Growing depth limit of <Emphasis Type="Italic">Zostera caulescens</Emphasis> in coastal waters along the Japan Sea coast of Honshu,Japan

There is a possibility that deep coastal marine macrophytes will be less critically affected by thermal stress associated with climate change and remain as refugia. Thus, information on them is expected to contribute to conservation of biodiversity in coastal areas affected by climate change. To document the deep-growing Zostera caulescens in relation to the light environment, field surveys were conducted in coastal waters of the central area along the Japan Sea coast of Honshu, Japan, and then the relationship between the light environment and seagrass depth limit was examined. In the coastal waters of Sado Island and Noto Peninsula, the presence of deep coastal communities of Z. caulescens was confirmed in the depth range of 20–25 m. Biomass and shoot density for Z. caulescens, ranging 34.1–51.3 g DW m^?2 and 83–112 shoots m^?2, were recorded at a depth of 20 m in Ryotsu Bay, Sado Island. For the genus Zostera including the deep-growing Z. caulescens, the relation between the extinction coefficient and seagrass depth limit was described by the fitted regression equation. The minimum light requirement of the deep-growing Z. caulescens was markedly lower than that of the shallow-growing Zostera marina.     

Antibacterial and hemolytic effects of aqueous and organic extracts from different tissues of sea urchin <Emphasis Type="Italic">Echinometra mathaei</Emphasis> on pathogenic streptococci

In this study, the antibacterial and hemolytic effects of the gonadal tissue, test, spines and Aristotle’s lantern of the sea urchin (Echinometra mathaei) were investigated. The aerobic Gram-positive pathogenic bacteria Streptococcus mutans and Streptococcus sobrinus were selected because of their importance in oral diseases. The samples of sea urchin were collected from an intertidal zone in the Persian Gulf and then dissected. Finally, the crude extracts of the gonadal tissue, test, spines and Aristotle’s lantern of the samples were separately prepared by the solvents, including phosphate-buffered saline (PBS), 96 % ethanol (Et) and 80 % acetonitrile (ACN). The antibacterial activities of the extracts were evaluated using the well diffusion method in two concentrations of 1500 and 600 µg well^?1. The antibacterial effects against streptococcal species were observed in the organic extracts (Et and ACN) of the gonads and test while the extracts of spines and Aristotle’s lantern showed no antibacterial activity. The hemolytic activity of the PBS, 96 % Et, and 80 % ACN extracts from the sea urchin were evaluated using horse red blood cells. Hemolytic activity was observed only in the 80 % Et and ACN extracts of the gonad and test. In conclusion, the organic extracts of gonadal tissue showed the most inhibitory activity on the growth of S. mutans and S. sobrinus.     

<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> from tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis> sp.) transmitted to a new host,bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>), in China

Several outbreaks of Streptococcus agalactiae infection of bighead carp (Aristichthys nobilis) were observed in China. The molecular epidemiology and pathogenicity of S. agalactiae in bighead carp and tilapia (Oreochromis sp.) is poorly understood. In the present study, we identified S. agalactiae strains isolated from diseased bighead carp using the API 20 Strep kit and 16S rDNA sequencing and determined whether these strains came from tilapia. Of the 46 identified S. agalactiae strains, 24 strains were successfully isolated from diseased bighead carps, 20 S. agalactiae strains were isolated from tilapia, and two S. agalactiae strains were isolated from tiger frog (Hoplobatrachus chinensis). The results of molecular typing, including multilocus sequence typing, molecular serotyping, surface protein gene detection, and virulence-related gene detection showed that the 44 strains from bighead carp and tilapia were highly similar, whereas different from tiger frog GBS strains. Remarkably, the bighead carp strain Hn1404 showed high virulence in bighead carp and zebrafish. Moreover, this strain was pathogenic to Nile tilapia (Oreochromis niloticus). In addition, comparative genomic analysis showed that isolate Hn1404 had a close relationship with the bighead carp and tilapia S. agalactiae strains. All the analyses of the genetic characteristics of bighead carp and tilapia strains showed that tilapia S. agalactiae strains could be transmitted to other fish species such as bighead carp.     

Long-term effects of stocking density on survival,growth performance and marketable production of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

Sea urchins were stocked at a density of 15 (D15), 30 (D30), 45 (D45) and 60 (D60) urchins/cage (0.3 m long × 0.2 m wide × 0.4 m high) in a laboratory culture environment for 16 months. The wet body weight (BW) and test diameter growth were monitored at 2-month intervals during the experiment. At the conclusion of the experiment, the surviving sea urchins were counted and the gonad wet weight (GW) and gonad color were measured. Specific growth rate (SGR) of body weight, survival rate (SR), gonad index (GI), gonad color difference (ΔE ₀₀), coefficients of variation (CV) of BW, GW, GI and ΔE ₀₀, total biomass yield (TY) and total gonad yield (TGY) per cage were calculated. Two marketable yield variables, graded according to gonad index, i.e., marketable biomass yield (MY) and marketable gonad yield (MGY), were also calculated. Coefficient of variation of final body weight (FW) and final test diameter (FTD) of sea urchins increased as the stocking density increased, indicating the existence of adverse social interactions. These adverse social interactions detrimentally affected FW, FTD, SGR, GW and GI (P < 0.01). Although SR decreased with the increasing densities, no statistical significant difference was detected. Sea urchins at D15 had the lowest gonad color difference (ΔE ₀₀) (P < 0.05). However, statistically equal CV of ΔE ₀₀ indicates this density effect was not a result of adverse social interactions. TY and TGY increased with increased density and can be described by the following equations: TY = 84.18X ^0.64, R ² = 0.999 and TGY = 24.16X ^0.38, R ² = 0.979. However, the MY and MGY were not significantly different among stocking densities. The results of this study demonstrate that in intensive culture S. intermedius at low stocking density can achieve high growth rate, gonad index and desirable color without decreasing the marketable yield. Farmers should choose to culture S. intermedius at low stocking densities.     

Gene expression of thyrotropin- and corticotrophin-releasing hormones is regulated by environmental salinity in the euryhaline teleost <Emphasis Type="Italic">Sparus aurata</Emphasis>

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65–81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments.     

Identification and characterization of microRNAs in the muscle of <Emphasis Type="Italic">Schizothorax prenanti</Emphasis>

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in the skeletal muscle of Schizothorax prenanti remain unknown. In this study, we analyzed the miRNA profiling in the skeletal muscle of S. prenanti at 30 days post-hatching (dph), 1 year, and 3 years by high-throughput sequencing. Two hundred twenty-nine unique miRNA types aligned to 201 independent pre-miRNA loci according to sequence similarity and 28 novel miRNAs were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in S. prenanti were highly conserved. Eight identified miRNAs validated using stem-loop qRT-PCR were differentially expressed in the process of skeletal muscle development of S. prenanti. This study provide not only an overall insight into the miRNA landscape in the skeletal muscle of S. prenanti but also a basis for further investigation of miRNA roles in skeletal muscle development of S. prenanti.     

Growth performance and biochemical composition of juvenile pearl oyster <Emphasis Type="Italic">Pinctada martensii</Emphasis> fed on artificial diets

The effects of three artificial diets (S1, S2 and S3) on survival, growth and biochemical composition of one-year-old pearl oyster Pinctada martensii were investigated. Six experimental groups (EG1, EG2, EG3, EG4, EG5 and EG6) and one control group (CG) were set up. EG1, EG2 and EG3 were solely fed on S1, S2 and S3, respectively. EG4, EG5 and EG6 were fed on mixed diets, as follows: S1 and Platymonas subcordiformis; S2 and P. subcordiformis; and S3 and P. subcordiformis, respectively. CG was fed on only P. subcordiformis. All groups were continuously fed for 60 days. Survival, growth and biochemical composition of soft tissues were compared across the groups. Results showed that survival rate, the absolute growth rate (AGR) and relative growth rate (RGR) of shell length did not differ significantly across the groups (p > 0.05). The AGR and RGR of total weight differed significantly among the groups (p < 0.05). AGR and RGR of shell length and total weight were the highest in EG5 and the lowest in EG1. Gross fat content showed insignificant differences among the groups (p > 0.05). However, gross protein content and ash content showed significant differences across the groups (p < 0.05). The gross protein of the groups solely fed on artificial diets was lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. The contents of other amino acids, total amino acids (TAAs) and essential amino acids (EAAs) showed significant differences across the groups (p < 0.05). TAA, EAA and delicious amino acids of the groups fed solely on artificial diets were lower than those of the groups fed on mixtures of artificial diets and microalgae or single microalgae. Results indicated that the artificial diet (S2) can serve as substitutes of microalgal diets for P. martensii.     

Alternative microalgal diets for cultivation of the fairy shrimp <Emphasis Type="Italic">Branchinella thailandensis</Emphasis> (Branchiopoda: Anostraca)

Fairy shrimp is known as a nutritional food for fish and crustaceans in aquaculture. In most hatcheries, the microalga Chlorella sp. appears to be the most common, suitable, and nutritious food to feed fairy shrimp. In this study, we attempted to determine other alternative algal diets for cultivation of fairy shrimp Branchinella thailandensis. Seven experimental diets including three treatments of dried Spirulina sp. at 0.75 (S1), 1.5 (S2), and 3.0 mg dry weight individual^?1 (S3); three treatments of Chlorococcum humicola at 5 × 10⁵ (Ch1), 1 × 10⁶ (Ch2), and 2 × 10⁶ cells mL^?1 (Ch3); and a control diet (Chlorella vulgaris at 1 × 10⁶ cells mL^?1) were fed to 5-day-old shrimp for 15 days. Evaluation of growth performance, egg production, survival percentage, and nutritional and carotenoid content of the experimental fairy shrimp revealed that Ch3 is the most suitable algal diet. Our results suggest that C. humicola is the best alternative food source for the cultivation of B. thailandensis. In addition, dried Spirulina powder is also a good choice when live algae are not available and can be used as an alternative feed in fairy shrimp cultures.     

Long-term fluctuations in mollusk populations before and after the appearance of the alien predator <Emphasis Type="Italic">Euspira fortunei</Emphasis> on the Tona coast,Miyagi Prefecture,northern Japan

The impact of Euspira fortunei as an alien predator on fluctuations in population densities of prey mollusks between 2001 and 2010 on the Tona coast, northern Japan, was investigated. This species increased dramatically from 2002 to 2004. In contrast, prey species such as Ruditapes philippinarum and Macoma incongrua decreased rapidly from 2001 to 2004, whereas Pillucina pisidium and Batillaria cumingii did not show a significant decrease during this period. Using a laboratory experiment, we were able to show that these decreases in the population densities of some species of mollusks but not other species were the result of species-selective predation by E. fortunei, as the experiment revealed that E. fortunei preferred to attack R. philippinarum and M. incongrua rather than P. pisidium, although the predator did expand its diet to include P. pisidium after it had consumed 16 of the 20 preferred prey available (i.e., R. philippinarum and M. incongrua). This case study suggests that invasive naticid predators have the potential to affect the population density and community structure of prey mollusks in recipient coastal ecosystems through predation.     

Effects of low-voltage constant direct current on plasma biochemical profiles and gene expression levels in crucian carp <Emphasis Type="Italic">Carassius carassius</Emphasis>

This study investigated the plasma biochemical profiles and gene expression levels of crucian carp Carassius carassius subjected to anesthetization with low-voltage constant direct current (DC) and tricaine methanesulfonate (MS222). Fish in the MS222 treatments and the DC treatments were exposed to either two concentrations of MS222 (100 and 200 mg/l) or constant DC with a voltage of 26 V, at a voltage gradient of 0.68 V/cm, respectively. The results showed that low-voltage constant DC immobilized fish more quickly, resulting in significant elevations of plasma cortisol levels compared to exposure of high or low doses of MS222. Electronarcosis led to an increased expression of heat shock proteins 70 (HSP70) as well as HSP90 compared to the treatment group with MS222. Also, electronarcosis enhanced mitochondrial respiratory chain gene (atp6, cox1 and nd5) and antioxidant enzyme gene (CAT, SOD and GSH-px1) expression levels. In conclusion, low-voltage constant DC can disturb fish plasma biochemical profiles and gene expression levels.     

Characterization and expression of <Emphasis Type="Italic">lin-28a</Emphasis> involved in <Emphasis Type="Italic">lin28/let-7</Emphasis>signal pathway during early development of <Emphasis Type="Italic">P. olivaceus</Emphasis>

Heterochronic lin-28 is a conserved RNA-binding protein that plays a key role in the timing of developmental events in organisms. As a crucial heterochronic gene, the protein controls developmental events of the second of four larval stages in Caenorhabditi elegans. Heterochronic let-7 miRNAs are often present in various species and highly conserved in sequence and biological function and are required for various biological processes. Previous studies showed that ten let-7 miRNAs were identified in the Japanese flounder (Paralichthys olivaceus) and that they were primarily expressed during metamorphosis. In this study, we clone and characterize the lin-28a gene from P. olivaceus and exhibit its dynamic expression pattern at different developmental stages and various adult tissues. The results show that the P. olivaceus lin-28a gene has high sequence similarity with other species and is highly expressed in the embryonic stage but weakly expressed in the larval stage. In addition, lin-28a overexpression causes cell proliferation and significantly promotes the levels of pre-let-7a and pre-let-7d while markedly depressing let-7a and let-7d expression in FEC (Flounder Embryonic Cell), which indicate that lin-28 possibly blocks the maturation of let-7 miRNAs. Additionally, lin-28a is identified as a target gene of let-7 miRNAs, and let-7 miRNAs directly regulate lin-28a expression by targeting its 3′ UTR. Taken together, lin-28a along with let-7 miRNA participates in a lin-28/let-7 axis pathway that regulates cell division and timing of embryonic and metamorphic events in P. olivaceus.     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.     

Influence of different microalgal diets on gonadal development of the carpet shell clam <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> broodstock

Studies considering broodstock conditioning of Ruditapes decussatus mostly used flagellates and diatoms for feeding, mainly Isochrysis galbana and Chaetoceros calcitrans, respectively. The present study evaluated the effect of different microalgal diets on gonadic maturation of R. decussatus including three microalgae species (C. calcitrans, Tetraselmis suecica, and Nannochloropsis oculata) which were used to prepare six diets: three monospecific and three bispecific mixed diets at a proportion of 1:1. Clams were daily fed at a ratio of 1% dry weight of algae/live weight of clam and unfed treatment was used as control. Results showed that clams fed C. calcitrans alone or mixed with T. suecica attained earlier full maturity with the highest response to spawning induction (after 34 conditioning days) and largest proportion in the partial spawning phase for both, followed by T. suecica alone after 48 days. In contrast, N. oculata alone or mixed diets demonstrated poor performance for gonadal maturation. Unfed treatment did not show any sign of maturation. The results indicated that T. suecica has high conditioning potential like C. calcitrans and their mixture gave the highest benefit. Therefore, T. suecica can be recommended for R. decussatus broodstock conditioning.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Feeding of the short-spined sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> on macroalgae and benthic animals

To examine the feeding ecology of the sea urchin Strongylocentrotus intermedius, two types of macroalgal diet (kelp Saccharina longissima and red alga Ptilota filicina) and four types of benthic animal diet [barnacle Balanus glandula, limpet Lottia cassis, free-living Perinereis aibuhitensis and P. aibuhitensis attached to a stainless steel wire (wired polychaete)], were separately or simultaneously provided for urchins of 5- to 60-mm test diameter (TD). Sea urchins of even 3-mm TD could graze the kelp, and began to graze the red alga, the wired polychaete and limpet by 5-mm TD, the barnacle by 8-mm TD and the free-living polychaete by 15-mm TD, respectively. These results suggest that the variety of macroalgae and benthic animals which the urchin can graze increases with the ontogeny of their feeding organs, motility and ability to catch animals. Both juvenile and adult sea urchins preferred to eat the polychaete as well as the kelp fronds. Feed conversion efficiency and daily growth rate of the small sea urchins fed benthic animals like the polychaete were higher than those fed macroalgae. The sea urchins are likely to grow better when feeding on animals than on macroalgae, even in habitats where they can access adequate quantities of kelp.     

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.     

Spontaneous polyploidization in critically endangered <Emphasis Type="Italic">Acipenser mikadoi</Emphasis>

We investigated the source of spontaneous polyploidization in the critically endangered Acipenser mikadoi. Fourteen sib progeny of A. mikadoi and 11 hybrids between an A. mikadoi female and a Huso dauricus male, all showing atypically high ploidy, were analysed. Parent assignment based on five highly polymorphic microsatellite markers confirmed spontaneous duplication of maternal chromosome sets via retention of the second polar body to be the source of spontaneous polyploidization. To our knowledge, this provides the first evidence of the maternal origin of spontaneous polyploidization in A. mikadoi. Factorial correspondence analysis of the multilocus microsatellite genotypes placed the parent fish of spontaneous polyploids in clearly delineated clusters of A. mikadoi and H. dauricus, and parent fish had mitochondrial control region haplotypes corresponding to their presumed species. Thus, parent fish were confirmed to be of pure genetic origin, and hybridization did not promote the observed spontaneous polyploidization.