cDNA cloning and complete primary structures of myosin heavy chains from brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso fast skeletal muscles

ABSTRACT:   The complete cDNA sequences encoding predominant types of myosin heavy chain (MYH) in the fast skeletal muscle were determined for brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso , which are used as materials for preparing high-quality surimi-based products. The cDNA consisted of 5973 and 5987 bp, respectively, and both encompassed an open reading frame encoding a polypeptide of 1936 amino acid residues. Brushtooth and wanieso lizardfish MYH showed the amino acid sequence identity of 92–93% to white croaker MYH, which was higher than that of 90% to walleye pollack MYH. The putative binding sites for ATP, actin, and regulatory and essential light chains in the subfragment-1 region of brushtooth lizardfish MYH exhibited a high identity with white croaker counterparts as well as the sequences of subfragment-2 and light meromyosin. In contrast, phylogenetic tree, constructed by the neighbor-joining method based on mitochondrial 16S rRNA gene, revealed that the two lizardfish species formed a cluster with walleye pollack, which was paraphyletic with white croaker. Therefore, a good reputation for lizardfish and white croaker to have a high thermal-gel forming ability seemed to be reflected by MYH rather than biological similarity as revealed by the mitochondrial 16S rRNA gene.     

Comparison in taste and extractive components of boiled dorsal muscle and broth from half-smooth golden puffer Lagocephalus spadiceus caught in Japan with those of the same fish imported

The taste and extractive components of boiled dorsal muscle and broth prepared from half-smooth golden puffer Lagocephalus spadiceus caught in Japan and from those imported from China were compared. In the sensory test, the first taste, elasticity, and saltiness of boiled muscle from Japanese (domestic) fish were higher than those of the imported fish, whereas the orthonasal fishy smell of the imported fish was higher than that of the domestic fish. The first taste, aftertaste, retronasal fishy smell, sweetness, saltiness, and umami of the broth prepared from dorsal muscle of the domestic fish were higher than those of the imported fish, whereas orthonasal fishy smell and bitterness of the imported fish were higher than those of the domestic fish. Most panelists preferred the overall taste of the domestic fish to that of the imported fish. The concentration of trimethylamine in the trichloroacetic acid extracts from boiled muscle and broth of the imported fish was higher than that of the domestic fish, suggesting that this substance contributes to the orthonasal fishy smell of the imported fish.     

Differences in polymer formation through disulfide bonding of recombinant light meromyosin between white croaker and walleye pollack and their possible relation to species specific differences in thermal unfolding

Fast skeletal light meromyosins (LMMs) of white croaker and walleye pollack were prepared in our expression system using Escherichia coli and determined for their polymer-forming ability and thermodynamic properties by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and differential scanning calorimetry (DSC), respectively. White croaker LMM formed dimer by heating at 80 degrees C and showed only a single peak at 32.1 degrees C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8, and 43.9 degrees C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM where Cys491 was substituted by alanine changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4, and 43.9 degrees C. However, this point-mutated LMM formed no dimer upon heating as in the case of native LMM. These results suggest that cysteine residue(s) participates in thermal gel formation of LMM when it locates in a suitable position of the sequence.     

Microsatellite and mitochondrial DNA analyses reveal no genetic difference between two pufferfish species torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and karasu <Emphasis Type="Italic">T. chinensis</Emphasis>

It has been reported that the nucleotide sequences of the mitochondrial and nuclear genes of Takifugu pufferfish torafugu T. rubripes and karasu T. chinensis show 99–100% sequence identity, indicating a very close relationship between the two species. To further investigate this genetic relationship, we compared genetic variation at four microsatellite loci and at the mitochondrial control region (CR) (561 bp) between groups of T. rubripes caught at two locations [TrG, caught in the Genkai Sea off Tsushima Island in 2003 (n = 50); TrS, caught in the Suwo Sea off Kita-Kyushu in 2008 (n = 50)] and T. chinensis caught at one location (TcK, caught off the east coast of Korea in 2004; n = 50). Analyses using microsatellite loci showed that genetic diversity index values of the TrG, TrS and TcK groups were 0.9505, 0.9350 and 0.9335, respectively, while values of genetic distance and genetic differentiation between TrG and TcK (0.0543 and 0.0189, respectively) were smaller than those between TrG and TrS (0.0857 and 0.0194, respectively). Analyses using CR for the same specimens showed that genetic distances were consistent with those obtained using microsatellite loci. These results, together with our previous observations, suggest that T. rubripes and T. chinensis are very closely related and possibly can be regarded as the same species.     

Thermal tolerance of a thermally selected strain of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> and the pedigrees of its F1 and F2 generations indicated by their critical thermal maxima

A thermally selected strain of rainbow trout has been established by selective breeding since 1966 in Miyazaki, Japan. In the present study, we compared the critical thermal maxima (CTMs), the temperatures at which organisms reach a predefined sublethal endpoint and lose their equilibrium, between a thermally selected and two normal (Donaldson) strains of rainbow trout. The CTM of one normal strain from Nikko (Nikko strain) acclimated to 20 °C (29.7 °C) was significantly lower than those of the thermally selected strain (30.0 °C) and the other Donaldson strain from Aomori (29.9 °C) (P?<?0.05). The F1 generations, F1T and F1N, were produced by crossing thermally selected strain females with Nikko strain males and Nikko strain females with thermally selected strain males, respectively. No significant difference was observed in the CTM between F1T [30.1?±?0.15 °C (n?=?30)] and F1N [30.1?±?0.16 °C (n?=?30)] (P?>?0.05) for fish acclimated to 20 °C, suggesting that the F1 offspring inherited the thermal tolerance trait from one thermally selected strain parent irrespective of whether it was the male or female. F2 offspring of F1T or F1N also showed the thermal tolerance trait. The coefficients of variation for CTM were also compared among all the datasets obtained in the present study and their values for F1 hybrids were lower than those of the parental generation of the Nikko strain (P?<?0.05). In contrast, the coefficients of variation of F2s were the same as those of their parental generation. Furthermore, the thermally selected strain and Nikko strain as a reference family provide a F2 generation for segregating phenotypes, which is required for in-depth genetic analysis of the thermally selected rainbow trout strain.     

Metagenomic analysis using 16S ribosomal RNA genes of a bacterial community in an urban stream,the Tama River,Tokyo

In an effort to determine genus- or species-level taxonomic profiles and diversity of bacterial consortia in the Tama River around urban Tokyo, next-generation sequencing technology targeting a 16S ribosomal RNA (rRNA) gene amplicon was employed. Metagenomic analysis performed by an Ion Personal Genome Machine after sequentially filtering samples through 5-, 0.8- and 0.2-μm filters yielded 1.48 Gb of 16S sequences (average 2.38 M reads/sample). The results indicated that half of the bacterial sequences belonged to Proteobacteria, followed by Bacteroidetes, Actinobacteria and Cyanobacteria. Flavobacterium (Bacteroidetes), possibly including a potential fish pathogen, was the most numerous genera in the Tama River metagenome, and accounted for?~?16% of assigned 16S reads, followed by Mycobacterium. Other dominant bacterial genera including Zoogloea, Sediminibacterium, Hyphomicrobium, Sphingopyxis, Thiothrix and Lysobacter, were thought to be associated with waste water and sludge. MiSeq metagenomic analysis revealed that environmental factors, particularly water temperature, influenced the bacterial composition throughout the year, with a strong negative correlation observed for Proteobacteria and a positive correlation for Bacteroidetes. In terms of bacterial genera, Flavobacterium was positively correlated with temperature, while Polaromonas, Pseudomonas and Bradyrhizobium were negatively correlated with this, suggesting dynamic change in the free-living bacterial population throughout the year and versatile adaptation strategies in relation to environmental factors.     

Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation.     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   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 .     

Structural and thermodynamic characterization of tropomyosin from fast skeletal muscle of bluefin tuna

ABSTRACT:   Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7–98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.