established by a standardized method for investigating sensitivity to chemicals, were significantly higher (12-29%) than that of females at least after 24h exposure in all the male neonates induced by juvenoids used in this study (P<0.05). This study demonstrated that the male daphnids induced by exposure to juvenile hormone and its analogs exhibit similar chemical tolerance.

Identification of alfonsino, Beryx mollis and B. splendens collected in Japan, based on the mitochondrial cytochrome b gene, and their comparison with those collected in New Caledonia

The sequences spanning 307 bp of the mitochondrial cytochrome b gene were determined for 45 sepcimens of Beryx splendens and seven specimens of B. mollis collected in Japan, resulting in identification of 11 and three haplotypes in the two species, respectively. The parsimony tree was constructed from the determined sequences and those registered into the GenBank data-base as species A and W of B. splendens collected in New Caledonia, featuring with two clades. The first clade comprised species W from New Caledonia and B. mollis in the present study, whereas the second one contained species A from New Caledonia and B. splendens in the present study. These results demonstrate a large geographic distribution for both B. splendens and B. mollis. Some of the haplotypes found in Japan were identical to those of New Caledonia for both B. mollis and B. splendens, suggesting levels of gene flow at the trans-oceanic scale.

Changes in mitochondrial fatty acid composition following temperature acclimation of carp and their possible effects on FoF1-ATPase activity

AbstractCarp undergo temperature acclimation of respiratory function by altering mitochondrial ATP synthase (FoF1-ATPase) both quantitatively and qualitatively (Itoi et al. 2003). To address such acclimation temperature-dependent changes of FoF1-ATPase activity, we investigated in this study the correlation between the fatty acid composition and FoF1-ATPase activity in fast muscle of thermally acclimated carp. The quantities of saturated fatty acids of mitochondria from carp acclimated to 10 °C were significantly lower than those of carp acclimated to 30 °C. While mono- and poly-unsaturated fatty acids tended to increase with cold acclimation of carp, the molar concentration of 16:0 aldehyde in mitochondria from the 10 °C-acclimated carp were less than those from the 30 °C-acclimated fish. The specific activities of FoF1-ATPase in the 10 °C- and 30 °C-acclimated fish mitochondria were calculated to be 167±22 and 56±10 nmol/min ⋅ mg mitochondrial protein, respectively, the difference being significant at P<0.005. Taken together, the increase in FoF1-ATPase activity in fast muscle mitochondria of carp after cold temperature acclimation may be closely related to the increase of unsaturated fatty acids in mitochondria. Abbreviations: BSA - bovine serum albumin; DHA - docosahexaenoic acid; EGTA - ethyleneglycol bis(2-aminoethylether)tetraacetic acid; EPA - eicosapentaenoic acid; FoF1-ATPase - mitochondrial ATP synthase; α-F1-ATPase - FoF1-ATPase α-subunit; β-F1-ATPase - FoF1-ATPase β-subunit; HEPES - 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; SDS - sodium dodecyl sulfate; SDS-PAGE - SDS-polyacrylamide gel electrophoresis.

Species identification method for Scombrops boops and Scombrops gilberti based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)-RFLP analysis using two enzymes, EcoNI and Mval, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR-RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops, suggesting that S. gilberti juveniles are rare in this area.

Population genetic structure of Scombrops boops (Percoid, Scombropidae) around the Japanese archipelago inferred from the cytochrome b gene sequence in mitochondrial DNA

The gnomefish (Scombrops boops) is a member of the percoid family Scombropidae, which includes a single genus and three to four species worldwide. Since little is known about the ecology of this species, here, sequencing analysis of the cytochrome b gene (1141 bp) in mitochondrial DNA detected 101 haplotypes from 186 individuals of S. boops collected from waters at seven localities around the Japanese archipelago. A single haplotype (Sb2) was the most abundant in the combined populations of S. boops from various localities. Genetic population structure analyses revealed no significant differences among these populations (Fst = − 0.0313–0.0195; Φst = − 0.0505–0.0615) with high haplotype diversity and low nucleotide diversity. This suggests that S. boops around the Japanese archipelago constitutes a single population, and indicates that the genetic structure of this population may be influenced by larval and egg dispersal in association with warm currents.