Effects of exposure to four endocrine disrupting-chemicals on fertilization and embryonic development of Barbel chub (Squaliobarbus curriculus)

The toxicities of 4 common endocrine-disrupting chemicals (EDCs), 17β-estradiol (E2), p,p′-dichlorodiphenyldichloro-ethylene (DDE), 4-nonylphenol (NP) and tributyltin (TBT), to sperm motility, fertilization rate, hatching rate and embryonic development of Barbel chub (Squaliobarbus curriculus) were investigated in this study. The duration of sperm motility was significantly shortened by exposure to the EDCs at the threshold concentrations of 10 ng L^?1 for E2 and TBT, 1 μg L^?1 for NP and 100 μg L^?1 for DDE, respectively. The fertilization rate was substantially reduced by the EDCs at the lowest observable effect concentrations (LOECs) of 10 ng L^?1 for E2 and TBT and 10 μg L^?1 for DDE and NP, respectively. Of the tested properties of S. curriculus, larval deformity rate was most sensitive to EDC exposure and was significantly increased by DDE at the lowest experimental level of 0.1 μg L^?1. Other EDCs increased the larval deformity rate at the LOECs of 1 ng L^?1 for E2, 10 ng L^?1 for TBT and 1 μg L^?1 for NP, respectively. Despite their decreases with the increasing EDC concentrations, the hatching rate and larval survival rate of S. curriculus were not significantly affected by the exposure to EDCs. The results indicated that all the 4 EDCs affected significantly and negatively the early life stages of the freshwater fish S. curriculus. Overall, E2 and TBT were more toxic than NP and DDE, while DDE might be more toxic to larval deformity rate than to other measured parameters. Thus, the 4 EDCs showed potential negative influences on natural population dynamics of S. curriculus. Our findings provided valuable basic data for the ecological risk assessment of E2, DDE, NP and TBT.     

Evaluation of Estrogenic Activity and Measurement of EDCs in Wastewater Treatment Plants

1IntroductionDuringthe past several years,concern hasincreasedover the potential pollution of watershed by estrogeniccompounds,including steroidal hormones fromhumanand ani mal sources.Effluents from wastewater treat-ment plants are sources of endocrine-d…     

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.     

Growth and antioxidant status of oriental river prawn <Emphasis Type="Italic">Macrobrachium nipponense</Emphasis> fed with diets containing vitamin E

A feeding trial was carried out to investigate the dietary vitamin E requirement of the oriental river prawn Macrobrachium nipponense (weight of 0.3–0.4 g) and its effect role on antioxidant activity. Prawns were fed with seven levels of vitamin E (0, 25, 50, 75, 100, 200, and 400 mg/kg diet) for 60 days. The results show that dietary vitamin E supplementation could significantly increased the prawn weight (P < 0.05). The activity of superoxide dismutase (SOD) in the hepatopancreas was significantly higher in prawns fed with diets supplemented with ≤75 mg/kg vitamin E than in those fed with diets supplemented with 100–400 mg/kg vitamin E (P < 0.05). The activity of catalase (CAT) in the hepatopancreas decreased significantly as dietary vitamin E supplementation increased (P < 0.05), and no significant difference was detected in glutathione peroxidase (GSH-Px) activity between different dietary groups (P >0.05). The contents of vitamin E in the hepatopancreas and in the muscle increased with increasing dietary vitamin E. There was a linear correlation between the vitamin E level in diet and that in muscle, and between the vitamin E level in diet and that in the hepatopancreas. All the above results indicated that dietary vitamin E can be stored in the hepatopancreas and muscle and lower both the activities of SOD and CAT in the hepatopancreas, suggesting that it is a potential antioxidant in M. nipponense. Broken line analysis conducted on the weight gains of prawns in each diet group showed that the dietary vitamin E requirement for maximum growth is 94.10 mg/kg.     

Analysis of phenotypic and genetic parameters for growthrelated traits in the half smooth tongue sole, <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Phenotypic and genetic parameters for growth-related traits in the half-smooth tongue sole, Cynoglossus semilaevis, were estimated in 22 full-sib families produced by normal and neo-male breeding stocks. As phenotypic males with female genotypes, neo-males are harmful in C. semilaevis aquaculture because they reduce overall production. The present study evaluated the difference in the growth-related traits: total length (TL), body weight (BW) and square root of body weight (SQ_BW) at the age of 570 days between normal and neo-male offspring (neo-males used as male parents). The difference in the proportion of females between normal and neo-male offspring was also assessed. Based on the linear mixed model, restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP) were used to estimate various (co)variance components and estimated breeding values (EBVs) of growth-related traits. As a result, all the mean values of the three studied traits were significantly larger in normal offspring than in neo-male offspring. Additionally, the female proportion was significantly larger in normal offspring than in neo-male offspring. Heritability was 0.128±0.066 2 for TL, 0.128±0.065 5 for BW and 0.132±0.062 9 for SQ_BW, all of which were low level heritabilities. The correlation coefficients of EBVs and phenotypic values of the target traits were 0.516 for TL, 0.524 for BW and 0.506 for SQ_BW, all of which were highly significant (P <0.01). Genetic correlations among TL, BW and SQ_BW were positive high (0.921–0.969) and higher than those of phenotype (0.711–0.748), both of which had low standard errors (0.063–0.123 for genotype, and 0.010–0.018 for phenotype). Compared with normal offspring, neo-male offspring have lower breeding values for each studied trait through EBVs comparison. Therefore, neo-male offspring should not be used as broodstock in a C. semilaevis breeding programs.     

Identification of Cytochrome P450 (CYP) genes in Zhikong scallop (Chlamys farreri)

Cytochrome P450 (CYP) superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop (Chlamys farreri) for CYP genes. In total, 88 non-redundant CYP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYP10 and CYP24 families. In comparison, protostomes (C. farreri, D. pluex, D. melanogaster) contained more CYP genes than deuterostomes (S. purpuratus and vertebrates) in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.     

QTL Detection for Albinism-Related Loci in Chinese Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The Chinese tongue sole (Cynoglossus semilaevis) is widely cultured in the coastal region of East Asia and has excellent economic value. However, the high albino rate of the breeding population has caused a significant loss to the aquaculture industry. To study the molecular mechanism of albinism, the present study used an albino Chinese tongue sole family to construct three simple sequence repeat (SSR) linkage groups, and draft a preliminary linkage map related to albinism. After albinism-related loci mapping, 18 albinism-related loci were detected under two models (containing 2407 genes) compared to the Chinese tongue sole genome. One of these loci, the tyrosinase related protein (tyrp2), which has been reported previously as an important gene regulating both eumelanin and phaeomelanin levels, was indicated to be the possible cause of albinism. Thirty-five Gene Ontology (GO) terms and 14 Kyoto Encyclopedia of Genes and Genome pathways were annotated via bioinformatic analyses. One GO term with protein tyrosine kinase activity, which contained 10 genes, was previously suggested to affect fish albinism. These results establish a foundation for further in-depth study of albinism in Chinese tongue sole.     

Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex class II antigens are important in vertebrate immune system.In the present study,the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole(Cynoglossus semilaevis),and its open reading frame(ORF) polymorphism was studied.The whole cDNA sequence was 992 bp in length,including the ORF with 717 bp.Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/amino acids in specific positions.Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA.Four Cyse-DAA alleles were observed in one individual,and three to five Cyse-DBA alleles were observed in each of the three detected individuals,which indicated that at least two loci existed in each gene.Moreover,in order to study the function of the alleles in resistance to infection,200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis.Fifty-six alleles were identified among the 40 individuals.Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA.Eighteen alleles were selected for studying their function in resistance to infection.Alleles Cyse-DAA*0201,Cyse-DAA*1101,Cyse-DBA*0401,Cyse-DBA*1102,Cyse-DBA*1801 and Cyse-DBA*2201 were identi-fied only in surviving individuals,while alleles Cyse-DAA*0901,Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals.This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/resistance in half-smooth tongue sole.     

Steroid sex hormone dynamics during estradiol-17β induced gonadal differentiation in Paralichthys olivaceus (Teleostei)

Steroid sex hormones, such as estradiol-17β (E2) and testosterone (T), are important regulators of sex change in fish. In this study, we examined the effects of E2 treatment on the dynamics of E2 and T during gonadal differentiation in the olive flounder Paralichthys olivaceus using histology and radioimmunoassay (RIA). Flounder larvae were divided into five groups (G0-G4), and fed with 0 (control), 0.2, 2, 20 and 100 mg E2/kg feed from 35 to 110 day post hatching (dph). Fish growth in the G1 and G2 groups was not significantly different from that of the control group (P>0.05), while fish in the G3 and G4 groups were less active and showed growth depression and high mortality. The gonads of fish in the G3 and G4 groups were smaller and surrounded by hyperplastic connective tissue. The frequency of females in the G0-G4 groups was 54.5%, 75.0%, 100%, 100% and 93.3%, respectively. The RIA analyses of E2 and T showed that T levels decreased during gonadal differentiation, and increased slightly at the onset of ovarian differentiation, while E2 levels increased gradually and peaked at the onset of ovarian differentiation in the control group. In the E2-treated groups, T levels decreased before the onset of ovarian differentiation. E2 levels were high on the 48 dph, but declined to a lower level on the 54 dph, and then increased gradually during gonadal differentiation. And a sharp increase of E2 levels were observed in all E2-treated groups at the onset of ovarian differentiation. The data suggest that T and E2 play important roles during gonadal differentiation, and an E2 dose of 2 mg/kg feed could induce sex reversal in P. olivaceus.     

Long-term effects of di-octyl phthalate on the expression of immune-related genes in <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Di-octyl phthalate (DOP) is widely used as a plasticizer in the plastics industry. As a result, DOP is often found in marine water ecosystems where many species are exposed to it. Our objective was to evaluate the effect of long-term (14 d) DOP exposure (2.6, 7.8, or 31.2 mg/L) on the expression of immunerelated genes in Tegillarca granosa. The expression of small heat shock protein (sHSPs) and tissue inhibitor of metalloproteinase (TIMP) were highest in clams exposed to 31.2 mg/L DOP on days 7 and 14. The relative expression of Tg-ferritin, superoxide dismutase (SOD), and metallothionein (MT) increased initially then decreased as the concentration of DOP increased. The hemoglobin of T. granosa (Tg-HbI) exhibited two distinct expression patterns at two time points. Our results suggest that the immune response of T. granosa against DOP pollution varies depending on the dose. Additionally, we identified some immune-related genes that are promising candidates for biomarkers of DOP.     

Sequence polymorphism of two major histocompatibility (MH) class II B genes and their association with Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex (MHC) class Ⅱ B molecules play an important role in the adaptive immune response in fish. Previous study has reported that two highly polymorphic class ⅡB genes, Cyse-DAB and Cyse-DBB exist in half-smooth tongue sole (Cynoglossus semilaevis). In this study, the polymorphism within exon 2 of the class Ⅱ B genes following bacterial challenge was evaluated. Two hundred C. semilaevis individuals were injected intraperitoneally with Vibrio anguillarum. Muscle tissue from the first 20 dead and 20 of the survivors was collected for genotyping. Sixty alleles from the 40 individuals were isolated, of which 32 belonged to Cyse-DAB and 28 belonged to Cyse-DBB. The rate of dN (non-synonymous substitution) was higher than that of dS (synonymous substitution) in the PBRs (peptide binding residues) of both class Ⅱ B genes. Conversely, the rate of dS was higher than dN in the non-PBRs and the complete exon 2 sequence. Thus, the results suggest that positive selection has occurred in the PBRs and purifying selection in the non-PBRs and exon 2. Thirteen class Ⅱ B alleles were used to study the association between alleles and resistance to infection. Though not significant, alleles Cyse-DAB*0601, Cyse-DAB*0706, and Cyse-DBB*0101, Cyse-DBB*1301 were only found in surviving individuals and may represent alleles that have resistance against V. anguillarum infection. Alleles Cyse-DAB*0701 and Cyse-DAB*1301 were significantly more prevalent in dead individuals than in surviving ones and may represent alleles that are associated with increased susceptibility to V. anguillarum infection.     

Effect of salinity on growth and first sexual maturity of Exopalaemon carinicauda (Holthuis, 1950)

To determine the optimal salinity for growth and first sexual maturity of ExopaIaemon carinicauda, the effects of salinity on growth and reproductive performance of early juvenile prawns of E. carinicauda were evaluated under laboratory conditions. Postlarvae from the same female broodstock were reared at six salinity levels （5, 10, 15, 20, 25, and 30） for 12 weeks. The specific growth rate （SGR） and survival rate （SR） under different salinity levels in the first 6 weeks were calculated and compared. SGR was significantly influenced by salinity. Prawns reared in salinity of 10 grew significantly faster （P〈0.05） than those reared in salinities of 5, 20, 25, and 30. However, ANOVA confirmed that there was no significant effect among the six salinity levels on SR. For the next 6 weeks, the body length （BL）, body weight （BW） at the first sexual maturity, and the age at median sexual maturity （As0） of females were measured and compared. Female prawns reared at salinity of 10 presented significantly shorter A50, but no significant differences among the six salinity levels for BL and BW at the first sexual maturity were observed. Based on the above information, the optimal salinity for growth and first sexual maturity of juvenile E. carinicauda is approximately 10.     

Expression profiles of sex-related genes in gonads of genetic male Takifugu rubripes after 17β-estradiol immersion

Estradiol treatment during early life stages of tiger puffer Takifugu rubripes induces feminization in genetic males. However, the ovaries in genetic males may revert to testes once estradiol treatment is halted.Therefore studies should investigate molecular mechanisms underlying ovary-to-testis recovery in genetic males after treatment. In the present study, tiger puffer were exposed to 10, and 100 μg/L 17 p-estradiol(E_2)from 15 to 100 days post-hatching(dph), then gonad phenotypes and expression profiles of six sex-related genes(cyp19a,foxl2, dmrtl, amh, sox9a, and sox9b) were characterized after the exposure. Results showed that both 10 and 100 μg/L E_2 induced ovarian development in genetic males at 100 dph. However, all ovaries induced by 10 μg/L E_2 first developed into intersexual gonads and subsequently reverted to testes after the exposure. As for treatment of 100 pg/L E_2, while the rest of the ovaries maintained morphological stability,percentages of intersexual gonads reached 38%-57%, and none were reverted to testes. Increased mRNA levels of cyp19 a,foxl2 and sox9b and decreased mRNA levels of dmrtl, amh, and sox9a were observed during the ovarian development in genetic males. While contrary gene expression profiles were detected during ovary-to-testis transformation. The mRNA levels of all the six genes were increased during the development of intersexual gonads. These results indicated that up-regulation of dmrtl, amh and sox9a is associated with initial ovary-to-intersexual transformation, and suppression of foxl2, cypl9a and sox9b is essential for complete ovary-to-testis recovery in genetic males. This research will help to trace the molecular processes underlying gonadal transformation in teleosts.     

Effect of dietary supplementation of vitamin C on growth,reactive oxygen species,and antioxidant enzyme activity of Apostichopus japonicus (Selenka) juveniles exposed to nitrite

Different amounts of vitamin C were added to diets fed to juveniles(2.5 ± 0.15 g) of sea cucumber Apostichopus japonic u s(Selenka) in an attempt to reduce the stress response of specimens exposed to nitrite stress. A commercial feed was used as the control diet and three experimental diets were made by supplementing 1 000, 1 500, or 2 000 mg vitamin C/kg diet to control diet separately in a 45-day experiment. Sea cucumbers were exposed to three different levels(0.5, 1.0, and 1.5 mg/L) of nitrite stress for 4, 8, and 12 h at four time intervals(0, 15, 30, and 45 d). Growth of the animals was recorded during the experiment. Reactive oxygen species(ROS)(i.e. hydroxyl free radical(-OH), malondialdehyde(MDA) and total antioxidant capacity(T-AOC)) and antioxidant enzyme activities(i.e., superoxide dismutase(SOD) and catalase(CAT)) were measured. Response surface methodology(RSM) was used to analyze the effect of multiple factors on ROS indices and enzyme activities. Weight gain(WG) and special growth rate(SGR) of vitamin C supplementation groups were significantly higher than those of control group( P 0.05). The levels of-OH and MDA increased under exposure time extending and nitrite concentration increasing, whereas T-AOC level decreased. SOD and CAT activities increased at 4 h and 8 h and decreased at 12 h. During the days in which the animal consumed experimental diets, the levels of-OH and MDA decreased and that of T-AOC increased. This result suggests that diets containing vitamin C could reduce the nitrite stress response in the animals and increase their antioxidant capacity. The multifactor regression equation of growth performance, ROS indices, and duration of feeding results suggest that vitamin C supplementation of 1 400–2 000 mg/kg diet for 29–35 days could reduce effectively the effects of nitrite exposure.     

Effects of varying densities on serum reproductive parameters in pen-reared juvenile female rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> farms

The primary goal of this study was to assess the effect of varying densities on serum reproductive parameters of immature rainbow trout Oncorhynchus mykiss. Experimental trout were maintained in intensive, pen-reared farms for 300 days in fresh water reservoirs. Initial densities were 4.6, 6.6, and 8.6 kg/m³ (40, 60, 80 ind./m³), indicated as SD1, SD2, SD3, and final densities were 31.1, 40.6, 49.3 kg/m³, respectively. A summary of the ovarian stages were observed by histological examination. Serum E₂ (estradiol), T (testosterone) were evaluated by radioimmunoassay and FSH (follicle-stimulating-hormone), LH (luteinizing-hormone), vitellogenin, 17α,20β-P (17α,20βdihydroxy4-pregnen-3-one) were measured by enzyme-linked immunosorbent assay. Our findings demonstrated that ovarian development were retarded (from stage III to stage IV) at highest rearing density (SD3) after 180 days of intensive culture (over 40.6 kg/m³). In addition, we observed an inverse relationship between serum reproductive parameters and rearing density. Furthermore, compared to serum reproductive parameters of SD1, E₂, T, FSH, vitellogenin, 17α,20β-P, GSI and LH of two higher density groups decreased firstly and significantly at 60 (over 15.9 kg/m 3 ), 180 (over 31.7 kg/m 3 ), 180 (over 40.6 kg/m³), 240 (over 36 kg/m³), 240 (over 36 kg/m³), 240 (over 45 kg/m³) and 300 (over 49.3 kg/m³) days, respectively. Comparing serum reproductive parameters within the same ovarian development stage of rainbow trout from varying densities revealed that higher population density also led to significantly lower overall serum reproductive parameters. Overall, this study presents the reproductive, endocrinological parameters of juvenile female rainbow trout at high rearing densities and indicates the need for rainbow trout (114.44±5.21 g, 19.69±0.31 cm) that are initially stocked at 6.6 or 8.6 kg/m³ should be classified and subdivided into lower density after 180 days of farming (not over 31.7 kg/m³).     

De-eutrophication of effluent wastewater from fish aquaculture by using marine green alga Ulva pertusa

The de-eutrophication abilities and characteristics of Ulva pertusa, a marine green alga, were investigated in Qingdao Yihai Hatchery Center from spring to summer in 2005 by analyzing the dynamic changes in NH ₄ ⁺ , NO ₃ ^? , NO ₂ ^? as well as the total dissolved inorganic nitrogen (DIN). The results show that the effluent wastewater produced by fish aquaculture had typical eutrophication levels with an average of 34.3 μmol L^?1 DIN. This level far exceeded the level IV quality of the national seawater standard and could easily lead to phytoplankton blooms in nature if discarded with no treatment. The de-eutrophication abilities of U. pertusa varied greatly and depended mainly on the original eutrophic level the U. pertusa material was derived from. U. pertusa used to living in low DIN conditions had poor DIN removal abilities, while materials cultured in DIN-enriched seawater showed strong de-eutrophication abilities. In other words, the de-eutrophication ability of U. pertusa was evidently induced by high DIN levels. The de-eutrophication capacity of U. pertusa seemed to also be light dependent, because it was weaker in darkness than under illumination. However, no further improvement in the de-eutrophication capacity of U. pertusa was observed once the light intensity exceeded 300 μmol M² S^?1. Results of semi-continuous wastewater replacement experiments showed that U. pertusa permanently absorbed nutrients from eutrophicated wastewater at a mean rate of 299 mg/kg fresh weight per day (126 mg/kg DIN during the night, 173 mg/kg in daytime). Based on the above results, engineered de-eutrophication of wastewater by using a U. pertusa filter system seems feasible. The algal quantity required to purify all the eutrophicated outflow wastewater from the Qingdao Yihai Hatchery Center into oligotrophic level I clean seawater was also estimated using the daily discharged wastewater, the average DIN concentration released and the de-eutrophication capacity of U. pertusa.     

Lipid oxidation and fatty acid composition in salt-dried yellow croaker (<Emphasis Type="Italic">Pseudosciaena polyactis</Emphasis>) during processing

Lipid oxidation in salt-dried yellow croaker (Pseudosciaena polyactis) was evaluated during processing with commonly used analytical indices, such as the peroxide value (POV), the thiobarbituric acid reactive substances (TBARS) value, and oxidative-relative lipoxygenase (LOX) activity. Additionally, fatty acids were analyzed using gas chromatography-mass spectrometry. Both POV and TBARS increased significantly (P < 0.05) at the rinsing stage. POV reached its peak value of 3.63 meq O₂ per kg sample at the drying stage, whereas TBARS constantly increased from 0.05 to 0.20 mg MDA per kg sample. Processing of salt-dried yellow croaker had an extremely significant (P < 0.01) effect on LOX activity. Twenty-six fatty acids were identified. Combined eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) content varied between (19.20 ± 0.37) mg g^?1 and (23.45 ± 1.05) mg g^?1. The polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) ratio in yellow croaker was 0.73–1.10, and the n-6/n-3 PUFA ratio was approximately 0.13–0.20. The contents of most fatty acids varied significantly (P < 0.05) during the different processing stages, and these differences were caused by lipid oxidation. C18:0, C16:1n7, C19:0, and C22:6n3 showed clear changes in principle component one of a principle components analysis. These fatty acids are potential markers for evaluating lipid oxidation in fish muscle because there was a significant correlation between these markers and TBARS and LOX activity (P < 0.05) with Pearson’s coefficients > 0.931.     

Biochemical response of the mussel Mytilus coruscus (Mytiloida: Mytilidae) exposed to in vivo sub-lethal copper concentrations

Many aquatic organisms are negatively affected by exposure to high copper concentrations.We investigated the biochemical response of the mussel Mytilus coruscus(Mytiloida:Mytilidae) to copper exposure.In vivo bioassays using M.coruscus and different copper concentrations were conducted.The activity of six biomarkers,namely superoxide dismutase(SOD),catalase(CAT),acid phosphatase(ACP),alkaline phosphatase(AKP),glutamic-oxaloacetic transaminase(GOT) and glutamic-pyruvic transaminase(GPT) were measured.Survival rates decreased with increased copper concentrations and exposure times.The LC50 values at 48,72,and 96 h exposure were 0.48,0.37,and 0.32 mg/L,respectively.Within digestive glands,CAT activity increased with increasing Cu concentrations.The activity of AKP showed no significant change,while the remaining four enzymes showed decreasing activity with increasing Cu concentrations.Within the gills,AKP activity increased when the Cu concentration was 0.05 mg/L,but showed no significant changes at higher concentrations.Activity of CAT and ACP within gills tended to decrease with increasing Cu concentration.The activity of SOD and GPT decreased at an exposure concentration of 0.2 mg/L.GOT activity within gills decreased at 0.1 mg/L and increased at an exposure concentration of 0.2 mg/L.Within the adductor muscle,AKP activity increased at 0.05 mg/L but did not change at higher exposure concentrations.ACP activity within adductor muscle tissue showed no change,while activities of CAT,GOT and GPT decreased with increasing Cu concentrations.SOD activity within the adductor muscle tissue significantly decreased at the 0.02,0.05 and 0.2 mg/L exposure concentrations.Our results show tissue specific differences for the six biomarkers in for M.coruscus.Our findings provide the basis for the establishment of reference activity levels against which biomarker changes can be estimated,and are essential preliminary steps in development of in vivo bioassays.