Quantitative loop-mediated isothermal amplification method for the detection of <Emphasis Type="Italic">Vibrio nigripulchritudo</Emphasis> in shrimp

Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60 min at 63°C. Quantitative analysis was then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification of the initial DNA concentration with a sensitivity of 10² copy numbers equivalent to 2.3 colony forming units/ml or 0.3 fg/μl. The LAMP assay was able to specifically detect two representative strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T _t) versus the log of bacterial number. A high correlation coefficient (R ² = 0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.     

Inheritance mode of microsatellite DNA markers and their use for kinship estimation in kuruma prawn Penaeus japonicus

The allelic inheritance mode of microsatellite DNA markers was examined using seven copulated wild females and their offspring. Five microsatellite loci, CSPJ002 *, CSPJ010 *, CSPJ012 *, CSPJ014 *, and CSPJ015 *, were used in the study. At almost all family/locus combinations, one sire was determined and distributions of genotypes in offspring were consistent with the Mendelian segregation ratio. Distributions of genotypes were consistent with the ratio after assuming a null allele at some loci. Consequently, the alleles of CSPJ002 * and CSPJ012 * were inherited following the Mendelian inheritance mode in every family; however, the null allele was expected in CSPJ010 *, CSPJ014 *, and CSPJ015 * in some families. Thus, these loci should be used carefully in population genetic analysis, but siblings could be detected in the dendrograms based on unweighted pair-group method using arithmetic averages (UPMGA).     

Determination of primary structure of amberjack myosin heavy chain and its relationship with structural stability of various fish myosin rods

The structural stability of fish myosin depends upon species and temperatures of water in which fish live. Primary, secondary, and quaternary structures of myosin heavy chain (MyHC) from three species of fish living at different temperature ranges have been compared with those of rabbit MyHC in order to investigate the differences in stability. Primary structure of MyHC, although being accessible for warm-water and cold-water fish (carp and walleye pollack), was not available in previous for tropical-water fish literature; so in this study primary structure of MyHC of the tropical-water fish amberjack has been determined by cloning and sequencing its cDNA. The MyHC has 1938 amino acid residues (AA), which are almost as much as as those of carp and walleye pollack. The amberjack MyHC is 91–95% homologous with other fish and rabbit MyHCs. There is a discernible difference between animal species with stable myosin rod (amberjack, carp, and rabbit) and walleye pollack with unstable rod. Stable rod species have a high probability of forming coiled-coil around the COOH-terminal end of the rod, while the pollack has a low coiled-coil formation probability. In addition, the average scores of the coiled-coil for myosin rod were rabbit (1.738) > amberjack (1.691) > carp (1.680) > walleye pollack (1.674) which correlated exactly with the observed stability. The results suggest that coiled-coil forming ability, particularly around the COOH-terminal end, directs structural stability of fish myosin rod.     

Extensive Screening for Edible Herbal Extracts with Potent Scavenging Activity against Superoxide Anions

To search for edible herbal extracts with potent antioxidant activity, we conducted a large scale screening based on the superoxide scavenging activity. That is, scavenging activity against superoxide anions were extensively screened from ethanol extracts of approximately 1,000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method. Among them we chose four edible herbal extracts with prominently potent ability to reduce the signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH, a spin adduct formed by DMPO and superoxide anion. They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit), and are allowed to be used as foodstuffs according to the Japanese legal regulation. The ESR-spin trapping method coupled with steady state kinetic analysis showed that all of the four extracts directly scavenge superoxide anions, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. Furthermore, polyphenol determination indicates that the activity is at least in part attributable to polyphenols. These results with such large scale screening might give useful information when choosing a potent antioxidant as a foodstuff.     

The effects of distance from coral reefs on seagrass nursery use by 5 emperor fishes at the southern Ryukyu Islands,Japan

Whilst the importance of seagrass beds as nurseries for coral reef fishes has been clearly recognized, the vast majority of early studies on fish nursery habitats emphasized the close proximity of the latter to coral reefs. To determine the potential nursery role of isolated seagrass beds, we investigated the degree to which juvenile emperor fishes (Lethrinidae) utilized seagrass beds in the presence/absence of adjacent coral habitats at Ishigaki Island (southern Japan), such fishes being known to use seagrass beds as nurseries. Seagrass beds in close proximity to coral habitats (distance between the two habitats of 50–200 m) had greater densities of lethrinid juveniles than those without adjacent coral habitats (2.5–4 km) for 3 different sites investigated, although a significant difference was obtained only for 1 site. Juveniles of Lethrinus atkinsoni, L. obsoletus, L. harak, and L. nebulosus were observed in seagrass beds with and without adjacent coral habitats, whereas L. ornatus occurred only in the former. Overall, most lethrinid juveniles utilize seagrass beds irrespective of the presence of adjacent adult coral habitat, suggesting that both types of seagrass beds would contribute to lethrinid adult populations. Therefore, management efforts for lethrinid populations should be applied not only to contiguous coral-seagrass habitat systems but also to isolated habitats.     

Effect of cultivation methods on nutritional enrichment of euryhaline rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

This study aimed at comparing fatty acid contents of rotifers cultured with different methods after nutritional enrichment in order to evaluate the rotifer quality produced by these methods. Rotifers were cultured using either a batch or a continuous culture. From the batch culture, three experimental subpopulations were used, sampled from the culture at 1, 24, and 48 h after rotifer inoculation. The continuous culture was performed with two tanks; one was for cultivation with continuous feeding and water supply (cultivation tank), and another was for harvesting from the cultivation tank by overflow (harvest tank). From the continuous culture, two subpopulations were used: one from the cultivation and one from the harvest tank. Nutritional enrichment was performed after each culture. Each population was enriched with Nannochloropsis oculata or a commercial enrichment diet. When the enrichment was performed with N. oculata on populations at 24 h after inoculation originating from either of the two tanks of continuous culture or the batch culture tank, a higher quantity of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was obtained from the two tanks of continuous culture. The same results were obtained when enrichment diet was used, this time including docosahexaenoic acid (DHA).     

Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

Relationship between fish growth rate and progression of artificially induced erythrocytic inclusion body syndrome in coho salmon, Oncorhynchus kisutch (Walbaum)

Abstract. Studies were conducted to clarify the relationship between growth rate and disease progression of erythrocytic inclusion body syndrome (EIBS) in artificially infected coho salmon, Oncorhynchus kisutch (Walbaum). In the diseased state, the haematocrit values decreased and the number of erythrocytes with inclusions was higher in faster growing fish. Rapid growth was accompanied by an abundance of immature erythrocytes, which had the greatest incidence of inclusion bodies.