Abstract:The half-smooth tongue sole, Cynoglossus semilaevis, is an increasingly important marine flatfish of potentially significant aquaculture value in China. It exhibits prominent sexually dimorphic growth in which females grow two to three times faster and larger than males. The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) plays a major role in embryonic development and sex determination. We evaluated the physiological function of a homologous Gadd45g gene on sex determination in C. semilaevis. Using RACE, we obtained a Gadd45g homologous gene from half-smooth tongue sole (named Gadd45g3). The full length Gadd45g3 was 1 147 bp, encoding 155 amino acid residues. A ribosomal_L7Ae domain was found in the sequence. Amino acid sequence analysis revealed that C. semilaevis Gadd45g3 shared 79%–88% identity with Gadd45g-like proteins in other fish and 58%–67% with Gadd45g in bony fish and higher vertebrates. The phylogenetic tree suggested that Gadd45g3 in C. semilaevis and Gadd45g-like in other fish were closely clustered in one branch because of their high similarity. Gadd45g-like genes were homologous with Gadd45g and typical for fish. They were produced from the additional fish-specific whole genome duplication. The ratio of Ka to Ks was used to diagnose the pattern of sequence evolution. The Ka/Ks for all comparisons of Gadd45g3 with homologous genes in other species was <0.3 suggesting that Gadd45g homologous genes are highly conserved. However, the Ka/Ks ratio for Gadd45g3 and Gadd45g homologous genes in mammals was higher than the former comparisons, suggesting that the function of Gadd45g3 in C. semilaevis varies from that of Gadd45g in mice. FISH results revealed that the Gadd45g3 gene is located on the autosome. Gadd45g3 expression was highest in the brain of both males and females, suggesting it is involved in the neurological development of C. semilaevis. Gadd45g3 expression was significantly higher in the testis of mature males and neo-males than in the ovary. In 20-day-old male larva, the expression of Gadd45g3 was markedly higher in the gonad than in same-aged females, whereas there was no difference in the level of gonadal expression between sexes in 95-day-old larva. Our results suggest that Gadd45g3 is a gender related gene that is necessary for testis maturation. Additionally, Gadd45g3 is involved in sex determination prior to gonadal differentiation. Moreover, Gadd45g3 also plays an important role in the early development of the ovary and testis.

Abstract:Insulin-like growth factor-I (IGF-I) is a key hormone in the vertebrate growth axis. Thus, the identification of polymorphic IGF-I gene molecular markers could aid in selection for desirable growth traits. We identified two polymorphic microsatellite loci with tetrad repeats in the IGF-I gene by polymerase chain reaction (PCR) and sequencing in common carp (Cyprinus carpio). One locus in intron1 was designated intron1189, and the other locus in intron2 was designated intron2310. The allele and genotype information for the two loci were obtained by polyacrylamide gel electrophoresis for all individuals from Heilongjiang carp (Cyprinus cario haematopterus) (YL) (n=263), a selected line of German mirror carp (Cyprinus carpio L. mirror) (JL) (n=229), and a cold-resistant line of Hebao red carp (Cyprinus carpio var. wuyuanensis) (HL) (n=255). We evaluated the association between the genotypes that constituted >3 percent of the frequencies of the two loci and growth-related traits. The allele numbers were 15 (intron1189) and 25 (intron2310), and both loci were high polymorphic (PIC>0.5). The number of genotypes that exceeded 3% of the frequency were 12 and 9 (YL), 12 and 10 (JL), and 10 and 10 (HL), respectively. The polymorphism at the intron1189 locus was significantly correlated with body length at 135 and 325 d, and body weight at 325 and 385 d in YL (P<0.05). There was a significant correlation between the polymorphism of intron2310 locus and body length and body weight during each detection period in JL (P<0.05). Fish with genotype 185/229 at intron1189 had the lowest body length at 135 and 325 d and the lowest body weight at 325 and 385 d in YL. Conversely, fish with genotype 205/221 at intron1189 had the highest body length at 135 and 325 d and the highest body weight at 325 d in YL. At intron2310, JL individuals with genotype 290/350 had the lowest body length at all time points and the lowest body weight at 135, 325, and 385 d. In contrast, body length and body weight were highest at these time in JL individuals with genotype 318/350. In summary, we isolated two microsatellite loci that have high polymorphism from introns of the IGF-I gene. These markers appear to be correlated with growth performance and should be further investigated to develop marker assisted selection breeding programs in common carp.

Abstract:We evaluated the correlation of scale genes (ant, eda, edar, fgfr) with four growth traits in common carp(Cyprinus carpio). A total of 155 microsatellite markers were selected from the homologous sequence of the carp genome. We used 116 progeny from a self-bred line of F1 derived from intercross lines of German mirror carp (Cyprinus carpio L.) and Amur wild carp (Cyprinus carpio haematopterus). We estimated the following genetic diversity parameters: allele number(No), effective allele number(Ne), observed heterozygosity(Ho), expected heterozygosity(He) using PopGene (Version 1.32). We used achi-square test to test for Hardy–Weinberg equilibrium. Of the 155 markers, 36 microsatellite loci were polymorphic with 2–4 alleles for each locus (86 alleles for the 36 loci). The mean number of alleles and the mean effective number of alleles were 2.388 9 and 2.209 4, respectively. The mean observed heterozygosity and expected heterozygosity were 0.624 5 and 0.529 2, respectively. The mean polymorphism information content (PIC) was 0.432 1 for the 36 loci, of which 23 loci had a moderate level of polymorphism (0.25≤ PIC<0.5) and 13 loci had a high level of polymorphism (PIC≥0.5). Our results suggest the population has a moderate level of polymorphism. Given that the microsatellite markers were located in the genes or their contexts, the level of polymorphism may be even lower. The majority (69%) of the 36 loci deviated significantly from Hardy–Weinberg equilibrium. The artificial selection pressure and development of the self-bred line may explain this phenomenon. Of the 36 markers, 10(28%), 7(19%), 7(19%), and 11(31%) were significantly associated with body weight, body length, body height, and body thickness, respectively. Eleven dominant genotypes were identified via the multiple comparisons. The higher screening rate of SSR markers tightly linked with growth traits demonstrated that the scale genes maybe associated with growth traits.

Abstract:The swimming crab, Portunus trituberculatus, is a commercially important fishery species and widely distributed on sandy and muddy bottoms in the coastal waters of Japan, Democeratic People’s Republic of Korea , China and Malaysia. It is a euryhaline crab species, surviving in wide-range salinity conditions, and water salinity condition influenced its artificial propagations significantly. Aquaporins (AQP) which belong to a major intrinsic proteins (MIP) family plays important roles in salinity stress response. In this study, aquaporins cDNA of Portunus trituberculatus was cloned by rapid amplification of cDNA ends (RACE) and named PtAQP. The full-length of PtAQP cDNA is 1 712 bp, including an open reading frame (ORF) of 771 bp which encodes a 256 amino acid polypeptide. Bioinformatics software analysis reavealed that PtAQP gene contains 6 transmembrane domains, 2 NPA structural units and conservative amino acid sequence which is matched to MIP family. Homologous analysis showed that PtAQP of Portunus trituberculatus has the highest homology to AQP1 of Callinectes sapidus. The quantitative real-time RT-PCR showed that PtAQP gene could be detected in all tested tissues of Portunus trituberculatus, with the highest expression level in stomach. After challenged by high and low salinity, the expression of PtAQP gene in gill and hepatopancreas of Portunus trituberculatus first fell then rase. Our results confirmed the osmotic regulation function of PtAQP gene and revealed the regulatory mechanism of PtAQP gene under salinity stress.

Abstract:We evaluated the effect of changes in salinity on four genes involved in salinity regulation (glycine transporter, zinc finger protein, heat shock protein 70, neuronal acetylcholine receptor) in Apostichopus japonicus using real-time quantitative PCR. The basal level of glycine transport gene expression was highest in the respiratory tree, followed by intestinal tissue and coelomic fluid. The level of zinc transporter and acetylcholine receptor gene expression was highest in the coelomic fluid, and expression was not detected in the respiratory tree. Expression of the Hsp70 gene was highest in the coelomic fluid, followed by the respiratory tree and intestine. Exposure to low salinity stress resulted in changes in glycine transporter gene expression in all tissues. Expression peaked in the intestine and respiratory tree after 72 h and 1.5 h, respectively. Conversely, expression in coelomic fluid decreased and was at its lowest level after 3 h. Expression of the zinc transporter gene also changed in response to low salinity stress. Expression peaked after 24 h in the intestinal tissue and after 6 h in the coelomic fluid. Neuronal acetylcholine receptor gene expression was initially down-regulated in the intestine during exposure to the low salinity stress. However, there was a significant increase in expression in the coelomic fluid at 48 h. The expression of Hsp70 mRNA peaked after 48 h in both coelomic fluid and respiratory tree and after 12 h in intestinal tissue. Quantitative analysis of the expression of these genes revealed significant differences among the different tissues and different exposure times. The expression profiles suggested that the four genes can be used as indicators of salinity stress. Given the changes in expression, the protein products of the four genes are likely involved in the regulation of metabolism as function proteins, or control the expression and the activity of the stress proteins as regulatory proteins, to improve the salinity stress tolerance of A. japonicus. Our results provide a foundation for studying the mechanism adaption of sea cucumber for the future study. Key words: Apostichopus japonicus; low salinity stress; glycine transporter; zinc finger protein; neuronal acetylcholine receptor; Hsp70

Abstract:Final oocyte maturation is a key step for successful spawning and fertilization. Like most other vertebrates, teleosts have full-grown postvitellogenic oocytes in the ovary that are physiologically arrested at the G2/M border in the first meiotic prophase and cannot be fertilized. Shortly before ovulation, the oocytes must become fully mature, which involves breakdown of the germinal vesicle(GVBD), chromosome condensation, assembly of the meiotic spindle, and exclusion of the first polar body. Meiosis is again arrested at the second metaphase. The time period from the resumption of meiosis to the second meiotic metaphase has been referred to as final oocyte maturation. Knowledge of the molecular events and the role of various factors involved in final oocyte maturation is a focus of research in the field of reproductive biology. Quantitative real-time PCR (qPCR) is often used to quantify transcript abundance in such studies; however the technique is subject to considerable experimental error and variation. A stably expressed housekeeping gene is typically used as a reference gene to normalize this variation. However an ideal universal reference gene that is stable under all experimental circumstances has not been described. Researchers should validate their reference genes before performing qPCR analysis. We documented the expression of six candidate reference genes: 18S rRNA(18S), 28S rRNA(28S), cathepsin Z (CTSZ), elongation factor 1-α(EF-1α), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and β-actin during 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) induced final oocyte maturation in common carp (Cyprinus carpio). We used a range of software to evaluate the expression stability of these reference genes. Analysis with Bestkeeper revealed that EF-1α had the lowest CV% (2.65) and STDEV (0.71) values, but had the highest Bestkeeper index value (0.956) among the six candidate reference genes, suggesting that EF-1α was the best reference gene in the present study. Analysis with geNorm revealed that EF-1α and CTSZ had the lowest M (gene expression stability) values which indicated that they were the most stable genes among the six. Additionally, the value of pairwise variation of these two genes (V2/3) was much lower than the default (0.15), suggesting that EF-1α and CTSZ should be used concurrently as a reference gene pair to normalize the expression of the target genes. Consistently, analysis with NormFinder and RefFinder demonstrated that EF-1α was the most stable gene among the six candidates. Hence, EF-1α can serve as a reference gene to normalize the expression of target genes during final oocyte maturation induced by DHP in common carp. The mRNA expression of luteinizing hormone receptor gene(LHR) during final oocyte maturation induced by DHP was normalized by 6 candidate reference genes respectively, and the relative expression was varied significantly depending on the reference gene that was used. Selection of a stably expressed reference gene is critical for all qPCR analysis to ensure accurate target gene mRNA expression information。

Abstract:Mitochondrial DNA (mtDNA) content is typically estimated as the copy number ratio of mtDNA to nuclear DNA(nDNA). However, the accuracy of mtDNA content measurement is affected by many factors, including the conformation of plasmid standards and the DNA template, the coexistence of mtDNA pseudogenes in the nuclear genome, and selection of both mtDNA and nDNA primer-pairs. To minimize the influence of these factors, an optimized method to quantify the mtDNA content in different tissues of Cynoglossus semilaevis was established using real-time quantitative PCR (RT-qPCR). First, two sets of candidate standards (the circular and linear plasmid) and three sets of DNA templates (enzyme digested, ultrasonic treated, and untreated) were prepared to evaluate the influence of the DNA template conformation. Additionally, four mtDNA and three nDNA primer pairs were also tested to determine their adequacy for the qRT-PCR analysis. The linear plasmid standard was more appropriate than the circular one because the super helical structure of the circular plasmid caused significant overestimation in RT-qPCR. There was no significant difference in the estimates of mtDNA content resulting from different DNA templates, suggesting that the DNA extracted by phenol-chloroform is suitable without any pre-treatment for extraction. The D-loop and ND1 primers yielded the same copy number, which was also the lowest among all the mtDNA primer pairs. The copy numbers for ATP6 and COII were 3.5 and 1.5 times higher than those from D-loop and ND1, respectively. The higher copy number of ATP6 and COII may be related to the co-amplification of homologous pseudogenes in the nuclear genome. Single copy nDNA loci ENC1 and MYH6 can be used as references for detecting cell numbers of diploids, and the precise mtDNA content per cell can be calculated using the formula: mtDNA content = 2×mtDNA copy number/nDNA copy number. In contrast, the mtDNA content value was lower when using the multicopy nDNA gene locus GAPDH as a reference. To evaluate the accuracy and stability of this optimized method, we measured the mtDNA content in four tissues (liver, kidney, spleen, and muscle) of C. semilaevis. D-loop and ENC1 primer pairs were chosen for the RT-qPCR, and the mtDNA content per cell was estimated using the method established in this study. There was no significant difference between triplicate repeats in each tissue (P>0.05), which suggests that the method has excellent repeatability. Furthermore, there was a significant difference in mtDNA content among the different tissues: 244–255, 156–172, 97–107, 86–89 copies per cell were detected in liver, muscle, kidney, and spleen, respectively.

Abstract:Turbot (Scophthalmus maximus L .) are a cold-water fish. Despite this, their growth rate is inhibited by exposure to low-temperatures in regions of China where cultured fish are reared in natural sea water that is <8℃ from December to March. To counter this, farmers extract deep well seawater or use heated natural seawater for cultivation. The extraction of a large amount of groundwater can result in a serious decrease in the water table and subsequent water shortage. Heating water is not only a waste of energy, but also it causes air pollution. Additionally, the costs of turbot cultivation increase significantly during this period. Improvements in performance traits and breeding of rapid growth varieties under low temperature conditions are important for two reasons: a reduction in pollution and ground water use and an increase in profitability. We evaluated the growth performance and heritability of growth traits in fish held for different durations at a range of low-temperatures (10.5–12℃). We used a total of 536 turbots from 25 family groups, including 13 full-sib families and 5 half-sib family groups (with 3 families derived from a cross of one dam with two sires and 2 families derived from a cross of one dam with three sires). During the 90-day study period, we measured the body weight (BW) and body length (BL) of each individual on days 18, 54, and 90. We calculated the SGR (specific growth rate) to describe growth performance. A general animal model was used to estimate the heritability of BW and BL after 54 and 90 days, with the initial body weight or initial body length (day 18) as a covariate. The specific growth rates (SGR) during days 18–54 and 54–90 were (0.869±0.181)%/d and (1.039±0.185)%/d (P<0.01), respectively. The specific growth rate between 54–90 d was 15.6% higher than in the previous period. The growth rate of turbot improved significantly with time and growth performance differed among families (P<0.01, one-way ANOVA). Turbot were able to adapt to low-temperature and prolonged holding at low temperature promoted cultivation of cold-resistant turbot. The heritabilities for body weight at 54 and 90 days were 0.440±0.129 and 0.548±0.150 (P<0.01), respectively; and for body length were 0.301±0.108 and 0.494±0.142 (P<0.01, t-test), respectively. The heritabilities for body weight and body length at 90 days were significantly higher than at 54 days. Our results suggest that the environmental effect decreases as duration of exposure to low temperature increases and a genetic effect can yield an increase in the growth of turbot. The heritability of body weight and body length increased with the duration of exposure to low-temperature, and the heritability of body length was higher than that for body weight. Thus, body length has greater utility as a breeding trait than body weight.

Abstract:Microsatellite DNA markers are increasingly being used to improve selective breeding programs. Currently, there is a need for molecular markers associated with traits for marker assisted selection in shrimp breeding programs. We analyzed the association between microsatellite DNA markers and body length, total body height, and body weight in an F2 family of Chinese shrimp (Fenneropenaeus chinensis) that were mated by artificial insemination. Eighty individuals from the F2 family were chosen for the normal distribution test. Nineteen microsatellite primer pairs were obtained to amplify the total DNA and genotype each locus. We calculated the mean number of alleles, mean expected heterozygosity, mean polymorphic information content. Genotypic differences of these microsatellites loci between the maximal high group and minimal low group were analyzed using SPSS11.5 software. One-way ANOVA was conducted to test the association between the individual marker alleles and the three economic traits. The body length, total body height, and body weight were normally distributed(P>0.05). The mean number of alleles, mean expected heterozygosity, and mean polymorphic information content were 2.470, 0.533 and 0.442, respectively. The microsatellite loci RS0683 and FC019 were significantly associated with body weight, body length, and total body height (P<0.05 or P<0.01). The most favorable genotypes for growth traits at the microsatellite locus RS0683 was AA (P<0.01) and the most unfavorable at locus FC019 was AA (P<0.05). We speculate that the genotype AA was positively associated with the three economic traits at locus RS0683 and the genotype AA was negatively associated with the traits at locus FC019. Our results suggest that selective genotyping integrates the advantages of BSA and marker-based analysis to improve our ability to detect an association between quantitative trait locus and markers. Two of the microsatellite loci had a significant effect on the three economic traits. Identification of a large number of DNA markers linked to QTL controlling the resistance to WSSV will contribute to the application of DNA marker-assisted selection during breeding of Chinese shrimp for intensive culture

Abstract:Brachymystax lenok is an important commercial fish that are now at risk of extinction due to over exploitation and environmental pollution in China. The development of culture techniques for supplementation would benefit from a better understanding of the endocrine cells in digestive tract and their role in ingestion, digestion, and absorption of food. We documented the localization, regional distribution, and relative frequency of endocrine cells in the digestive tract of B. lenok. We used 6 mammalian antisera, including 5-hydroxytrptamine(5-HT), somatostatin(SS), pancreatic polypeptide(PP), gastrin (GAS) and substance P(SP) to locate the endocrine cells in 1-, 2-, and 3-year-old individuals by immunohistochemical SABC. 5-HT immunoreactive(-ir), SS-ir, and PP-ir cells were detected in the esophagus and throughout the stomach, including the stomachus cardiacus, stomachus fundus, and stomachus pyloricus of all age classes (except the esophagus of one-year old fish). The highest density of 5-HT-ir and SS-ir cells was in stomachus fundus of three-year old B. lenok(P<0.05). The density of PP-ir cells was significantly higher in the stomachus pyloricus of 2-year-old individuals than in 1- and 3-year-old fish (P<0.05). However, these three kinds of endocrine cells were not detected in the foregut, midgut, hindgut, or rectum. GAS-ir cells, GLU-ir cells, and SP-ir cells were not detected in any part of the digestive tract of 1–3-year-old B. lenok. As the endocrine cells of other teleosts, the 5-HT-ir , SS-ir, and PP-ir cells of B. lenok were divided into two types: open type with cytoplasmic process and closed type without a cytoplasmic process. In summary, 5-HT-ir, SS-ir, and PP-ir cells mature in the gastrointestinal tract of B. lenok after 1 year, and have the same regulatory function at this time as in adults. The density of these cell increased with age.

Abstract:We evaluated the effects of water temperature (12, 16, 20, 24, 28, and 32℃), salinity (5, 10, 15, 20, 25, and 30 ppt) and pH (7.2, 7.7, 8.2, 8.7, 9.2 and 9.7) on oxygen consumption and ammonia excretion in artificially propagated Mugil cephalus juveniles (0.21±0.03 g). The seawater temperature, salinity, and pH were maintained at (19±1)℃, 30, and 7.7, respectively, during the acclimation period. Dissolved oxygen was measured by Winkler`s iodometric method and ammonia was measured using the hypobromite oxidation method. Water temperature had a significant effect on both oxygen consumption rate YO and ammonia excretion rate (YN) and the co-relationship was expressed by the quadratic equations YO=0.0256X2+0.2191X0.1054 (P<0.05) and YN=0.0054X2+0.0441X0.0082(P<0.01), respe- ctively. The oxygen consumption rate and ammonia excretion rate of M. cephalus juveniles increased as water temperature increased and was highest at 24℃, after which the rates decreased. A one-factor analysis of variance revealed that salinity had a significant effect on the oxygen consumption rate (P<0.01). When the salinity was within the range 5–30, the oxygen consumption rate decreased, increased, then decreased again as salinity increased. Conversely, the ammonia excretion rate first increased, then decreased, and was expressed as Y=0.0013X2+ 0.0027X+0.047 (P<0.01). A one-factor analysis of variance revealed that pH also had a significant effect on both oxygen consumption rate and ammonia excretion rate. The oxygen consumption rate and ammonia excretion rate first increased, then decreased as pH increased. The co-relationship was expressed by the quadratic equations Y=0.02583X2+0.198X+0.0775 (P<0.01) and Y=0.02583X2+0.198X+0.0775 (P<0.01), respectively. Water temperature and pH had significant effects on the suffocation point (P<0.01). The salinity also had a significant effect on the suffocation point (P<0.05), but not on the time to suffocation (P>0.05). Our results provide insight into the bioenergetics of M. cephalus juveniles, and provide useful technical parameters for seedling rearing and live transportation.

Abstract:The Pacific white shrimp, Litopenaeus vannamei, originated in Pacific coastal waters but are now widely farmed in inland China and are one of the most important farmed aquatic species world-wide. Leucine plays an important role in the maintenance of nitrogen balance, energy metabolism, blood glucose concentration, growth hormone and hemoglobin concentration, and also stimulates protein synthesis in muscle tissues. Thus, a deficiency of leucine in diets can cause severe biochemical malfunction, including growth retardation. Until now however, the optimal leucine requirements of white shrimp were unknown. We evaluated the leucine requirements of juvenile Pacific white shrimp Litopenaeus vannamei (Boone) in low-salinity water(0.6−0.8 g/L). Six diets containing fish meal, peanut meal and crystalline amino acids with different concentrations of L-leucine(15.95, 17.95, 19.95, 21.95, 23.95, and 25.95 g/kg dry diet) were formulated to contain 400 g/kg crude protein. A total of 1 000 healthy shrimps (initial body weight: 0.38±0.004 g) were randomly distributed into 18 rectangular fiberglass aquariums (280 L, 3 aquaria per diet, 30 shrimps per aquarium) and fed one of the diets for 56 d. Weight gain rate increased significantly with increasing leucine concentration up to 23.95 g/kg dietary leucine (diet L11, P<0.05). The diets containing higher leucine concentrations had no effect on growth performance (P>0.05). Group L11 had the highest protein efficiency ratio (PER), body protein deposition (BPD), hemolymph total protein (TP), and the lowest feed conversion ratio (FCR), hemolymph aspartate aminotransferase (AST), alanine transaminase (ALT) activities, and hemolymph urea nitrogen (HUN) content. The PER, BPD, and TP of group L11 were significantly higher than those of group L7, whereas the FCR, AST, ALT, and HUN of group L11 were significantly lower than those of group L7(P<0.05). The survival of group L7 was lower than that of group L9 and L10 (P<0.05), but there was no significant difference among the other groups(P>0.05). Our results suggest that a lack of dietary leucine decreases growth performance and survival of Pacific white shrimp. A broken-line regression calculated using weight gain rate indicated that the optimal dietary leucine requirement for L. vannamei reared in low-salinity water was 24.80 g/kg leucine of dry diet, corresponding to 61.99 g/kg leucine of dietary protein.

Abstract:We evaluated the effects of dietary supplement with herbs on growth performance and plasma biochemical characteristics of Amur sturgeon (Acipenser schrenckii). A compound herbal additive, containing Poria, Radix Paeoniae Alba, Herba Houttuyniae, and Radix et Rhizoma Rhei, was added into the basal feed of Acipenser schrenckii. Amur sturgeon were assigned to one of four treatment groups (n=8 fish/group, initial body weight: 137.61±22.26 g) and fed a diet supplemented with 0% (control), 0.5%, 1%, or 2% of the herbal additive for 8 weeks. Each treatment group was conducted in triplicate. Fish were fed to apparent satiation three times daily. We measured growth, plasma biochemical profiles, plasma hormone levels, and immune-related indicators. The body weight of each fish was measured every thirteen days during the experimental period. The weight gain rate(WGR), specific growth rate(SGR), and digestibility (D) were significantly higher in the 0.5% group than in the control group (P<0.05). However, the SGR and WGR of fish fed diets supplemented with 1% and 2% herbal additive were not different from those in the control group (P<0.05). The highest acid phosphatase (ACP) activity was observed in fish fed a diet supplemented with 1% herbal additive (P<0.05). Plasma total protein and globulin contents were significantly higher in both the 0.5% and 1% groups than in the control group (P<0.05). Plasma concentrations of cholestenone (CHOL) and glycerin trilaurate (TG) decreased significantly in fish fed diets supplemented with the herbal additive(P<0.05). Plasma alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) increased significantly in fish fed 2% herbal additive(P<0.05). Supplementation with herbal additives reduced plasma cortisol levels in the 0.5% group but increased plasma T4 levels significantly in both the 0.5% and 1% groups (P<0.05). Our results suggest that supplementation with herbal additives at a 0.5% level can improve growth performance and alleviate stress in Acipenser schrenckii. Furthermore, the medicinal plants used in this study reduced the level of blood lipid in Amur sturgeon fed the formula feed.

Abstract:Virtual population analysis (VPA) is one of the most powerful techniques available to estimate the abundance of fish stock and study the dynamics of harvested fish populations. As a rule of thumb, VPA is commonly applied in fisheries stock assessment on the assumption of fishing mortality is constant throughout the year. However, the reality of fishery is complicated, and few fisheries will conform to the assumption. A cohort can be classified different fishing stage in terms of instantaneous rate of fishing mortality, e.g. fishing season and non-fishing season. And more importantly, many countries have initiated fishing moratorium measure to protect and restore the fishery resources within the context of the worldwide fisheries collapse. Therefore, we improved and extended the conventional VPA by incorporating the effect of fishing staging, or staging VPA to meet the challenge of fisheries characteristics with fishing season, non-fishing season or fishing moratorium in this paper. Based on the differences fishing mortality during the stage at one year, we analyzed and discussed the performances of four scenarios on the results of assessments using simulated fisheries data. The simulation study suggests that conventional VPA model will leads to 6%–33% biases due to ignoring or incorrect stages in fisheries stocks assessment. We have proved that the staging VPA overcame the shortage of conventional method which based on cohort and the results from this model is more close to the reality owing to it can be used to estimate not only continuous fishing fisheries but discrete fisheries also. This work highlights a need for a more careful treatment of the times of start and end of a fishing season in fish population models. The natural mortality rate and fishing mortality are two important parameters in staging VPA application. Some of the problems of data collection and parameters estimation that will be encountered while implementing staging VPA method are also discussed. In general, considering the staging VPA is appropriate for the reality of the fisheries, we concluded that this method is more reliable for fish stock assessments compared with the conventional VPA.

Abstract:Identifying the patterns of how stream fish assemblages vary spatially and how environmental factors influence fish assemblages is critical for fish species conservation and management. We evaluated the spatial patterns in fish assemblages at a river-network scale and documented the correlations between fish assemblages and local habitat features and tributary spatial position using data collected from 94 segments within headwater streams (1st-, 2nd-, and 3rd-order streams) of the Wanhe watershed at Dabie Mountain during July and August 2013. A total of 6 288 specimens representing 42 species, 13 families and 4 orders were collected, among which species of the family Cyprinidae comprised 52.38% of total species richness. Both local habitat (i.e., canopy cover, water temperature, conductivity, substrate size, and heterogeneity) and spatial position variables (i.e., stream order, link, downstream link, and confluence link) varied significantly with a decline in elevation. We observed significant variation in fish diversity, but not abundance, among the 1st-, 2nd-, and 3rd-order streams with higher species richness occurring in higher ordered streams. However, there was no variation in species richness and abundance among the three tributaries (i.e., the Changhe, Wanshui, and Qianshui streams). Both habitat (i.e., elevation) and spatial variables (i.e., stream order and downstream link) had a significant influence on fish species richness, but fish abundance was only influenced by local habitat (i.e., elevation). There was substantial overlap in assemblage structures across the three ordered streams, and an obvious nested pattern was observed for assemblage structures from 3rd-order to 1st-order streams in turn. Additionally, fish assemblage structures were affected by both habitat (i.e., elevation) and spatial variables (i.e., confluence and downstream links). Our results suggest that local fish assemblages in the headwater streams of the Wanhe watershed are determined by both local and spatial processes. Thus, both local habitat conditions and river-network connectivity should be considered in combination when framing schemes for the conservation and management of fish species in this study area.

Abstract:The demand for seafood is expected to rise substantially as both the standard of living and human population size continue to increase. Stock enhancement programs will likely help fill some of the projected gap in supply as well as replenish severely depleted or recruitment-limited fisheries. At present, stock enhancement programs have been widely implemented in the coastal waters of the Yellow Sea and Bohai Sea, China, including a program to enhance populations of the swimming crab (Portunus trituberculatus). However, little effort has been made to evaluate the effectiveness of these stock enhancement programs, particularly for species that are not suitable for physical tagging, such as the swimming crab. We conducted a quantitative analysis to assess the recapture rates of swimming crabs using survey data collected in May, June, July, and August of 2010 and 2011. We compared survey results before and after the release of swimming crabs in the waters off southern Shandong Peninsula. The wild population was relatively rare: only 0.9 individuals were caught per site per hour prior to release of cultured crabs in late May, 2010. Capture rates increased to 26.08 individuals per site per hour 10 days after the release of ~110.15 million seedling swimming crab, and the supplemented stock accounted for 96.55% of the total catch. The stock density was 1.70 individuals per site per hour during surveys in August, and the age-0 swimming crabs accounted for 64.29% of the total catch. The total yield of swimming crab was 3 108 tons in 2010, the average body weight was 232.3 g, and the recapture rate was 7.54%. In 2011, the density of the wild population of swimming crab was 1.70 individuals per site per hour before supplementation in late May, but increased to 29.14 individuals per site per hour 10 days after release of ~131.32 million seeding swimming crab, of which the supplemented stock accounted for 94.17% of the total catch. In August, the density had declined to 1.00 individual per site per hour and age-0 individuals represented 70.00% of the total catch. The total annual yield was 2 896 tons, the average body weight was 226.0 g, and the recapture rate was 6.43%. Our results suggest that the stock enhancement effort was more successful in 2010 than 2011. Our results also suggest that the water quality (temperature, transparency, salinity, nutrient composition and concentration) and food supply off southern Shandong Peninsula were suitable for stock enhancement of swimming crab, evidenced by the high recapture rates in 2010 and 2011. However, the poor water quality in Jiaozhou Bay and Huangjiatang Bay was associated with relatively low recapture rates

Abstract:Artificial reefs provide a substrate for macroalgae to form artificial algal beds. We surveyed the sessile macroalgae community in four artificial reef zones(ARZ) of Jiaming, Beihai, Shengkun, and Tonxin in Shuangdao Bay in June and August, 2013. These reefs were constructed in 2006, 2006, 2010, 2008. We documented the composition of the macroalgal communities and the interactions between the communities and environmental factors. Ten transects (50 cm × 50 cm) were set randomly in each ARZ. All the macroalgae in a transect were collected by a SCUBA diver. Additionally, underwater videos were taken to provide additional information. Environmental factors (temperature, light intensity, pH, DO, salinity, depth, and transparency) were recorded at the time of collection. The samples of algae were identified, measured, and weighed in the laboratory. We identified 9 species, including 5 species of Rhodophyta, 2 species of Phaeophyta, and 2 species of Chlorophyta. Rhodophyta was the most abundant genera, accounting for 93.69% of the biomass in June and 85.26% in August. The macroalgal communities differed significantly between the surveys in June and August, with a dissimilarity score of 79.65%, based on analysis of similarities (ANOSIM). Over 90% of the dissimilarity was contributed by five species, Chrysymenia wrightii, Heterosiphonia japonica, Gracilaria textorii, Sargassum muticum, and Ulva pertusa. The macroalgal community composition was similar at Jiaming and Tongxin (R = 0.059 in June and 0.217 in August, P>0.1, ANOSIM). The biomass increased from 164.99 g/m2 (June) to 243.829 g/m2 (August), whereas the percentage cover decreased from 64.53%–91.20% (June) to 52.53%–72.53% (August), except in the Shengkun ARZ. The dominant species was H. japonica in June and C. wrightii in August. Multidimensional scaling (MDS) analysis and clusters based on Bray-Curtis similarities revealed that the ARZs could be divided into four clusters, one containing Jiaming, Beihai, and Tongxin in June, one containing these three ARZs in August, one containing Shengkun in June, and one containing Shengkun in August. Based on principal component analysis (PCA) and redundancy analysis (RDA), water temperature, salinity, and benthic light intensity have the most influence on macroalgae community composition. Temperature was the most important factor, accounting for 45.30% of the variation in the communities. The communities in June consisted primarily of cold and warm temperate species whereas those in August consisted primarily of subtropical and warm temperate species. The relationship between salinity and community structure may be a function of the large amount of rainfall that occurred in August, whereas the effect of salinity might be negligible in other circumstances over a year. The transparency depth was ca. 2.5 m in June, but less than 2.0 m in August. Thus, light may be a limiting factor for the distribution of macroalgae. The ARZ in Shengkun had the lowest biomass, potentially a function of low light levels due to its depth. Additionally, blooms of fouling organisms (Spongiidae, Ciona intestinalis and Obelia sp.) in August affected the percentage rate. The appearance and abundance of Phaeophyta is representative of the maturity of the macroalgal communities. However, Phaeophyta was only found at the Beihai ARZ, a site that was deployed ca. seven years previously. Given the poor underwater light conditions, we speculate that the macroalgal communities in Shuangdao Bay require >7 years to mature

Abstract:The mudskipper(Boleophthalmus pectinirostris) is an amphibious fish, adapting to a wide range of temperature and salinity. It inhabits the mudflats of intertidal zone and the mangrove forests in China, Korea, Japan, Vietnam and Malaysia. In China, it is mainly found in coastal mudflats of Zhejiang, Fujian, Taiwan, Guangdong and Guangxi provinces. Due to its economic importance, this species is widely cultured in southeast China. In the past 30 years, its natural resource has decreased gradually due to overfishing, loss and fragmentation of its natural habitats and environmental pollution. Genetic diversity of this species has been previously studied, but the samples were only obtained from a certain range of its habitats. In this study, amplified fragment length polymorphism(AFLP) markers were used to analyze the genetic diversity of 8 B. pectinirostris populations from the coastal waters of Northwest Pacific, in an attempt to illustrate its phylogeographic patterns and genetic structure. The findings of this study will provide insights into the future fishery management and conservation for B. pectinirostris. Instead of the traditional method polyacrylamide gel electrophoresis (PAGE), QIAxcel DNA High Resolution Kit Gel Cartridges was used to perform the AFLP analysis. Among the 126 individuals from the 8 populations in Japan, Korea, Vietnam and Guangxi, Guangdong, Fujian, Taiwan, and Zhejiang provinces of China, 186 loci between 15 bp and 500 bp were detected using 6 selective amplification primers(E-AGC/M-CTG, E-AGC/M-CCA, E-ATG/M-CCA, E-ATG/M-CTT, E-ACT/M-CAC, E-AGG/M-CTT), and 160 of which were polymorphic (86.02%). The amplified bands ranged from 25 to 36 for each primer combination. On the other hand, the detecting rates of each primer varied from 52.0% to 96.42%, with E-ACT/M-CAC showing the lowest detecting rate of 52.0% while other primers were all above 82%. The proportion of polymorphic loci from 8 populations varied from 27.96% to 57.53%, with the highest one for Korea population and the lowest one for Guangdong(China) population. The polymorphic loci proportion of B. pectinirostris were much lower than those of other fish species. Overfishing, habitats loss and environmental pollution may be the reasons for the low genetic diversity of B. pectinirostris. Nei’s distances between populations were from 0.038 to 0.151. The highest genetic distance was observed between Taiwan and Guangxi populations while the lowest was observed between Zhejiang and Guangdong populations. The pairwise comparisons of Fst ranged from 0.175 to 0.459, all of which were statistically significant(P=0). NJ tree constructed on the base of genetic distances between populations showed that the 8 populations were divided into two groups (A and B). Group A included Japan, Korea and Taiwan populations while Group B included the populations from Zhejiang, Fujian, Guangdong, Guangxi Provinces and Vietnam. The analysis of molecular variance showed that 15.48% of the total genetic variance was contributed by the among groups variation (Fct=0.15, P<0.01), 62.06% within populations variation (Fst=0.38, P=0) and 22.46% among populations within groups variation (Fsc=0.27, P=0). Coefficient of population differentiations (Fst) was 0.385 7 and the geneflow (Nm) from Gst was 0.796 4, which were comparatively lower than those of most other fish species. Also, strong genetic divergence between the populations of B. pectinirostris was found based on the pairwise comparisons of Fst and Nm. In comparison with the other marine migration fish, B. pectinirostris live in burrows on the mudflats of intertidal zone and do not have the feeding and spawning migration habits in their life cycle. Accordingly, their dispersal ability is very poor, leading to genetic differentiation between their populations. Since Japan, Korea and Taiwan are islands or peninsula, the habitats of B. pectinirostris populations in these locations are geographically different from those of the continental populations. Hence, genetic exchanges among them are very difficult to realize.

Abstract:Spinibarbus sinensis is one of the most important freshwater fish species in the upper reaches of the Yangtze River. Unfortunately however, the stock of the wild population declined sharply in recent years. There is an urgent need to understand the population structure of the fish to aid development of conservation measures and allow for continued exploitation of this important species. We collected 288 individuals from three geographic areas: Jialingjiang River, Chishuihe River, tributaries of the upper reaches of Yangtze River, and Qingjiang River, a tributary of the middle Yangtze River. The morphological variation and biology of the three populations was investigated using traditional morphological (one-way analysis of variance, cluster analysis, principle component analysis, and discriminant analysis) and truss network analysis. The three populations of S. sineisis differed significantly(P<0.05) for 29 morphological proportional characters(e.g., TL/BL, FL/BL, BW/BL). A cluster analysis indicated that the morphological charactersof S.sineisis in Jialing River were most similar to S. sineisis in Chishui River. The principal component analysis yielded eight principal components with a cumulative contribution ratio of 62.93%. The discriminant analysis suggested that these three populations could be divided. The discriminant functions of the three populations of S. sineisis were established based on 16 morphological characters that differed significantly among the populations. The total accuracy of discrimination of S.sineisis from the three populations was 99.31%. Our results suggest that there are significant morphological differences among S.sinensis populations in the three sampling areas in the upper reaches of Yangtze River basin

Abstract:A La Niña event is one of the strong signals of global climate anomalies. Understanding the consequences of La Niña event to marine ecosystems is important for improving fisheries stock assessment in the East China Sea. We compared species composition and the species diversity in winter between a normal year (2004) and a La Niña year (2008) in an offshore area of the East China Sea in winter using data collected from fisheries-independent bottom trawl survey. The number of observed species increased from 45 to 61 after the occurrence of La Niña. The number of warm water species decreased whereas warm temperature species increased. Though the dominant species composition only varied slightly, the stock density per station changed significantly. The stock density per station of warm water species such as Trichiurus japonicus decreased significantly whereas the stock density of warm temperature species such as Larimichthys polyactis tended to increase. Between the December of 2004 and 2008, the species richness indexes (R) increased from 1.56 to 1.92, the Shannon-Wienner diversity indexes (H′) increased from 1.23 to 1.42, and the Peilou evenness indexes(J′) increased from 0.51 to 0.57. There were no significant differences between the diversity indices accross the whole study area between 2004 and 2008. If the study area was divided into two parts, there was significant difference in the diversity indices between a normal year and a La Niña year in the southern region but not the northern region. The fishing pressure remained relatively stable during this period so the variability in fish community structure can likely be attributed to the change in climate in the East China Sea. The pattern and mechanism of the eco-response to the occurrence of La Niña events varies with the domain. Interdisciplinary studies are needed to improve our understanding of such events in the ecosystem.

Abstract:We evaluated the relationship between the rate of fishing success and the sinking performance of purse seines operated by a Chinese tuna purse seine fleet from 2006 to 2012. The 95% confidence intervals for maximum sinking depth (D) and average sinking speed (S) of the middle part of gear ranged from 195.3 m to 209.3 m and from 0.181 m/s to 0.192 m/s, respectively. The data obtained from at-sea observations indicate there is a significant difference in the fishing success among the different sinking speeds. Fishing success increased rapidly from 20.0% to 66.7% with an increase in the sinking speed. However, there was no relationship between fishing success and sinking depth. An initial run of the full logistic regression model revealed that the sinking depth is not a significant factor of fishing success (P>0.01), i.e. the probability of fishing success depends only on the sinking speed of the fishing gear. Moreover, fishing successes at different speeds predicted by the model were similar to the at-sea observations. Our results suggest that more effort should be made to accelerating the sinking speed but not deepen the sinking depth of fishing gear.

Abstract:Antarctic krill (Euphausia superba)(hereafter krill), are one of the most important components of the Antarctic ecosystem food web. Area 48 of the Commission on the Conservation of Antarctic Marine Living Resources (CCAMLR) is in the Atlantic sector of the Southern Ocean, and is the primary fishing ground for commercial krill fishing fleets. We evaluated the changes in catch rate over time in this region using average catch rate data derived from a database which covers CCAMLR subareas 48.1 (50°W−70°W, 60°S−65°S), 48.2 (30°W−50°W, 57°S−64°S), and 48.3 (30°W−50°W, 50°S−57°S) from 1982 to 2011 using the Mann–Kendall(M–K) method. The average nominal CPUE was ~5 t/h in 1982 and increased to 10 t/h by 2011 in Area 48. The standard deviation for these estimates was very large and almost equal to the mean. Nominal CPUE fluctuated periodically, but the linear trend growth per year was 0.221 t/h. Subarea 48.1, the primary fishing area, had a linear trend growth per year of 0.088 t/h. Subarea 48.2 accounted for the biggest proportion of the increase in Area 48, increasing at a rate of 0.323 9 t/h per year. The rate of increase in subarea 48.3 was 0.261 t/h per year. In the past 30 years, the krill catch rate in area 48 increased slowly at first, then rapidly up to 1999, before undergoing a significant change after 2001. After the late 1980s, and concomitant with a decrease in the southwest Atlantic sector krill resource, catch rates followed an upward trend as fishing technology improved. The mutation point in area 48 in 1999 occurred at the same time as the Japanese fleet catch rate increased significantly, accounting for more than 70% of the global catch. The mutation points of catch rate occurred in 2001 and in 2004 in subarea 48.1, though they were not significant. There was retraction of sea ice area from 2001 to 2003 resulting in a rebound in krill populations and a subsequent increase in catch per unit effort (CPUE) in 2004. There were two mutation points of catch rate in subarea 48.2, the first one occurred in 1995–1996, and was a downward change point that did not pass the α=0.05 significance level. The second event occurred in 1996–1997 and passed the α=0.05 significance level in 2000. The mean density of post-larval krill during 1976–2003 in the southwest Atlantic sea was lowest in 1994, resulting in lower harvest during 1995–1996. Recruitment restored normal and the catch rate mutation points in subarea 48.2 appeared in 1996–1997. The mutation points of catch rate occurred in 1992 in subarea 48.3, and passed the α=0.05 significance level in 1996. After the breakup of the former Soviet Union in 1991, the size of the Russian and Ukrainian trawler fleet declined sharply because fuel subsidies were canceled. The catch by the former Soviet Union before 1992 was about 84% of the world total, but after 1992 the global catch was made predominantly by vessels from Japan, which has a CPUE more than twice that of the former Soviet Union’s fleets. At a spatial level, the fishing effect dominates the three subareas, but the impact of sea ice coverage is more significant when closer to the shelf of the Antarctic Peninsula.

Abstract:A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for detection of infectious haematopoietic necrosis virus (IHNV). A set of specific primers were designed based on the IHNV L-protein sequence. IHNV RNA was used as a template for RT-LAMP in a reaction mixture containing reverse transcriptase and Bst DNA polymerase. The RT-LAMP products were observed by the naked eye by adding SYBR Green I fluorescent dye. Positive samples exhibited a fluorescent green color whereas negative samples exhibited a reddish brown color. The RT-LAMP assay had no cross reactivity with infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). The RT-LAMP assay was highly sensitive, and could detect < 10 pfu IHNV RNA. We confirmed these results using 2% agarose gel electrophoresis. In summary, we developed a sensitive, specific, low cost, and simple RT-LAMP method for rapid field diagnosis of IHNV. The method can achieve high throughput detection, without relying on any special equipment.

Abstract:The startle response is a response to sudden, startling stimuli, such as sudden noise or threatening disturbance. Fast escape is a common type of stress response behavior in teleost and amphibian larvae. Pufferfish (Takifugu obscurus), an important aquaculture species in southern China, typically exhibit an energy-dissipative inflation behavior in response to a stressor. It is unknown whether pufferfish have a fast escape capacity and under what kind of stressful situation. Understanding the stress responses and neuro-mechanisms of avoidance behavior provides a theoretical basis for improving aquaculture practices. We exposed larval pufferfish and zebrafish (Danio rerio) to a sudden sound stimuli (100 Hz, 110 dB) and recorded their behavior using a high-speed (1 000 frames per second) digital camera. Both species attempted to escape when exposed to sudden auditory stimuli. However, the probability of exhibiting fast escape behavior was significantly lower in pufferfish than in zebrafish, and the latency of the behavior was significantly longer in the former than in the latter(P<0.01). However, the behavior probability was significantly increased and the latency reduced in pufferfish by treatment with a GABAA receptor inhibitor(P<0.05). The Mauthner cell in the hindbrain is believed to be the commander neuron of this behavior. Together with the reticulospinal neurons (e.g., Mid2cm and Mid3cm), Mauthner cells form the brainstem escape network that regulates the fast escape behavior. Our histological evaluation revealed that the morphological characters of the Mauthner cells in pufferfish differed from those in zebrafish and most other fish. The cells were oval-shape, and the dorsoventral/mediolateral axial ratio was significantly smaller than that in the zebrafish. In the pufferfish medulla, we were unable to define neurons that are readily visible in zebrafish, including the Mid2cm, Mid3cm, or the synaptic connections between the eighth nerve and Mauthner cell. Our results suggest that the Mauthner cell in pufferfish was less developed and the fast escape behavior may not be as important as in zebrafish. Instead, the pufferfish has developed a unique behavior characterized by sudden inflation and floating on the surface of the water as a defense mechanism under sudden stressful stimulation. Our results provide a basis for further physiological study of the neural mechanism underlying the stress response in pufferfish.

Abstract:We evaluated the effects of substrate type [no substrate (control), concrete, sand, sand and mud, and gravel] on survival, growth, and behavior of Fenneropenaeus chinensis under laboratory conditions in a semi-intensive culture mode (SICM) and intensive culture mode (ICM). In the SICM, substrate type had no effect on survival (S) and molting frequency (MF) (P>0.05), though S and MF were lowest in the control and highest in the sand and mud substrate treatment (100% and 12.9 %/d, respectively) in the ICM. Substrate type significantly affected the specific growth rate (SGR) of F. chinensis reared in SICM and ICM (P<0.05). In the SICM, the SGR was highest in the sand and mud substrate treatment, and was 1.39 times higher than the SGR in the control. In the ICM, the SGR (1.94 %/d) was highest in the control, but there was no significant difference between the control, sand, and sand and mud substrate treatments (P>0.05). Substrate type had a significant effect on the behavior of F. chinensis (P<0.05). In the SICM, the feeding ratio (FR) and locomotion rate (LR) were all highest in the sand and mud substrate treatment before and after feeding, however, attacking frequency (AF) was highest in the sand substrate treatment before feeding and in the control treatment after feeding, peaking at 4.53 times/(shrimpּh−1). There was no significant difference in AF between the control, sand and mud substrate, and gravel substrate treatments (P>0.05). In the ICM, the FR and AF were highest in the sand substrate treatment before feeding and there was no difference in LR among the substrate treatment groups (P>0.05). The FR was highest in the control and lowest in the gravel substrate treatment after feeding. LR and AF were highest in the sand and mud substrate treatment. Our results suggest that the addition of substrate enhanced the growth and survival of F. chinensis in culture. F. chinensis exhibited behavioral differences when reared over different substrate types and preferred the sand and mud substrate.