Genetic difference between Ezo-awabi <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and Kuro-awabi <Emphasis Type="Italic">H. discus discus</Emphasis> populations: Microsatellite-based population analysis in Japanese abalone

ABSTRACT:   This study documents the genetic population relationships between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus by means of the microsatellite technique with an emphasis on the extent of genetic difference between Ezo- and Kuro-awabi populations. Eight markers were employed to screen five populations each collected from Ezo- and Kuro-awabi habitats. All eight loci showed polymorphisms in all populations (number of alleles per locus, 11.1–14.1; averaged expected heterozygosity, 0.64–0.70). The number of loci that yielded significant genetic heterogeneities (allele frequency distribution and/or pairwise θ ) between Ezo- and Kuro-awabi populations was larger than that generated in comparisons between populations within Ezo- and Kuro-awabi habitats. According to the bootstrap neighbor-joining trees constructed on the basis of two genetic distance measures ( D _A and D _ST), the 10 populations fell into two clusters of populations (Ezo-awabi and Kuro-awabi habitat groups), and the results of hierarchical amova testing also supported the neighbor-joining clustering. The outcomes presented here suggest that the microsatellite markers used in this study are potentially an efficient means to differentiate Ezo- and Kuro-awabi populations.     

Ontogenetic habitat shift in abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>: a review

Ecological literature on abalone Haliotis discus hannai populations is reviewed to identify processes and factors driving ontogenetic shifts in habitat. Abalone recruitment is related to the algal community type, with abalone shifting with growth from deeper crustose coralline algal (CCA) habitats to shallower kelp bed habitats via algal turfs. Timing of the habitat shifts is linked to ontogenetic changes in diet, from benthic diatoms to a diet dominated by macroalgae. Susceptibility to predation appears to change, concomitant with the shift from cryptic microhabitats during early-life stages to more exposed habitats during latter-life stages. Interstitial spaces between cobbles and boulders in the CCA habitat are considered to be important for reducing predation risk. The loss of CCA nursery habitat through sedimentation and macroalgal overgrowth likely negatively affects abalone recruitment. Preservation of diverse algal communities, including suitable habitats for the different abalone growth stages, is critical for successful abalone recruitment.     

Dietary value of gametophytes and juvenile sporophytes of the brown macroalga <Emphasis Type="Italic">Eisenia bicyclis</Emphasis> for juvenile abalone <Emphasis Type="Italic">Haliotis diversicolor</Emphasis>

The dietary value of juvenile stages (gametophyte and juvenile sporophyte) of the brown macroalga Eisenia bicyclis for post-larval and juvenile abalone Haliotis diversicolor of 2.0–6.5 mm in shell length (SL) was examined and compared with that of a benthic diatom, Nitzschia sp., in laboratory experiments. Most abalone actively fed on these diets, but there were large variations in the growth rate among the diets and among the growth stages of abalone. Growth rates of abalone fed on Nitzschia sp. were highly variable within each growth stage, but showed no clear differences among growth stages. In contrast, in abalone fed gametophytes or juvenile sporophytes, growth rates linearly increased as abalone grew. Growth rates of >60 μm SL/day were observed in juveniles of >3 mm SL fed gametophytes, and juveniles of >5 mm SL fed juvenile sporophytes. These results indicate that the dietary value of the juvenile stages of E. bicyclis for the abalone changes as they grow, and with growth juvenile abalone begin to efficiently utilize gametophytes and juvenile sporophytes in that order.     

Purification and some properties of alanine racemase from the marine gastropod <Emphasis Type="Italic">Cellana grata</Emphasis>

ABSTRACT:   High levels of free d -alanine were found in the muscle of a marine gastropod Cellana grata that inhabited the intertidal zone, and alanine racemase activity was detected in the muscle. The authors purified alanine racemase from the muscle of C. grata to characterize its enzymological properties. The molecular mass of the enzyme was estimated to be 40.5 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 41.4 kDa by gel filtration, suggesting that the enzyme was monomeric in nature. Kinetic experiments, performed using the purified enzyme, revealed that the Lineweaver-Burk plot for d -alanine as a substrate resulted in a K _m value of 20.4 mM, and the value for l -alanine was 43.0 mM. Of the several types of amino acids tested, alanine was found to be the specific substrate for the enzyme. In the measurement of alanine racemase activity, exogenous pyridoxal 5'-phosphate (PLP) was not required for the enzyme activity; however, aminooxyacetic acid, hydroxylamine and phenylhydrazine, which inhibit PLP-dependent enzymes, strongly inhibited the enzyme activity. These results suggest that the enzyme is PLP-dependent. This is the first report on the purification and some properties of alanine racemase in a marine gastropod.     

Complete nuclear ribosomal DNA sequence analysis of Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Pacific abalone Haliotis discus hannai (Haliotidae, Gastropoda) is an economically important shellfish species in northern China. The complete nuclear ribosomal DNA (nrDNA) of Pacific abalone was amplified, sequenced, and analyzed. The length of the nrDNA was determined to be around 10.7 kb, and to contain, in order, small subunit ribosomal RNA (nrSSU) genes (1871 bp), internal transcribed spacer (ITS, 759–762 bp), large subunit ribosomal RNA (nrLSU) gene (3411 bp), and an intergenic spacer (IGS, 4624–4654 bp). The SSU and LSU regions were almost identical in different individuals, and show little variation from those of other abalone species. The two different variations of the ITS2 region were presented, and this phenomenon also existed in other species. A phylogeny tree was constructed, based on ITS region sequence datasets, to determine the evolutionary relationships of abalones. Abalones have two major subclades, mainly distributed in the North Pacific, Europe and Australia. The IGS region of the nrDNA was sequenced and analyzed for the first time. Several repeat fragments were present upstream of the sequence, and were significantly different between individuals (93.86% sequence identity). The complete nrDNA sequence will be useful for the classification, identification, phylogeny, germplasm management, and breeding of this shellfish.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Use of thraustochytrid <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. as source of lipid and fatty acid in a formulated diet for abalone <Emphasis Type="Italic">Haliotis asinina</Emphasis> (Linnaeus) juveniles

The effects of using thraustochytrid Schizochytrium sp. as source of lipid and fatty acids in a formulated diet on growth, survival, body composition, and salinity tolerance of juvenile donkey’s ear abalone, Haliotis asinina, were investigated. Treatments consisted of diets either containing a 1:1 ratio of cod liver oil (CLO) and soybean oil (SBO) (Diet 1) or thraustochytrid (Diet 2) as source of lipid and fatty acids at 2 % level. Natural diet Gracilariopsis heteroclada (Diet 3) served as the control. No significant difference in growth was observed in abalone fed Diet 3 (SGR: 5.3 % BW day^?1; DISL: 265 μm day^?1) and Diet 2 (SGR: 5.2 % BW day^?1; DISL: 255 μm day^?1). Survival ranged from 78 to 85 % for all treatments and was not significantly different from each other. A 96-h salinity stress test showed highest survival of 84 % in abalone fed Diet 2 compared with those fed diets 1 and 3 (42 %). The high growth rate of abalone fed Diet 2 and high tolerance to low salinity could be attributed to its high DHA content (8.9 %), which resulted to its high DHA/EPA ratio of 10.5 %. These fatty acids play a significant role in abalone nutrition. The fatty acid profile of abalone meat is a reflective of the fatty acid profile of the oil sources in the diet. The present study suggests that the use of Schizochytrium oil in lieu of CLO and SBO can support good growth of abalone which is comparable with abalone fed the natural seaweeds diet.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

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.     

Establishment of a model for chemoattraction of <Emphasis Type="Italic">Symbiodinium</Emphasis> and characterization of chemotactic compounds in <Emphasis Type="Italic">Acropora tenuis</Emphasis>

Corals harbor symbiotic dinoflagellates, Symbiodinium spp., acquired from surrounding environments. Because Symbiodinium are present at low densities in the water column, corals may attract these symbionts using chemotactic compounds. To examine whether corals contain chemotactic compounds, we established an assay to measure the chemotactic activity for Symbiodinium using an extract of the coral Acropora tenuis, a major reef-building coral in Japan. Our assay revealed that Symbiodinium strain NBRC102920 (clade A), which is taken up by juvenile A. tenuis polyps, is attracted to crude A. tenuis extracts. We found that the chemotactic compounds are water-soluble, heat-labile macromolecules and that the chemotactic activity was inhibited by N-acetyl-d-glucosamine (GlcNAc). We separated the GlcNAc-binding fraction (Fr-ActL) and identified it as the most plausible candidate for the chemoattractant, since the chemotactic activity of the crude A. tenuis extract appeared to be mainly attributable to the activity of Fr-ActL and was also inhibited by the addition of GlcNAc. These results indicate that chemoattraction is mediated via the binding of Symbiodinium to Fr-ActL.     

Isolation and characterization of <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> causing infection in farmed <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> in China

In August 2008, a massive epizootic occurred among Portunus trituberculatus reared together with Palaemon carincauda Holthuis and Sinonovacula constricta in the seawater pond of Ningang farm, Rudong district, Jiangsu province, China. The disease occurred in crabs from juveniles to adults, and the mortality rate reached 30–40%. The diseased crabs exhibited lethargy, hepatopancreas turgidity, and elevated levels of turbid hemolymph. A gram-negative, rod-shaped bacterium (designated as strain LGS-1) was isolated from the ascitic fluid of the diseased and moribund crabs and was confirmed as the causative agent by our infectivity study. We conducted morphological and biochemical characterization of LGS-1, and sequenced the 16S rRNA gene, which led us to identify the bacterium as Vibrio metschnikovii. Drug sensitivity tests showed that this pathogenic bacterium is sensitive to florfenicol, orfloxacin, and SXT, but completely resistant to antibacterial drugs like gentamicin, erythrocin, and acheomycin. This is the first report on Vibrio metschnikovii as a virulent pathogen for Portunus trituberculatus.