Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   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, Eco NI and Mva I, 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.     

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.     

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.     

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.     

Effects of stocking density on intermediate culture of the razor clam Ensis arcuatus (Pharidae: Bivalvia)

Commercial production of most bivalve species involves a phase of intermediate cultivation during which juveniles are grown under protected conditions until they can be transferred to the final grow‐out location. Consequently, the aim of this study was investigating the effect of density on growth and survival in the intermediate culture of the razor clam Ensis arcuatus in raft. Two series of experiments were performed, using in the first experiment two holding systems: 5‐L plastic bottles and PVC cylinders, both perforated, covered with a 1 mm mesh net to prevent razor clam escape and a 10‐cm layer of coarse grained sand (300–1200 μm grain diameter). In this trial, two stocking densities were tested: 0.15 and 0.30 kg m^?2. In the second one‐two densities were essayed (0.62 and 1.24 kg m^?2) in the PCV cylinders. In the first experiment after 27 of trial higher growth in cylinders at low density was observed. At the end of this trial all juveniles died in the 5‐L plastic bottles and higher growth and survival were recorded in the cylinders at low density. In the second experiment, significant differences in growth and survival were found on day 70, with the best results at low density. Five‐L plastic bottles must be rejected as holding system for the intermediate culture of E. arcuatus and the PVC cylinders at low densities could be a good alternative. At the end of the experiment razor clams reached the adequate size for their transfer into the grow‐out system.     

Comparison between α-LNA and DHA in early developmental stages of <Emphasis Type="Italic">Takifugu obscurus</Emphasis> and <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   Rearing experiments were conducted to investigate the essential fatty acid requirements in the early developmental stages of river puffer Takifugu obscurus and tiger puffer T. rubripes using two n-3 series unsaturated fatty acids, α-linolenic acid (18:3n-3, α-LNA) and docosahexaenoic acid (22:6n-3, DHA), under salinity of 30 and 18.5–20.3°C. River and tiger puffer larvae used in this study were 15 and 14 days old after hatching, and their average body weights were 30.1 and 20.8 mg, respectively. The results on fatty acid requirements of these two species were evaluated from fish growth, survival, fatty acid composition of the fish body and activity test results. The DHA groups of both river and tiger puffer exhibited better survival and weight gain. However, there was no difference in the mean final body weights of river puffer between two dietary groups. Also, the DHA group of tiger puffer showed better results in the recovery test from anesthetic condition than that obtained in the LNA group. In an examination of the fatty acid compositions of the whole body, the LNA group containing no dietary DHA resulted in 0.5% DHA in tiger puffer and 1.1% DHA in river puffer . These results suggest that α-LNA from Artemia converted to eicosapentaenoic acid (20:5n-3, EPA) and to DHA successively by their fatty acid metabolism. Symptoms following essential fatty acid deficiency were not observed in any experimental groups. As river puffer did not represent a significant difference in the dietary effects between α-LNA and DHA treatment groups, its essential fatty acid requirement was assumed to be somewhat closer to that of the freshwater fishes in comparison with that of marine fishes, including tiger puffer.     

Hybridization success of three common European cyprinid species, <Emphasis Type="Italic">Rutilus rutilus,Blicca bjoerkna</Emphasis> and <Emphasis Type="Italic">Abramis brama</Emphasis> and larval resistance to stress tests

ABSTRACT:   Hybridization success at early developmental stages and larval resistance to osmotic, thermal and fasting tests in roach Rutilus rutilus , silver bream Blicca bjoerkna , common bream Abramis brama and their F1 hybrids were investigated. Results revealed that hybrid survival rates were similar to parents. At the eyed embryo stage, however, a maternal effect was observed as a general trend during hatchling and larval stages. After these stages, hybrids displayed a higher survival rate than their parents. Under stress tests, no survival was observed after 40 min for osmotic and thermal shocks and after 24 days for the prolonged fasting test in these species and their F1 hybrids. The median survivals of hybrids were intermediate between the two parents. For total mortality, hybrids were also affected by a maternal effect but to the advantage of the hybrids.     

Optimum temperature and salinity conditions for growth of green algae <Emphasis Type="Italic">Chlorella ellipsoidea</Emphasis> and <Emphasis Type="Italic">Nannochloris oculata</Emphasis>

ABSTRACT:   The effects of temperature and salinity on growth of green algae Chlorella ellipsoidea and Nannochloris oculata were determined to compare the optimum culture conditions. A four-temperature (15, 20, 25, and 30°C) × three-salinity (10, 20, and 30) factorial design with triplicates was applied. Specific growth rate (SGR), maximum density, and duration to reach maximum density of C. ellipsoidea were significantly affected by both temperature and salinity. The highest SGR was observed in C. ellipsoidea at 25°C and salinity 10, but the maximum density was very low. The highest maximum density was achieved in C. ellipsoidea at 15°C and 10. The slope constant of the linear relationship between semilogarithmic growth of C. ellipsoidea and day of culture was highest at 15°C and 10. The SGR and duration to reach maximum density of N. oculata were significantly affected by both temperature and salinity. However, maximum density of N. oculata was significantly affected by temperature, but not salinity. The highest maximum density was achieved in N. oculata at 25°C and 30, but SGR was significantly lower than that of N. oculata at 25°C and 10. The slope constant of the linear relationship between semilogarithmic growth of N. oculata and day of culture was highest at 25°C and 30. Based on these results, the condition of 15°C and salinity 10 seemed to be optimal for maximum density of C. ellipsoidea , and the condition of 25°C and 10 and 30 for SGR and maximum density for N. oculata , respectively.     

Induction of (<Emphasis Type="Italic">Z</Emphasis>)-8-heptadecene in marine green alga <Emphasis Type="Italic">Bryopsis maxima</Emphasis> by mechanical wounding

ABSTRACT:   After mechanical wounding of the marine green alga Bryopsis maxima in a phosphate buffer, one main organic component was released to the solution. The organic compound was characterized by a molecular ion at 238  m/z by gas chromatography–mass spectrometry (GC-MS) and was deduced as 8-heptadecene by the fragment pattern. To identify the stereochemistry of 8-heptadecene, synthetic ( Z / E )-8-heptadecene was coinjected to the solution of wounded B. maxima . Comparing retention times and mass spectra with the synthetic sample, the target hydrocarbon obtained from wounded B. maxima was coincident with ( Z )-8-heptadecene.     

Identification of juvenile abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis> based on number of open and sealed respiratory pores

ABSTRACT:   Changes in the number of respiratory pores (open pores) and their imprints (sealed pores) in post-larvae and juveniles were observed and compared for four abalone species Haliotis diversicolor , H. discus discus , H. madaka , and H. gigantea . The first open pore was evident at a shell length (SL) of 1.5 mm in H. diversicolor , 1.9–2.0 mm SL in H. discus discus , and 2.3–2.4 mm SL in H. madaka and H. gigantea . The number of open pores in H. diversicolor gradually increased with growth, with four to five pores at 2.5–18.0 mm SL and five to six pores at 18.0–27.0 mm SL. The other three species maintained four to five open pores after they reached 3.4–4.5 mm SL. The total number of open and sealed pores (TNP) was greater in H. diversicolor than in the other species at the same SL. Juvenile H. diversicolor were identified among field-caught abalone by the difference in the relationship between SL and TNP (SL–TNP relationship) and also by the monoclonal antibody reaction method. The results of the two methods were in perfect agreement, indicating that our method using the SL–TNP relationship is reliable for the identification of H. diversicolor .     

Difference in the level of paralytic shellfish poisoning toxin accumulation between the crabs <Emphasis Type="Italic">Telmessus acutidens</Emphasis> and <Emphasis Type="Italic">Charybdis japonica</Emphasis> collected in Onahama,Fukushima Prefecture

ABSTRACT:     The difference in paralytic shellfish poisoning (PSP) toxin accumulation in the crabs Telmessus acutidens and Charybdis japonica was investigated at Onahama, Fukushima Prefecture, from 2002 to 2005. The level of toxin accumulation in the hepatopancreas of T. acutidens corresponded to that of mussels when examined on a yearly basis. In 2003, some crabs had a high toxicity of approximately 1000 MU, which compares to one-third of the human minimum lethal dose. Therefore T. acutidens should be monitored as a vector species of PSP toxin. The toxin profile of T. acutidens was also investigated. Because an increase in highly toxic species of PSP toxins with a decrease in low toxic species, such as N -sulfocarbamoyl-11-hydroxysulfate toxins, was not clearly observed between consecutive samples, toxin transformation in T. acutidens was considered to have a minimal impact on toxicity. PSP toxins were also detected in several specimens of C. japonica , but the highest toxicity was only 7.4 MU/g in the hepatopancreas. Because C. japonica is widely distributed in the coastal waters of Japan, investigation of toxin accumulation in the crab should be continued, including its investigation in different areas of water from Onahama, where the causative dinoflagellates of PSP occur in different conditions.     

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.     

Culture possibilities of the razor clam Ensis arcuatus (Pharidae: Bivalvia)

This study investigated razor clam Ensis arcuatus culture from larval rearing to commercial size to assess it's aquaculture potential. Broodstock was spawned several times in hatchery facilities to demonstrate that larval availability is not a constraint for hatchery production. Larval culture lasted 20 days, showing an average survival of 14.35%. The razor clam seed reached a size of 28.5 mm at 4 months. Seed culture is feasible; however, it is constrained by the fact that razor clams need to be buried. Higher survival was observed when culturing razor clam seed with a substrate (82–83%), irrespective of the substrate grain size, while containers held without a substrate showed a significantly lower survival (56.5%). We showed that it is possible to rear E. arcuatus juveniles in bottles, with survival as high as 94–95% being achieved. The length increase of the hatchery‐produced E. arcuatus over 600 days was described by the equation Ln L=4.5+(?141.17/t), where L is length in mm and t is time in days. On‐growing showed adequate survival rates and it took 2–3 years to reach commercial size.     

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.     

Size selectivity of escape holes in conger tube traps for inshore hagfish <Emphasis Type="Italic">Eptatretus burgeri</Emphasis> and white-spotted conger <Emphasis Type="Italic">Conger myriaster</Emphasis> in Tokyo Bay

ABSTRACT:   In Tokyo Bay, conger tube-trap fishers voluntarily deployed escape holes of at least 13 mm diameter to reduce the by-catch of young conger smaller than the marketable size of 35 cm total length. Comparative fishing experiments in Tokyo Bay were carried out using tube traps with seven hole diameters (3, 9, 13.5, 14, 15, 17, and 19 mm), to obtain the size-selectivity of the escape-hole size for inshore hagfish Eptatretus burgeri and white-spotted conger Conger myriaster . On the assumption of the geometrical similarity in the combination of body g and hole perimeter m at a given retention probability, a single master curve s ( R ) = exp(−13.52 + 11.31 R )/(1 + exp [−13.52 + 11.31 R ]) was estimated for the two species in terms of relative R  =  g / m using the Share Each LEngth's Catch Total (SELECT) process based on a multinomial distribution. Almost all conger and hagfish with a girth equivalent to the hole perimeter were able to pass through the hole. Thus, the two species have a high ability to escape through a narrow space by squeezing the body through the hole in the rigid PVC tube. Optimum escape-hole size is discussed from several objectives for conger tube-trap fishery management, e.g. reduction of hagfish by-catch. This study also specifies the conditions for pooling data when fishing experiments using more than two fishing gears are replicated.     

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.     

Dietary value of benthic diatoms for post-larval abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis> associated with feeding transitions

ABSTRACT:   The feeding behavior and growth of post-larval Haliotis diversicolor with initial shell lengths (SL) of approximately 500 μm (Exp. 1-1 and 1-2), 800 μm (Exp. 2), and 1200 μm (Exp. 3) were studied in a laboratory setting while they fed on four species of benthic diatom Achnanthes longipes , Cocconeis sublittoralis , Cylindrotheca closterium , and Navicula ramosissima . Exp. 1-1 and 1-2 revealed no marked differences in post-larval growth rates (mean 24–39 μm SL/day) among the diatom species. However, marked differences in growth rates among the species were revealed in Exp. 2 and 3. Three species, A. longipes , Co. sublittoralis, and Cy. closterium , produced faster growth (Exp. 2 mean 29–51 μm/day, Exp. 3 mean 36–44 μm/day) than N. ramosissima (Exp. 2 mean 18 μm/day, Exp. 3 mean 23 μm/day). Post-larvae fed N. ramosissima had lower digestion efficiency (42.8%) than those fed other diatom species (90.7–100%). Diatom extracellular substances appeared to be principally used from post-settlement to 800 μm SL, and diatom cell contents were required to produce rapid growth of larger post-larvae (>800 μm SL). It is likely that the availability of each diatom for post-larvae was affected by diatom morphology, attachment strength, frustule strength, and post-larval size.     

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.     

Genetic population structure of <Emphasis Type="Italic">Nibea albiflora</Emphasis> in Yellow Sea and East China Sea

ABSTRACT:   The population genetic structure and level of gene flow of Nibea albiflora from the Yellow Sea and the East China Sea were examined with a 479-bp segment of a mtDNA control region. In total, 65 samples were collected from three locations and 37 haplotypes were obtained. Mean haplotype diversity and nucleotide diversity for the three populations ranged from 0.9130 ± 0.0308 (Zhoushan) to 0.9926 ± 0.0230 (Xiamen), and from 0.0073 ± 0.0043 (Qingdao) to 0.0099 ± 0.0057 (Xiamen). Analysis of molecular variance and pairwise F _ST revealed little genetic structure between the Yellow Sea and the East China Sea in N. albiflora . But based on the exact test of differentiation, the null hypothesis that N. albiflora within the Yellow Sea and the East China Sea constitutes a panmictic mtDNA gene pool was rejected. This might be caused by the broad spawning areas but not by the Yangtze River outflow. Mismatch distribution revealed that N. albiflora has undergone population expansion, possibly before the last 85 000–170 000 years. The existence of high gene flow between stocks in the studied area was supported by our results. Annual migrations, larval drift in the ocean currents, and recent range expansion could be the reasons for little genetic structure in the studied area.