Abstract:Ptychobarbus dipogon is one of the most important endemic fish species in the upper and middle reaches ofthe Yarlung Tsangpo River. However, P. dipogon populations have declined sharply in recent years and their biology ispoorly understood. To better understand the biology of P. dipogon and their relationship with management, we describethe relationships between standard length and body weight and age, verify annual growth of lapilli otolith annuli, andestimate P. dipogon growth. The age structure and growth characteristics of P. dipogon were investigated by examining956 specimens collected in the Yarlung Tsangpo River during 2008–2009 and 2012–2013. Standard lengths were146–569 mm in females, 167–506 mm in males, and 78–297 mm in some undetermined specimens. Otolith annuliformed from March–June each year. Estimated ages were 3–24 years in females, 3–13 years in males, and 2–6 years inundetermined specimens, respectively. The standard length–body weight relationships were described by W= 2.494 ×10−5Ls2.877 for females and W= 2.790×10−5Ls2.856 for males. The von Bertalanffy growth equations were: Lt= 606.9[1−e−0.114(t+0.163)] and Wt=2538.4[1−e−0.114(t+0.163)]2.88 for females and Lt=496.3[1−e−0.162(t−0.018)] and Wt=1391.1[1− e−0.162(t−0.018)]2.86for males. The growth inflexion points were 9.1 years for females and 6.5 years for males, and the corresponding standardlengths and body weights were 396.0 mm and 743.6 g for females and 322.5 mm and 406.3 g for males, respectively.Our results show that the P. dipogon population tended to be younger. Climate changes, biological invasion, andover-exploitation could be the main factors threatening P. dipogon stocks. The sound management policies proposed inthis study should be implemented to conserve P. dipogon populations.

Abstract:A complete diallele crossing experiment involving the Bohai, Denmark, Donaldson, Norway, and Californiastrains as well as self-breeding within each strain was carried out to evaluate general combining ability,specific combining ability, and reciprocal, maternal, and line-bred effects for body length and body weight inrainbow trout at four time points. The comparative analysis showed that the 20 corresponding cross combinationsand five strains from self-breeding were significantly different (P<0.05). Only a few genetic effects were significant,but the genetic effect was not the same among the different traits and time points. The Bohai and Donaldsonstrain cross combination had the fastest increases in body weight and body length, and length was significantlydifferent when compared with the self-breeding results. Combining the Donaldson strain with other strains lead tosignificantly higher body weights at 249 and 397 days of age. A significant maternal effect of the Denmark strainoccurred at 249 days of age. The maternal effect of the Donaldson strain on the length trait was significantly thelargest at 249 and 397 days of age. The cross combination of the Bohai and Donaldson strains was the most efficientamong all cross combinations, particularly for the weight trait. The effects of cross combination of the Bohaiand Donaldson strains on the length trait were positive (0.30, 0.03, 0.17, and 0.30) with a maximum at 771 days ofage. In conclusion, the Donaldson strain showed the highest growth performance of the rainbow trout offspring.The combination of the Bohai and Donaldson strains was the best and should be used as the basis for a breedinggroup.

Abstract:Saline-alkaline water use has broadened the development of Chinese fisheries. Breeding new varieties ofalkaline-resistant fish is a fundamental task, and elucidating fish alkaline-resistant mechanisms, identifying candidategenes, and exploring their functions are crucial. Decoding genome information promotes the use of molecular markersand selection of candidate genes. Leuciscus waleckii (Dybowski) is highly tolerant to high salinity and alkalinity andsurvives in Dali Lake (Inner Mongolia) with alkalinity up to 53.57 mmol/L (pH 9.6). Therefore, this fish species is agood subject to investigate the evolutionary mechanism of high salinity and alkalinity adaptation. L. waleckii in DaliLake differ from their freshwater counterparts because they are characterized by a breeding migration in which theyspawn in freshwater and grow in alkaline water. Marked differences are observed during acute-phase alkaline tolerancebetween the alkaline population from Dali Lake and the freshwater population from the Songhuajiang River, indicatingthe genetic heritability of alkaline tolerance. However, the mechanism of high tolerance to alkalinity remains unknown,and very few physiological and genetic studies have been performed. Thus, we prepared a hybrid F2 system (alkalineDali Leuciscus × freshwater Songhua Leuciscus). A large number of candidate genes are associated with alkaline-resistance traits, and microsatellites, single nucleotide polymorphisms, and other molecular markers have been appliedin fish population genetics studies. We used the gene recombination principle to select molecular markers closelyrelated to alkaline-resistance traits and located functional genes using molecular markers. In this study, we examinedand genotyped alkaline tolerance in 77 hybrid F2 individuals using 39 simple sequence repeat (SSR) markers, including19 expressed sequence tag EST-SSRs. The individuals were divided into three groups of 43, 20, and 14 individualsbased on their alkaline tolerance. Fisher’s exact probability test was used to examine the associations between markersand the trait. The results showed that two markers were strongly linked to alkaline tolerance (P<0.05), and one was anEST-SSR. Sequence alignment of that EST-SSR showed that it was highly homologous with the hypoxia inducible factorHIF-3 gene of Wuchang bream (Megalobrama amblycephala) and the HIF-4 gene of grass carp (Ctenopharyngodonidellus). These genes are involved in the response to hypoxia and increased hypoxia tolerance in fish. The approachwas to select a functional gene using molecular markers, localize the functional gene, and narrow the range ofcandidate genes to lay the foundation for breeding new varieties of alkaline-resistant fish. In-depth study of this functionalgene will be conducted in the future. Our results suggest further analyses of candidate alkaline-responsive genes,which will help in the understanding of teleost adaptation under extreme environmental stress and ultimately benefitfuture breeding for an alkaline-tolerant fish strain.

Abstract:Edwardsiella tarda is a significant pathogenic bacterium that causes considerable economic losses in flounderaquaculture. We created 47 flounder families using parental fish resistant to E. tarda and screened them long termthrough cross-fertilization, self-crossing, and gynogenesis experiments during April–June 2014. We infected 39 flounderfamilies with E. tarda from June–October. The half lethal bacterial concentration was determined to be 80 in eachfamily using a small number of flounder in a preliminary experiment. Survival rate was 1.19%–51.19% (mean, 20.29%).Seven families with survival rates >30% were most able to resist E. tarda; the nine families with survival rates of20.29%–30% had normal resistance to E. tarda; and the 23 families with survival rates < 20.29% were least resistant toE. tarda. Family 1406 had the best resistance, which was self-crossed with 1005. The families with the best resistancewere all descendants of families 1005, 09104, and 0915. Family 09104 was a descendant of family 0768, which wasresistant to Vibrio anguillarum, and was self-crossed with family 1005 and a Korean group. Family 0915 wascross-fertilized with Korean and Japanese groups. These disease-resistant flounder could be popularized as new strainsto reduce the occurrence of virulent ascitesosis disease.

Abstract:Tilapia feature asynchronous oocyte development and batch spawning. We conducted this study to furtherunderstand ovarian maturation and the physiological mechanisms involved in non-synchronous spawners, such as tilapia.Changes in sex steroid hormones (estradiol-17β[E2] and progesterone [P]), vitellogenin (Vtg) levels, and VtgmRNA expression were studied during ovarian development in Oreochromis niloticus using histological, enzyme-linked immunoassay, and quantitative reverse transcription-polymerase chain reaction methods, respectively. Theresults showed that the gonadosomatic index (GSI) of O. niloticus paralleled ovarian development and peaked at stage V.Serum E2 levels increased significantly beginning at stage II, peaked at stage IV, then decreased significantly at stage V,whereas P levels increased gradually beginning at stage II, peaked at stage V, and decreased significantly at stage VI.Hepatic Vtg content increased initially, decreased, and then peaked at stage IV. Serum and ovarian Vtg contents increasedgradually beginning at stage II, peaked at stage V, and decreased significantly at stage VI. Hepatic Vtg mRNAexpression peaked at stage III and decreased gradually atstage IV. Ovarian Vtg mRNA expression was relatively lowerand peaked at stage V. These results indicate that E2 and P play different roles during the early and late ovarian developmentalstages, respectively. A close correlation was detected between changes in Vtg content in different tissues andovarian maturation.Vtg mRNA from the liver and ovary were involved in Vtg synthesis; the liver is the major syntheticorgan active during the yolk accumulation phase, but ovarian Vtg synthesis was relatively low.We provide some basicdata to further explore the endocrine regulatory mechanisms in non-synchronous spawning fish.

Abstract:The order Clupeiformesis in the Class Osteichthyes, and many of these species are of significant economicvalue. The mitochondrial cytochrome c oxidase subunit 1 (CO I) gene sequences of seven species pertainingto six genera in Clupeiformes collected from Chinese coastal waters were amplified using a polymerase chain reactiontechnique. The homologous sequences of other species of Clupeiformes from GenBank were also includedin this study. A total of 273 single sequences from 90 species pertaining to 41 genera in Clupeiformes were barcodedusing the CO I gene and then sequenced and compared to verify the feasibility of using DNA barcoding toidentify species. Clustal X and MEGA6.0 were used to calculate base composition, sequence substitutions, andgenetic distance. The results indicated that mean T, C, A, and G contents were 28.3%, 28.3%, 24.2%, and 19.2%,respectively, indicating that base composition was biased.The nucleotide sequences revealed base substitutions,including 71 conversion sites and 46 transversion sites. Most appeared on the third codon, including 62 conversionsites and 45 transversion sites. The second codon did not have conversions or transversions. The conversion andtransversion ratio reflects evolutionary rate; if the probability of transversion is greater, the conversion and transversionratio is smaller, and the evolutionary rate is faster. The evolutionary rate of the third codon was the fastest,followed by the first codon, and the second codon was the most stable. DNA barcoding can be used to identifyspecies if the distance between congeners is much larger than that between conspecifics and different individualsof the same species are clustered in the same phylogenetic tree branch. The congeneric and conspecificgeneticdistances were 0.131 and 0.003, respectively, and the congeneric distance was 41 times that of the conspecificdistance. However, some congeneric distances were too small or the conspecific distances were too large, leadingto different names for the same species and intraspecific differentiation. All conspecifics clustered together in thephylogenetic tree, except Dorosoma cepedianum and Dorosoma anale, suggesting that these may be the same species.The phylogenetic analysis showed that 97.8% of the fish were monophyletic. In summary, the DNA barcodesof clupeiform fish complied with the requirements for species identification, and the neighbor-joining tree basedon the CO I gene identified species more accurately. The phylogenetic results show that the CO I gene was suitablefor clarifying primary taxa and provided some reference value for the phylogenetic relationships of senior taxa.

Abstract:The Sox family of transcription factors is involved in a variety of developmental processes, including sexdetermination and gonadal differentiation. The high-mobility-group (HMG) domains of all Sox proteins have highlyconserved primary structures, and all appear to be capable of binding to the same target DNA sequence ofAACAA(A/T)G. The Sox gene family is divided into 10 subgroups (designated A–J) based on sequence similarity andgene organization. Sox19 is a member of the group B Sox genes, though it differs from other members in possessing anintron. The Sox B group is further divided into the B1 subgroup, which encodes transcriptional activators, and the B2subgroup, which encodes repressors. Based on sequence and phylogenetic analyses, Sox19 has been assigned to the B1subgroup. Interestingly, the Sox19 gene has always been considered fish specific but the full length cDNA encodingSox19 has only been cloned in Danio rerio, Takifugu rubripes, and Dicentrarchus labrax, and little is known about theexpression patterns in other species. In this study, we successfully cloned the full-length Sox19 cDNA sequence of theChinese giant salamander (Andrias davidianus), which is the largest extant amphibian, and used quantitative real-timepolymerase chain reaction (PCR) analysis to detect Sox19 gene expression in different tissues. We cloned a 1290-bpSox19 gene cDNA, including 270 bp of the 5′-untranslated region (UTR) sequence, 858 bp of the open reading frame(ORF), and 162 bp of the 3′-UTR sequence. The ORF encoded 285 amino acids, of which a 71 amino acid protein wasfrom the N-region and a 39–109 amino acid region was the highly conserved HMG-box. Bioinformatics software wasapplied to analyze the A. davidianus Sox19 protein, which is hydrophilic. A subcellular localization analysis showed that96% of the protein was present in the nucleus, possibly without the transmembrane region or signal peptide, 24.91%was in the α-helix, 3.16% was in the extended strand, 2.11% was in the β-corner, and 69.82% was in the random coil.The tertiary structural prediction revealed a α-helical structure as the functional site. The A. davidianus Sox19 proteinencoded 285 amino acids, which is less than that in fish. Therefore, fish have more similar “AAAA” polymers thanthose of the A. davidianus (150 amino acids), but A. davidianus has an additional “QNLV” at the C-terminal end, suggestingthat the Sox19 gene may have lost the lactamine oligomer during evolution and mutated to a more elaborate andcomplex transcriptional regulatory region. The amino acid sequence analysis revealed that the putative A. davidianus-Sox19 protein had 55%, 58%, and 64% identity with T. rubripes, D. rerio, and D. labrax, respectively. The phylogeneticanalysis further demonstrated that A. davidianus split off earlier based on the Sox19 gene data, revealing a more primitiveevolutionary status. The Sox19 gene could have evolved through ancient fish-specific duplication of Sox3, althoughSox19 is expressed in the developing lens and central nervous system. We analyzed Sox19 expression in the gonad,heart, kidney, and bowel of adult A. davidianus to confer the functions of the protein by fluorescence quantitative PCR.The results show the highest Sox19 gene expression level in the heart, suggesting that Sox19 helps maintain heart function.The high expression level in the gonads shows that Sox19 may be involved in gonadal development and differentiation.Sox19 was also expressed in the kidney and intestine, indicating that Sox19 plays a role maintaining kidney andbowel functions in A. davidianus. The Sox19 gene expression levels in A. davidianus were not the same as those in thefish species investigated. Our results provide insight into the role of the Sox19 gene in A. davidianus and further ourunderstanding of Sox19 gene expression in amphibians. The Sox19 gene could be the earliest molecular marker of embryogenesisin A. davidianus.

Abstract:Portunus trituberculatus is the primary marine crab species cultured in China. Molting is vital for growth,reproduction, and regeneration in crustaceans. Molting is regulated by a complex molecular mechanism but the detailsremain unclear and require further research. Calmodulin (CaM) is a conserved multifunctional protein that regulatesCa2+ and glycogen metabolism, influences cell division and movement, and is involved with neurotransmitter synthesisand release. Choosing and cloning a CaM gene to investigate the functions and roles of CaM during molting depends onthe transcriptome data. P. trituberculatus CaM cDNA was cloned using rapid amplification of cDNA ends and namedPTCaM. The PTCaM full-length cDNA was 1981 bp, including a 450-bp open reading frame that encoded a 256 aminoacid polypeptide, with a molecular mass of 16.8 kD and an isoelectric point of 4.09. A bioinformatics analysis revealed thatPTCaM has a highly conserved sequence with a typical Ca2+ EF-hand binding domain of the EF superfamily. The EF-handis a calcium binding motif with two active canonical EF hands. Ca2+ binding induces a conformational change in theEF-hand motif leading to activation or inactivation of target proteins. A homology analysis showed that PTCaM had thehighest homology with Eriocheir sinensis and Drosophila melanogaster. A phylogenetic analysis showed that PTCaM wasin the same branch with Litopenaeus vannamei, E. sinensis, and Procambarus clarkia. A real-time quantitative polymerasechain reaction analysis of different molting stages showed that PTCaM was expressed significantly differently in all tissuestested during molting, which reveals a molting function for PTCaM. PTCaM expression in the eyestalk and gill weredownregulated during the post-molt stage, suggesting that PTCaM may participate in preparation for molting. PTCaMexpression was upregulated in heart, hemocytes, and muscle during the pre-molt stage, suggesting that PTCaM may regulateion transport, particularly that of Ca2+. PTCaM expression was downregulated in most tissues after eyestalk ablation,compared to that in normal control tissues. PTCaM may cooperate with molt inhibiting hormone or ecdysone because single-eyestalk ablation has less of an inhibiting effect on ecdysone synthesis.

Abstract:Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the most serious diseases of shrimp,and has a wide host range. IHHNV is the smallest of all known shrimp viruses and is a non-enveloped, linear, single-strandedDNA virus in Brevidensovirus Densovirinae Parvoviridae. This disease causes serious economic losses worldwide. IHHNV ishighly pathogenic and is associated with high Litopenaeus stylirostrismortality rates. IHHNV causes chronic infection, slowgrowth, lesions in the forehead sword, antenna, and the cephalothoracic and abdominal shell of Litopenaeus vannamei. SinceIHHNV was first identified, many studies have established diagnostic methods, and epidemiological surveys have been conducted,but the pathogenic mechanisms of IHHNV infection remain relatively unknown. In this study, we identified putativehost cell receptors for the IHHNV capsid protein (CP) in L. vannamei gill membranes. DNA was extracted fromIHHNV-infected L. vannamei (Sheyang isolate) and preserved at the Microbiology and Immunology, Preventive VeterinaryLaboratory of Nanjing Agricultural University. The IHHNV CP gene was amplified by polymerase chain reaction, and theIHHNV CP BL21-pET-28a-CP prokaryotic expression vector was prepared. The protein was expressed primarily in the precipitate.The CP in the precipitate was purified using a HisTrap™ HP column and then used to prepare a polyclonal antibody.The antibody titer reached 1︰51200 after three immunizations. A Western blot analysis demonstrated that the polyclonal CPantibody bound specifically to CP. Then, gill membrane proteins from uninfected L. vannamei were obtained from a homogenateand ultracentrifuged to obtain a uniform gill membrane protein distribution. A virus overlay protein binding assay(VOPBA) and a His pull-downassaywere carried out to localize the IHHNV CP receptors. VOPBA is a classic method used toidentify virus receptors and has been used to identify the host cell receptors for White Spot Syndrome Virus and Yellow HeadVirus. The suspected protein strips were analyzed by mass spectrometry. Results of the two assays indicated that signal transducerand activator of transcription (STAT), heat shock protein 90 (HSP90), prophenoloxidase-2, and the Na+/K+-ATPase alphasubunit participate in IHHNV infection. These four proteins have various biological functions, such as interferon signaltransduction, protection against stress, defense, and maintenance of osmotic balance. These proteins may interact with theIHHNV CP and are associated with penetration and the cytopathic effects caused by IHHNV. Few studies have evaluated putativeIHHNV CP receptors, probably because of the lack of a mature shrimp cell line. We used traditional VOPBA to localizethe CP receptors from L. vannamei gill membrane proteins. Taken together, STAT, HSP90, prophenoloxidase-2, and theNa+/K+-ATPase alpha subunit are involved in IHHNV infection. These results will lay the foundation to identify the IHHNVinvasive mechanism. The specific effects of the four proteins remain to be studied in detail.

Abstract:Zinc (Zn) is an essential trace element in fish. It functions as a cofactor in several enzyme systems and isinvolved in many important physiological processes, including improved growth and development, immune function,and defense against free radicals. Signs of impaired growth, increased mortality, cataracts, short body dwarfism, andlow tissue Zn may occur in fish fed a Zn-deficient diet. In contrast, excessive dietary Zn can negatively affect the nutritionalstatus of other elements, such as iron, cadmium, and magnesium. Therefore, it is necessary to ensure minimaldietary levels to meet the requirement and be certain that levels do not exceed the maximal limits, which can affectgrowth and other responses in fish. Blunt snout bream (Megalobrama amblycephala) is in the Cyprinidae family, whichis one of the largest fish families in the world. Production of cyprinids reached about 0.71 million tons in 2011, which isthe seventh most freshwater-cultured fish species in China (Bureau of Fisheries of the Ministry of Agriculture , China,2012). The blunt snout bream has become an important and popular freshwater species. The dietary zinc requirementforblunt snout bream is reportedly 20–184 mg/kg. We conducted a 12-week growth experiment to quantify the optimumdietary Zn requirement of juvenile blunt snout bream and evaluated its effects on antioxidant responses in this species.Seven experimental diets were formulated to contain graded levels of Zn (7.4 mg/kg, 20.3 mg/kg, 32.1 mg/kg,51.0 mg/kg, 84.4 mg/kg, 169.7 mg/kg, and 332.4 mg/kg diet, supplied as ZnSO4·7H2O. Casein and gelatin were used asdietary protein sources). Each diet was assigned to three replicate groups of 40 fish [initial body weight, (3.6±0.1) g]cultured in 500-L aquaria (r=0.9 m, h=0.75 m). The results showed that the rate of weight gain increased linearly withthe increase in dietary Zn level, then remained nearly unchanged in the high Zn treatment groups (≥32.1 mg/kg). Specificgrowth rate and fish Zn content showed a similar tendency with rate of weight gain. Whole body moisture contentof blunt snout bream decreased significantly in the supplemented groups compared to that in the un-supplementedgroup (P<0.05), whereas whole body crude protein content increased significantly in the supplemented groups comparedto that in the un-supplemented group (P<0.05). No significant difference was observed in the food conversion rateamong the treatments (P>0.05). The serum chemistry analysis showed that dietary Zn had significant effects on contentsof total protein, urea nitrogen, high density lipoprotein cholesterol, total cholesterol, and triglycerides (P<0.05), but noeffect on albumin content or alkaline phosphatase activity (P>0.05). Hepatic malondialdehyde content decreased significantly(P<0.05), whereas superoxide dismutase and catalase remained unchanged with increases in the dietary Znsupplement rate (P>0.05). A broken-line regression analysis showed that the optimum dietary Zn requirement of bluntsnout bream was 32.6 mg/kg for maximum growth and 47.6 mg/kg for maximum fish Zn concentration. These resultsincrease our knowledge of the zinc requirement in blunt snout bream to enhance the feed quality and discover the versatilefunctions of Zn in blunt snout bream.

Abstract:Fatty acyl-CoA Δ9 desaturase (FAD9) is a membrane-bound enzyme anchored in the endoplasmic reticulumthat plays an important role regulating cell membrane fluidity and fatty acid metabolism. The FAD9 sequencehas two transmembrane domains and three amino acid motifs. Purifying the FAD9 protein can be difficultbecause of its membrane structural characteristics. Fish species frequently express FAD9 heterologously. TheChinese mitten crab, Eriocheir sinensis, is an important aquatic species. We designed primers based on the FAD9cDNA sequence from E. sinensis (Accession Number: JQ693685) to obtain the opening reading frame (ORF). TheORF was subcloned into the pCold-TF DNA prokaryotic expression vector to generate the pColdTF-fad9 recombinantexpression vector, which was transformed into E. coli BL21(DE3)pLysS, and FAD9 was expressed successfullyin E. coli BL21(DE3)pLysS using IPTG induction. The sodium dodecyl sulfate-polyacrylamide gel electrophoresisresults showed that the recombinant protein had an approximate molecular weight of 95.10 kD, whichwas consistent with the theoretical molecular weight, and that it was mainly detected in the supernatant. We testeddifferent IPTG concentrations (0.1 mmol/L, 0.3 mmol/L, 0.5 mmol/L, 0.8 mmol/L, 1.0 mmol/L, and 1.2 mmol/L),induction temperatures (35℃, 25℃, 18℃, and 15℃), and induction times (2 h, 5 h, 10 h, 15 h, and 20 h). Theresults showed that when the IPTG concentration was 0.1 mmol/L, expression of the recombinant protein was significantlylower than that at the other IPTG concentrations. Temperatures of 15–18℃ and an induction time of 20 hwere optimal. The highest recombinant protein expression was detected when the IPTG concentration was 0.3 mmol/L,with a 20-h incubation at 15℃. The fusion protein was identified by purification and western blotting. The proteincontained a 6×His-tag, so we used His-tag nickel ion affinity chromatography to purify the protein and ananti-6×His-tag antibody for western blotting. The results showed that the pColdTF-fad9 recombinant protein wassuccessfully expressed in E. coli, and western blotting revealed that the pColdTF-fad9 recombinant protein wasspecifically recognized by the 6×His antibody, indicating that the recombinant protein had antigen activity. Ourstudy provides basic methods to purify and detect the activity of E. sinensis FAD9 and will promote further studyon the functions of FADs.

Abstract:Strongylocentrotus intermedius was introduced to China from Japan in 1989. It is the only sea urchin speciescultured in China due to its short spines and high gonad quality. Family selection is being applied to genetically improvegrowth rates and gonad quality of S. intermedius. Families from different environments (water temperature,stocking density, illumination, and culture facility) rank differently during family selection, suggesting a family by environmentinteraction. However, the diet by family interaction, which is one of the most important environment factors,has not been examined. In this study, two different diets (artificial feed and Undaria pinnatifida) were fed to six familiesof S. intermedius to examine the effects of diet, family, and the diet by family interaction on final body weight(FBW), specific growth rate (SGR) for body weight, feed conversion rate (FCR), and change in the coefficient of variation(ΔCV). At the end of the 60-day experiment, we found no significant diet by family interaction effect on FBW orΔCV. Sea urchins fed U. pinnatifida grew significantly larger and had more uniform FBW than those fed artificial feed(P < 0.01). Significant differences were detected in both FBW and ΔCV among families. These differences suggest thatfamily selection is an effective genetic method to improve FBW and uniformity. We compared the FCRs of S. intermediusfamilies fed macro-algae and artificial feed for the first time. Significant diet by family interaction effects weredetected on both SGR and FCR (P<0.01). Sea urchins in all families fed U. pinnatifida had larger SGR and FCR(P<0.01). Both SGR and FCR were significantly different among families fed the different diets (P<0.05). These differencessuggest that both SGR and FCR can be improved genetically using family selection. The families fed the twodiets were the same when ranked by SGR, suggesting that the interaction effect on SGR was caused by variations in thefamilies between diets. However, family ranking using FCR was the opposite of that for SGR. Re-ranking the familiesshowed that FCR was different based on diet. In conclusion, our results suggest that growth rates, FCR, and uniformityof S. intermedius were improved genetically by family selection. In addition, diet significantly lowered growth rate,FCR, and uniformity when family selection was applied. Breeders should consider the family by diet interaction whenFCR is the target trait.

Abstract:A 68-d feeding trial was conducted to investigate the effects of replacing fish oil with dietary wheatgerm oilon growth, body composition, serum biochemical indices, and lipid metabolic enzymesin juvenile Cynoglossussemilaevis Günter [initial body weight, (3.70±0.01) g]. Six isonitrogenous and isoenergetic experimentaldiets were formulated with 0, 20%, 40%, 60%, 80%, and 100% replacement of the fish oil with wheat germ oil.Survival rate was similar among the fish fed the different diets (P>0.05). Specific growth rate and the protein efficiencyratio decreased with increasing replacement level; the 80% and 100% replacement groups were significantlylower than the control group (P<0.05), whereas the feed conversion ratio showed a reverse trend [80% and100% replacement groups were significantly higher than control group (P<0.05)]. No significant differences wereobserved in the viscerosomatic index, hepatosomatic index, or condition factor (P>0.05). Liver fat content increasedwith increasing replacement level, whereas whole fish fat content decreased; whole fish water content inthe 80% and 100% replacement groups was significantly higher than that in the control group (P<0.05). Muscleand liver C18: 3n-3 and C18: 2n-6 concentrations increased significantly with increasing wheat germ oil level (P<0.05),whereas C20: 5n-3 and C22: 6n-3 concentrations decreased significantly (P<0.05). No differences in serum glutamic-pyruvate transaminase, glutamic-oxaloacetic transaminase, or alkaline phosphatase (P>0.05) were detected.Serum triglyceride and total cholesterol levels decreased with increasing replacement level; the 80% and 100%replacement groups were significantly higher than the control group (P<0.05). No significant difference was foundin serum lysozyme activity (P>0.05), but serum malondialdehyde content decreased. Hepatopancreas lipoproteinlipase, hepatic lipase, fatty acid synthase, and intestinal lipase activities increased with increasing replacementlevel. These results suggest that wheat germ oil can replace 60% of fish oil without significantly affecting growthof juvenile C. semilaevis Günter. Wheat germ oil promoted body fat metabolism and inhibited lipid peroxidationbut significantly changed fatty acid composition in tissues.

Abstract:Norfloxacin is an efficacious broad-spectrum antibiotic that has been used widely to prevent and controlfish bacterial diseases. However, no comprehensive study has examined the toxicity of norfloxadin to aquaticanimals. Siberian sturgeon is the principle sturgeon species cultured in China and has high scientific research andproduction value. Quinolones occasionally cause an abnormal spinal column in Siberian sturgeon when used tocontrol bacterial diseases. Thus, we investigated the accumulation rule and toxicity of different norfloxadin dosagesand exposure times in Siberian sturgeon. Norfloxacin was given orally to experimental fish at dosages of 0,30 mg/kg, 50 mg/kg, and 100 mg/kg for 3–5 d. Blood, liver, kidney, and cartilage were collected for 24–240 h. Thequantities of drug retained in the four tissues were determined, and histological sections of the liver, kidney, andcartilage were observed. The results showed that the concentrations of norfloxacin in the four Siberian sturgeontissues increased gradually with increasing norfloxadin dosage and time in a trend of kidney > cartilage > liver >serum. Significant differences in liver, kidney, and cartilage drug concentrations were detected when norfloxadinwas given to fish at the same dosage but for different durations. These differences disappeared after withdrawal for240 h. The histology indicated that the liver and kidney were damaged when norfloxadin was given to fish at 50mg/kg for 5 d, and the damage appeared to be more serious at 100 mg/kg for 3–5 d. Damage to the liver, but notthe kidney, recovered gradually during the 240 h withdrawal. The number of chondrocytes near the perichondriumdecreased in cartilage when norfloxadin was given at 100 mg/kg for 3–5 d but recovered gradually during the 240h withdrawal. These results suggest that the norfloxadin dosage should be no more than 50 mg/kg and that exposuretime should be < 5 d to prevent liver, kidney, and cartilage damage. Based on these results, norfloxadin exhibitedpotential damage to the liver, kidney, and cartilage in Siberian sturgeon, suggesting that the dosing regimenmust be strictly followed in sturgeon aquaculture. Further study is needed to describe norfloxadin metabolism,residue, and damage repair. Suitable Chinese herbal medicines could be used to promote cartilage differentiationand repair.

Abstract:The petroleum hydrocarbon concentrations in seawater were 0.05 mg/L, 0.30 mg/L, 0.50 mg/L, and 1.00 mg/Lseparately. Three replicates were designed for each treatment. The experimental period for the scallop, Chlamys farreri,was 30 days. Sampling occurred at hours 0 and 12 and days 1, 3, 6, 10, 15, 21, and 30. The gills, digestive gland, andhemocytes were stored at −80℃ and were evaluated within 24 h. The results showed that none of the biomarkerschanged significantly in the 0.05 mg/L group. Alkaline phosphatase (ALP), superoxide dismutase, and glutathione peroxidaseactivities increased significantly in the gills and digestive glands of the 0.30 mg/L and 0.50 mg/L groups(P<0.05), whereas these activities were similar to or lower than control levels in the 1.00 mg/L group. Biomarkers in thegills changed more significantly than those of the digestive gland. Hemocyte membrane stability did not change significantlyin the 0.05 mg/L group but was lower than that in the controls and other groups later in the experiment(P<0.05). Hemocyte stability was lower in the groups exposed to higher petroleum hydrocarbon concentrations.Theseresults suggest that C. farreri can detoxify low concentrations of petroleum hydrocarbon, such as 0.05 mg/L. No effecton hemocyte stability was observed over the short term in the 0.30 mg/L and 0.50 mg/L groups, but oxidative damageoccurred later in the experiment, which decreased membrane stability. The C. farreri biomarkers changed significantlyin the 1.00 mg/L petroleum hydrocarbon concentration group and oxidative damage to the gills increased and hemocytemembrane stability decreased substantially after 3 d. Antioxidant and ALP activities changed significantly in the gillscompared to those in the digestive gland at the same petroleum hydrocarbon concentration, so the gills were more sensitiveto petroleum hydrocarbon than the digestive gland. The changes in the biomarkers showed dose- and time-effectcharacteristics; thus, these biomarkers may be useful to evaluate oceanic oil pollution.

Abstract:Cryptocaryon irritans is a holotrichous marine ciliate protozoan that causes “white spot disease”, which isone of the most devastating parasites of Larimichthys crocea in Fujian Province. No safe drug strategy has been identifiedto prevent and cure “white spot disease”. To investigate the expression and prognostic value of macrophage migrationinhibitory factor (MIF) in C. irritans diseased L. crocea tissues, MIF expression was detected by immunohistochemistryof intestine, spleen, head kidney, and liver specimens of pathologically identified and healthy L. crocea tissues.None of the negative controls reacted, but MIF staining was intense in all L. crocea tissues infected with C. irritansdisease. The MIF staining rates for the intestines, liver, spleen, and head kidney were 54%, 80%, 86% and 90%,respectively. The high MIF expression levels in diseased tissues indicate that MIF plays an important role in homeostasisof C. irritans infected with L. crocea.

Abstract:Fecundity of Coregonus ussuriensis, collected during October and November 2013–2014 in the HeilongjiangRiver, China, was studied. Fish age was determined by scales, fork length, body weight, net body weight, and ovarianweight. The ages of the specimens were 5+–8+ years. Individual absolute fecundity was determined by the gravimetricmethod and was used to calculate relative fecundity per centimeter, relative fecundity per gram, gonadosomatic index,relative fatness, and other indices. Individual fish absolute fecundity was 1.161×104–5.921×104 eggs (mean, 3.027×104eggs). Relative fecundity per centimeter was 307.13–1119.37 eggs/cm (mean, 622.5 eggs/cm. Relative fecundity pergram was 19.81–56.98 eggs/g (mean, 34.62 eggs/g). The relationships between absolute fecundity and fork length, bodyweight, and age were regressed with a power function, linear equation, and a parabola. The relationships between relativefecundity per centimeter and fork length, body weight, and age were described by a power function and a powerfunction and linear equation. Relative fecundity per gram was not correlated with fork length, body weight, or age butwas correlated with gonadosomatic index and ovarian weight, using the power function as the best fit. A multiple regressionanalysis revealed that individual absolute fecundity was significantly correlated with ovarian weight and forklength, while relative fecundity per centimeter and relative fecundity per gram were significantly correlated with ovarianweight and gonadosomatic index. The correlation between individual fecundity and ovarian weight was higher thanthat with the gonadosomatic index based on the partial correlation coefficient.

Abstract:The veined rapa whelk, Rapana venosa, (Valenciennes 1846), which is in the family Muricidae, is a largepredatory marine gastropod native to the coasts of China, Korea, and Japan. In the mid-1940s, it invaded the Black Seaand is now known worldwide as a notorious invasive species. In China, R. venosa is an economically important molluscanspecies, but the wild resource has decreased dramatically in the last 20 years because of over-exploitation anddeterioration of environmental conditions. Large-scale production of whelks has not been accomplished; therefore, understandingthe reproductive cycle of this species is crucial for establishing aquaculture techniques for seed production.In the current study, the reproductive cycle of R. venosa in Jinghai Bay, Shandong Province, China was investigatedfrom March 2012 to February 2013 in relation to environment factors. Water temperature at the sampling site showed atypical seasonal pattern of the temperate zone, with a unimodal peak in August and a minimum in January. The chlorophylla concentrations exhibited a similar pattern, with a peak observed in September and a minimum in January. Thechlorophyll a concentrations were significantly related to seawater temperature. Gonadal development of R. venosa wasobserved using tissue slices stained with hematoxylin and eosin, and the gametogenic stages were categorized into earlyactive stage (stage I), late active stage (stage II), ripe stage (stage III), partially spawned stage (stage IV), and recoverystage (stage V). Gametogenesis was initiated in September, continued slowly during the winter, followed by rapid gonaddevelopment during spring and summer. Most individuals were ripe between May–June, and gametes were releasedMay–August. The spawning peak occurred in July, and the gonads recovered August–November. R. venosa had synchronizedgonadal development in both sexes, and no hermaphrodites were found. Among 1093 specimens, 552 werefemales and 541 were males, resulting in a balanced sex ratio. The condition index and oocyte diameter clearly variedseasonally; increased at the beginning of gametogenesis, reached a peak at gonad maturity, and then declined after gameterelease. This is the first study to establish the gametogenic pattern of R. venosa along on the coast of ShandongProvince, China. The data obtained from this study will be useful for sustainable management of wild stocks and futureaquaculture of this species.

Abstract:Scomber japonicus is one of the primary pelagic fish in the East China Sea and Yellow Sea and has continuedto maintain a high yield in recent years. Inter-decadal changes in the reproductive biology characteristics ofS. japonicus must be investigated to determine the cause for the continued high production under strong fishingpressure. The inter-decadal changes in reproductive stock structural characteristics, such as the fatness index, femaleto male ratio, and fork length at maturity, were evaluated based on data collected from 1054 S. japonicussamples collected during three periods from 1960 to 2012 in the East China Sea. The results show that the agestructure, dominant age group, fatness index, and the minimum and mean fork length sat maturity of there productivestocks tended to decrease with time. The 2 to 7-year age and the 2 to 5-year age groups were dominant in the1960s, where as the 1 to 4-year age and 1 to 2-year age groups were dominant in the early 21st century. Mean forklength and mean body weight decreased by 47.04 mm and 247.39 g, respectively, from the 1960s to the early 21stcentury, which were decreases of 13.98% and 44.17%, respectively. The fatness index decreased by 0.14, whichwas a decrease of 11.48%. Mean fork length at maturity in males and females decreased by 48.07 mm and 45.70 mm,respectively, which accounted for decreases of 14.30% and 13.58%, respectively. The female to male ratio wasnear 1: 1 (P>0.05), but showed an increasing trend. A large number of age-1 fish joined the reproductive stock inthe early 21st century, compared with the previous two periods, which increased the number of reproductive stocks.No significant difference in fork length at maturity was detected between males and females (P>0.05), but minimumand mean fork lengths at maturity in males were slightly larger than those in females. S. japonicus has followedsome self-adjusting mechanisms to maintain population continuity. For example, reduced age at maturityand increases in the gonadosomatic index and the percentage of females have improved relative fecundity andreduced egg size in face of increasing fishing pressure during the past 50 years. We propose three fishery managementstrategies to maintain population stability and sustain the fishery based on the long-term changes in thebiological characteristics of the S. japonicus reproductive stocks in the East China Sea. The first is to control thepelagic fishery fishing effort and establish fishing boat and net standards. The second is to set up spawning fishreserves on the main spawning grounds. Special closed fishing periods should be implemented during larval feeding.The third strategy is to implement a total allowable catch management system for S. japonicus.

Abstract:The Portuguese oyster, Crassostrea angulata, is one of the most important shellfish species in China andoccurs mainly in the intertidal zone of the south Yangtze River. It is widely cultured in Zhejiang and Fujian provincesand many of its seeds are collected from natural areas. Molecular genetic data can provide valuable insightsinto C. angulata management and conservation. In the present study, we investigated the population geneticstructure and demographic history of C. angulata from the coast of Zhejiang (Yueqing) and Fujian (includingNingde, Langqiao, Fuqing, and Putian) provinces by analyzing the partial mitochondrial cytochrome c oxidasesubunit 1 (CO I) sequences in 183 samples. All populations were characterized by high genetic diversity.Forty-four polymorphic sites were identified, and 39 haplotypes were found. The mean genetic and mean nucleotidediversity values were 0.8524 and 0.00406, respectively. Despite high levels of genetic diversity within thepopulations sampled, no significant genealogical branches corresponded to the sampling locality. Analysis of molecularvariance indicated that most of the genetic variation was attributed to within-group variation (91.94%,P=0.0003). Population pairwise ΦST values were low (ΦST = −0.01193~0.11486), suggesting frequent gene flowamong the populations in this area. However, low but significant genetic differentiation was observed between theNingde samples and the other four samples from four sites, which was presumably due to the relatively closedgeophysicallocation of Ningde. Ningde is surrounded by many bays, islands, and gyres, which act as natural barriersthat may have hindered dispersal of oyster larvae. The haplotype network of CO I sequences with stellateradiation structure suggests no clear geographical differentiation. A Bayesian phylogenetic tree was characterizedby a staggered distribution of haplotypes and some small branches supported with moderately high bootstrap values.Significant expansion of C. angulata populations is suggested based on results from neutral tests and themismatched distribution; the actual time that expansion likely started in 2.5×105–2.1×105 years ago. Historicalrecolonization (though population range and demographic expansion during the late Pleistocene) and the currentgene flow (larval dispersal interacting with oceanographic processes, such as ocean currents) of adult sedentary C.angulatamay be responsible for the poor genetic differentiation. Our results will help in understanding the historicalprocesses of intertidal species and help disentangle the complex interactions among various factors thatgenerate the phylogeographical patterns and genetic diversity of C. angulata in the East China Sea.

Abstract:Enhancement and release of Larimichthys crocea has become more and more important because of continualdecline in its resources. The traditional method of marking was not suitable for the larvae of Larimichthyscrocea. In order to effectively evaluate itseffect of the enhancement, it has been a top priority to build a scalemethod of marking which is suitable for the larvae of Larimichthys crocea. The paper studied the effects of hexahydratestrontium chloride on otolith marking, growth and survival of Larimichthys crocea, aiming to improve theapplicability of this marking technique in tracing Larimichthys crocea for a better evaluating of its effect of theenhancement. The experiment selected 4 groups’ the larvae of Larimichthys crocea that were hatchery-reared, eachgroup had 2000 larvae of Larimichthys crocea. Two groups were immersed by hexahydrate strontium chloride, andothers were used as control groups. Before the experiment, we respectively selected 50 larvae in 4 groups to comparetheir difference of body length and body weight. During the experiment, we recorded the number of deathsper day so that we compared their difference of survival rate. After they were experimented 7 d and 50 d, we randomlyselected 15 larvae in 4 groups, respectively. We compared their difference of growth by biological determination,and we used laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to analyze theotolith of strontium distribution features. The results showed that fish exposed to increased levels of strontium hadhigher strontium-calcium ratio in the given part of the otolith relative to those of control and wild fish. Thehigher strontium-calcium ratio in the given part of the otolith was located by 0.65 to 0.80 mm far away from thecore area of the otolith. The strontium-calcium ratios in the given part of the otolith for the marked, control andwild fish were (3.58±1.09) mmol/mol, (1.73±0.08) mmol/mol, and (1.09±0.35) mmol/mol, respectively. They hadextremely significant difference (P<0.01). The higher otolith strontium-calcium ratio could be used as elementalfingerprint in identifying marked fish. In addition, there were no significant difference in survival rate amongmarked and control fishes by the statistical test (P>0.05), indicating that immersion in water enriched in strontiumdid not negatively affect the survival rate of marked fish. In terms of growth, after experimented for 7 d, the averageof body length and body weight of marking group and control group were (19.43±2.67) mm and (0.11±0.05) g,and (19.64±1.99) mm and (0.12±0.04) g, respectively. After experimented for 50 d the average of body length andbody weight of marking group and control group were (44.76±4.15) mm, (1.82±0.55) g, and (43.26±4.71) mm,(1.62±0.53) g, respectively. The body length, body weight and their relationship were tested according to statisticalmethods. We found that there were no significant difference (P>0.05), implying that strontium did not negativelyaffect the growth of marked fish. Our results suggested that immersion in water enriched with strontiumcould be an effective means of marking hatchery-reared Larimichthys croceafor stock enhancement. The meanscould provide solid fundament for the evaluation about scientific and reasonable enhancement and release.

Abstract:The class Scyphomedusae currently comprises 5 orders, 19 families, and about 202 species. These jellyfishare important pelagic predators at the center of concern about the “rise in gelatinous zooplankton” and degradationof coastal ecosystems. Previous phylogenetic relationship studies among Scyphomedusae species reliedprimarily on morphological data. However, morphology-based phylogeny and classification of this group are difficultdue to the high diversity of morphological characters, and the topic remains controversial. Large molecularlevel advancements have yielded interesting results among the Scyphomedusae taxa. Nemopilema nomurai,Rhopilema esculentum, Cyanea nozakii, and Aurelia sp. are four common macro-jellyfish species in the Bohai andYellow seas. In this study, the nuclear ITS-5.8S rDNA gene fragments from polyps, ephyrae, and adults of thesespecies were amplified using polymerase chain reaction techniques. Homologous sequences of other Scyphomedusaejellyfish from GenBank were also included in this study. The genetic information indices, including basecomposition, sequence variation, and Kimura two-parameter genetic distance were examined. Phylogenetic treeswere constructed based on the maximum-likelihood and Bayesian inference methods to determine the phylogeneticrelationships among Scyphomedusae jellyfish. Significant length variations in the ITS-5.8S rDNA region wereobserved (length, 675–833 bp). A total of 886 aligned base pairs, with 507 variable sites and 197 parsimony informativesites, were detected in the four jellyfish species. Inter-individual internal transcribed spacer region(ITS)1 polymorphisms were observed in most of the Scyphomedusae jellyfish species(four families and nine species).Repeats in microsatellite ITS loci play an important role in length heteroplasmy. The genetic distancesamong families, species, and individuals were 0.295–0.491, 0.024–0.812, and 0–0.099, respectively. The phylogenicanalysis showed that the topologies of the maximum-likelihood and Bayesian inference trees were somewhatdifferent and different from morphological taxonomy. In conclusion, ITS is an appropriate barcoding tool to identifyjellyfish species and classify young jellyfish. Furthermore, ITS1 was highly divergent among different Scyphomedusaejellyfish and could be an appropriate marker for molecular systematic studies at the species level.Additional in-depth studies with more samples are needed to further clarify the position of Scyphomedusae jellyfishin jellyfish taxonomy.

Abstract:Fish aggregating devices (FADs) are anchored and drifting objects deployed in the ocean to gathertropical tuna and other pelagic fish species. These devices support thousands of fishing vessels worldwide. Thesefloating objects increase catch and catchability of tunas, but large-scale use of FADs raises the possibility of apotential negative effect on tuna stocks and the pelagic ecosystem: (1) possible deleterious alterations in the normallife history of tunas associated with FADs, i.e., schooling, feeding, fitness, growth, migration, natural mortality,etc., which would result in loss of genetic quality; (2) other ecological effects associated with FAD fishing thatperturb the balance in the pelagic ecosystem by increasing by-catch of non-target species, such as miscellaneousbony fish, sharks, rays, and sea turtles. However, the ecological impact of FADs assessed in different areas usingseveral different approaches is controversial. This review summarizes studies on the ecological impact of FADsover the past 30 years, introduces the latest developments by domestic fisheries scientists and industry, and discussesdirections for future research. We aimed to provide a better understanding for future studies and potentialFAD management options.

Abstract:Nine different proportions of a group of bacteria, including Shewanella haliotis, Bacillus cereus, andAeromonas bivalvium were isolated from the gut of Pacific white shrimp, Litopenaeus vannamei. The shrimp werefed fodder with 109 cfu/g compound bacteria for 28 d using an orthogonal experimental design, and the effects ongrowth and innate immunity of the shrimp were assessed. Ammonia-nitrogen was adjusted to 26.67 mg/L withNH4Cl after 16 d, and ammonia-nitrogen resistance of the shrimp was tested. The results show that shrimp growthimproved after the 28 d probiotic feeding regime, compared to that of the control (P<0.05), and the 6︰1︰3 proportionwas better than the others. These results suggest that the compound bacteria promoted growth through secretionof nutrients and improved feed digestibility. Moreover, the positive effects of the compound bacteria oninnate immunity and ammonia-nitrogen resistance of L. vannamei were eventually expressed as improved growth.The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and total superoxide dismutase (T-SOD)were also enhanced compared to those in the control (P<0.05), but the effects were different. The 2︰3︰3, 4︰2︰3,and 6︰1︰3 proportions increased ACP activity significantly more than the others (P<0.05), and the 4︰2︰3 and6︰1︰3 proportions increased ALP activity significantly more than the others (P<0.05). The 2︰1︰1 proportionincreased T-SOD activity significantly more than the others. No changes in shrimp serum lysozyme activity weredetected in any of the bacteria-tested groups compared to the control. The results that serum ACP, ALP, and T-SODactivities were enhanced compared to the control show that the different proportions of bacteria positively affectedshrimp innate immunity. In addition, the 4︰3︰1 and 6︰3︰2 bacterial proportions significantly enhanced ammonia-nitrogen resistance in Pacific white shrimp (P<0.05). These results show that the compound bacteria hadpositive effects on ammonia-nitrogen resistance of L. vannamei and suggest that ammonia-nitrogen resistance isassociated with the immune system.