Abstract:In this study, we used Pelodiscus sinensis as a model to study the germ cells differentiation in reptiles. Firstly, we cloned P. sinensis dazl cDNA fragment of 1 007 bp, containing a 197 bp 5' untranslated region (UTR), a 33 bp 3' UTR and an open reading frame (ORF) for 258 amino acid residues. The predicted P. sinensis Dazl was maximally 96% identical with Chrysemys picta bellii Dazl protein, 75% identical with Mus musculus Dazl protein. The RT-PCR result showed that the P. sinensis dazl mRNA was absent in all somatic tissues, but abundant in adult ovary and testis. Chemical in situ hybridization revealed that the P. sinensis dazl mRNA was exclusively expressed in germ cells but barely detected in somatic cells, and displayed a dynamic distribution expression pattern in germ cells during gametogenesis. In adult testis, the dazl mRNA signals were strongly dispersed in primary spermatocyte and secondary spermatocyte, weakly in spermatogonia, barely detected in spermatids. In adult ovary, the dazl mRNA signals were strong in early primary stages of oocytes and displayed a uniform distribution; as oocytes grew into larger size, the dazl mRNA signals were concentrated in the perinuclear cytoplasm and became weak as vitellogenesis further developed. In conclusion, the findings in this study suggested that dazl gene would play important roles in germ cells differentiation during gametogenesis in P. sinensis. The characterization of dazl gene in P. sinensis would provide the basis for further invstigations into reproductive development in turtles or reptiles.

Abstract:In order to investigate the effects of dietary fish oil replacement with blending vegtable oils on the fattening performance, physiological metabolism and biochemical composition of adult female swimming crab (Portunus trituberculatus), five isonitrogenous and islipidic fattening diets were formulated to replace 0%, 25%, 50%, 75% and 100% of fish oil (defined as Diet 1-Diet 5) with blending vegtable oils (soybean oil∶rapeseed oil=1∶1). The five fattening diets were fed to female P. trituberculatus (150±25) g for 45 days. The results showed as follows: ① Dietary fish oil levels had no significant effects on survival rate, weight gain rate, gonadosomatic index and hepatosomatic index, meat yield and total edible yeild of adult female P. trituberculatus. ②There were significant differences in the triglyceride (TG) content, malate dehydrogenase (MDH) lipase (LPS) and lipoprotein lipase (LPL) activities in the hepatopancreas among five diet treatments; the highest levels of TG and LPS were detected in Diet 5, and the highest MDH was detected in Diet 1, while the highest LPL was detected in Diet 2. The highest TG and TC were detected in Diet 3 and Diet 4, respectively. ③ The hepatopancreatic peroxidase (POD) activity of the crabs in Diet 1 was significantly higher than that of Diet 2 and Diet 3, while the Diet 4 had the highest activities of hepatopancreatic alkaline phosphatase (ALP) and acid phosphatase (ACP). The highest and lowest values of total superoxide dismutase (T-SOD) activity in the serum were detected at treatments of Diet 2 and Diet 1, respectively, while the highest serum ALP activity was detected in Diet 1. ④ Dietary fish oil replacement has significant effects on the contents of crude protein and total carbohydrate in muscle, and the highest levels of crude protein and total carbohydrate in the muscle were dected in Diet 3. There were significant differences in the contents of total lipid and total carbohydrate in the hepatopancreas among five treatments, and the higher levels of them were detected for Diet 3 and 4 treatments; moreover, the highest ovarian crude protein content was also detected in Diet 3 treatment. ⑤ Dietary fish oil replacement had significant effects on the fatty acid profile of muscle, ovary, and hepatopancreas; tissue n-6 polyunsaturated fatty acid (n-6 PUFA) increased with increasing dietary fish oil replacement level, while the contents of n-3 PUFAs and highly unsaturated fatty acid (HUFA) showed a decreasing trend. In conclusion, dietary fish oil replacement by vegetable oils had no siginificant effect on survival rate and fattening performance of adult female P. trituberculatus, but promote the TG, crude lipid and n-6 PUFA accumulations in the hepatopancreas as well as the POD and phosphatase; 50% fish oil replacement level could increase the crude protein conetent of ovaries and muscle. These results indicated the dietary optimal fish oil replacement with blending vegetable oils could be 50% for the fattening of adult female P. trituberculatus.

Abstract:This experiment discussed the immunomodulatory effect of MMO in vitro, which used Meretrix meretrix oligopeptides (MMO) as raw material and RAW264.7 cells as experimental models. The research set up control group, positive medicine group (lipopolysaccharides, LPS), low concentration MMO group, medium concentration MMO group, and high concentration MMO group. Cell activity was detected by methyl thiazolyltetrazolium (MTT) colorimetric method, cell cycle was detected by flow cytometry, and cell morphology change was observed by hematoxylin-eosin staining (H.E) and cell phagocytosis ability was detected by neutral red phagocytic experiment. And immunofluorescence labeling was used to observe the expression of iNOS in cells. The contents of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β) in cell supernatant were detected by nitrate reductase analysis and enzyme-linked immunosorbent assay (ELISA). The results showed that MMO group exhibited increased proliferation and differentiation of RAW264.7 cells. When the concentration of MMO was 250 μg/mL, it obviously enhanced the phagocytosis capacity, increased the G1/G0 phase and G2/M phase cells in the cell cycle, induced NO release of iNOS, and promoted the secretion effect of IL-6 and IL-1β. All the phenomena were similar to LPS for RAW264.7 cells. Therefore, it is proved that MMO has the effect of promoting the activation of RAW264.7 cells and the potential of enhancing the non-specific immunity.

Abstract:Based on 69 samples of Loligo chinensis and 100 samples of Uroteuthis duvauceli collected in Luchaogang in November of 2015, the paper studied the species identification between L. chinensis and U. duvauceli by principal component analysis, stepwise discriminate analysis and Fourier analysis. Results showed that there were highly significant differences in the 12 morphological parameters of beak between two species. There were also significant differences between male and female U. duvauceli. The total successful discrimination rate for L. chinensis and U. duvauceli by stepwise discriminate analysis was 88.2%. The total successful discrimination rate for L. chinensis and U. duvauceli based on the result of principal component analysis was 90.1%. The total successful discrimination rate for male L. chinensis and male U. duvauceli was 83.3%. The total successful discrimination rate for female L. chinensis and female U. duvauceli attained 97.8%. The total successful discrimination rate for L. chinensis and U. duvauceli based on the upper beak by Fourier analysis was 83.3%. The total successful discrimination rate for L. chinensis and U. duvauceli based on the lower beak by Fourier analysis reached 95%.

Abstract:In order to understand the structure and expression profiles of TNFSF6 gene in Gadus macrocephalus, the full-length cDNA of TNFSF6 was cloned and analyzed using the bioinformatics tools, and quantitative real-time PCR (qRT-PCR) was used to examine the TNFSF6 mRNA expression levels in various tissues of adult G. macrocephalus, at different development stages of larvae, and during PCNNV outbreak. The results showed that TNFSF6 cDNA contains a 5'UTR of 315 bp, a 3'UTR of 500 bp, and an ORF of 573 bp encoding a protein of 190 amino acids. The results revealed that TNFSF6 was expressed in various tissues and it was higher in the spleen and gill. QRT-PCR indicated that the transcript level of TNFSF6 on 15, 20, 37, and 40 d post hatching was 0.28, 0.15, 0.12, 0.13 times of that on 5 d respectively. Compared to the control, the transcript level of TNFSF6 decreased significantly during virus outbreak on 77 d, however, there was an upward trend of TNFSF6 expression on 24 and 46 d respectively. The results indicated that TNFSF6 plays an important role in the early development stages and in responding to PCNNV outbreak.

Abstract:In order to suppress the growth of Pseudo-Nitzschia sicula and N. frustulum appearing during the cultivation of filaments of Scytosiphon lomentaria in our lab, the effects of germanium dioxide (GeO₂) at 1.00−4.00 μg/mL on the growth and development of filaments of S. lomentaria and the growth of epiphytic diatoms were studied in 3 different kinds of co-cultured systems. Results indicated that: ① the suitable concentrations of GeO₂ for co-cultured systems of filaments and diatoms were 1.00−2.50 μg/mL, at which the percentage decreases of diatoms were all above 67.33%±5.18% after 14-day co-culturing, meanwhile the filaments of S. lomentaria were in good conditions and good developments. 2.00 μg/mL was the most suitable concentration of GeO₂ since the filaments of S. lomentaria, under that condition, had the highest daily average growth rates which were all higher than 11.00% in 3 kinds of co-cultured systems, and ratios of sporangial branchlets as well as average diameter of sporangia were 57.47%±5.31% and (24.55±1.01) μm, respectively, which were not significantly different from those in control groups. ② Although diatoms were suppressed more effectively when the concentrations of GeO₂ was up to 3.50 and 4.00 μg/mL, the growth of filaments, the ratios of sporangial branchlets and average diameter of sporangia after inducing were also influenced negatively. What is more, filaments were dead when the concentration of GeO₂ reached 4.00 μg/mL. ③ N. frustulum is more sensitive to GeO₂ than P. sicula. After 14-day co-culturing, the percentage decreases of N. frustulum were between (82.10%±2.40%)−(96.35%±0.79%) at any concentration of GeO₂, which were higher than the percentage decreases of P. sicula under same conditions. Moreover, on the 14th day in co-cultured systems of filaments of S. lomentaria, P. sicula and N. frustulum, there was negative correlation between percentages of N. frustulum in the two species of diatoms and concentrations of GeO₂.

Abstract:This study aimed to develop a new type of low-salt and healthy compound salt agent for pickled tilapia (Oreochromis sp.) fillets processing technology. In this paper, the effects of different additions of sodium chloride and potassium chloride, sodium malate and white sugar on the sensory evaluation, salt content, water content, proteolysis index and texture of marinated tilapia fillets were investigated by single factor experiment with fresh tilapia fillets respectively, and optimization of mixing parameters of compound salt agent by Box-Behnken Response Surface method. Results indicated the effect of potassium chloride, sodium malate, sodium chloride on the quality of tilapia fillets was significant. The best processing technology of compound salt agent pickled tilapia fillets were as follows: the addition of potassium chloride was 2.67%, sodium malate was 1.32%, sodium chloride was 9.1%, white sugar was 0.5%. Under these conditions, the actual sensory evaluation was 89, the actual salt content was 2.81%, and the relative error of the target prediction value was 2.39%, indicating that the experimental model could be used to predict the actual value. Preliminary results indicate that compared with the control group, the sodium content of tilapia fillets decreased by 22.92%, which provided a new fast pickling processing technology of low salt and low sodium for the processing of tilapia.

Abstract:Artificial reef is an important part of marine ranching, and the scientific site selection is a necessary step of artificial reef area construction. By columnar sampling in the alternative region of Ma’an Archipelago waters in Shengsi, Zhejiang Province, physical properties including particle size, moisture content, natural density, and mechanical properties of penetration strength and shear strength were analyzed. The results showed that the sediments were mainly composed of sand, silty sand and clay, and the main component is sandy silt. The bottom sediments of different regions are different because of their various particle sizes, porosity, natural density, etc., so the bearing capacity of sediments also is different. Correlation analysis between the physical properties and mechanical properties showed that the moisture content of sediment and penetration strength, shear strength of failure are very strong in linear negative correlation, and the correlation coefficients are −0.67 and −0.64, respectively. These correlations could be used as an important reference for artificial reef sites suitability assessment. After the regression equation between sediment moisture content and particle size was established, it can provide a scientific basis for the analysis and research of sediment bearing capacity on a larger scale, also can reduce the cost of the investigation of sediment properties greatly.

Abstract:Based on 780 Sthenoeuthis oualaniensis samples collected from September to October 2012, the fishery biological characteristics of medium form and dwarf form populations were compared and studied, which provided basic data for the management and protection of squid fishery resources in the South China Sea. The mantle length (ML), body weight (BW), gonadosomatic index (GSI) and feeding factor (FF) of the samples were determined according to the marine survey standard, and the number of growth increments of statolith was determined by grinding. The results showed that the ML range of medium form is 79-266 mm, dominant in 111-145 mm, and vary with latitude insignificantly; ML range of dwarf form group is 56-118 mm, dominant in 66-95 mm, and increases slowly with latitude. BW range of medium form is 17.47-957.20 g, dominant in 45.01-120.00 g; BW range of dwarf form is 5.81-76.56 g, dominant in 5.01-50.00 g; BW of medium form and dwarf form both increase with latitude. All of the relationships between ML and BW of female and male of medium form and dwarf form could be described by power functions. Sex ratio (female/male) of medium form is 0.80∶1 and maturity ratio (immature/mature) is 1.12∶ 1; sex ratio (female/male) of dwarf form is 3∶1, and maturity ratio (immature/mature) is 0.83∶1. GSI of medium form is 1.71 and 1.31 in female and male, respectively; GSI of dwarf form is 4.07 and 4.11 in female and male, respectively. FF of medium form is 5.07 and 3.54 in female and male, respectively; FF of dwarf form is 1.89 and 2.75 in female and male, respectively. The range of statolith age of medium form is 30-135 d, and that of dwarf form is 44-81 d. Based on the captured date, hatching periods of medium form and dwarf form are dominant in July and August, respectively. Results indicate that the size of medium form is bigger than that of dwarf form, maturing and hatching are not in synchronization in medium form and dwarf form, and maturing and hatching of female of dwarf form are earlier than those of medium form. The results also indicate that the sea areas at 7°, 14° and 16° north latitude are the feeding grounds of squid populations in the South China Sea.

Abstract:According to previous studies, we found that HcTyp-1 gene may be involved in the formation of nacre color in shell of Hyriopsis cumingii. Primers were designed based on the cDNA sequence of HcTyp-1 gene in H. cumingii, and 8 SNPs were obtained by comparative screening and the correlation between SNPs and nacre colour-related traits was analyzed. Moreover, the location of HcTyp-1 gene on genetic map was studied. The study of the relationship between these polymorphic sites and shell nacre color traits found that, C+3057T showed a significant difference in the “a”; G+2985T and T+3006C had significant difference of L, b and a, dE respectively. Moreover, A+2834C, C+2919T and G+2986T all had significant difference in L, a and dE. Linkage disequilibrium analysis showed that there were 6 SNPs in high linkage disequilibrium except C+2912T and C+2983A. The result of haplotype analysis indicated that the frequency of T2 and T4 in the white mussels was significantly higher than that of purple group, but T6 and T8 were higher in purple group. The results suggested that the polymorphic loci in HcTyp-1 could be potential genetic markers for future molecular selection of different nacre color in H. cumingii. Furthermore, we mapped HcTyp-1 gene on LG16 of the previous linkage map, which will lay the foundation for the further study of the molecular mechanisms about this gene.

Abstract:The purpose of the present study is to reveal the genotype by environment (G×E) interactions on growth and shell color traits in the white-shell strain of Pacific oyster (Crassostrea gigas). The specimens of the white shell strain of C. gigas under six-generation of selection as parents were used to construct full-sib families following the method of nested design. All families were divided into two batches and grown in two environments, Rushan and Rongcheng. Linear mixed model and REML method based on an animal model were applied to estimate genetic parameters of white shell C. gigas at the age of 11 months. The best linear unbaised prediction (BLUP) method was used to estimate breeding values for shell height and L*, and superior families were selected based on comprehensive estimated breeding values. The results showed that heritabilities for growth and shell color traits in Rushan ranged from 0.14±0.08 to 0.62±0.18, while these were different in Rongcheng, varying from 0.01±0.03 to 0.78±0.19, which indicated that G×E interactions might be present as scale effects. After integrating the data in two different environments, heritabilities for growth and shell color traits ranged from 0.02±0.02 to 0.51±0.09. However, the estimates of heritabilities might be over-estimated because maternal/common environmental effects and dominance effects were included in the estimation model due to absence of some families and convergence problem. Genetic correlations for all growth and shell color traits between two environments, ranging from -0.47±0.40 to 0.75±0.18, were less than 0.8. This suggested that G×E interactions in the form of re-ranking of families across environments was apparent. It will be necessary to select lines that are suited to particular sites. The top 20 offspring in the rank of comprehensive estimated breeding values derived from different families in two different environments, indicating that the different families performed differently across the different rearing sites. The families G1 and G21 performed better in Rushan area, while the G4, G22 and G5 were most excellent families in Rongcheng area, and familiy G2 had high adaptability to both sites. The information obtained in this study will benefit genetic improvement of the white shell strain of C. gigas.

Abstract:In order to study the correlation between EGFR gene and growth traits (shell length, shell width, shell height and body weight), we analyzed the variation of EGFR intron 1 sequence from Sinonovacula constricta by using direct sequencing. There were 17 SNP sites in intron 1, named SNP1–SNP17 respectively. Based on Chi-square test, the 13 sites were fitted to Hardy-Weinberg equilibrium, and the 10 sites showed moderate polymorphism through polymorphism detection (0.25<PIC<0.5). The correlation between SNPs and growth traits (shell length, shell width, shell height, and body weight) were further analyzed using a General Linear Model and multiple comparisons, and the results showed that a total of 16 SNP sites were significantly associated with shell length, shell width, shell height and body weight. The results suggest that EGFR gene could be used as potential genetic marker for molecular breeding. The study also laid a foundation for further research on its growth function.

Abstract:Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix. In order to study the function of MMPs in the immune defense of Apostichopus japonicus, the full-length cDNA sequence of the matrix metalloproteinase 16 gene, named Aj-MMP-16, was cloned using RACE method. The sequence characteristics and function of Aj-MMP-16 were preliminarily analyzed. The results showed that the full-length cDNA of this gene was 2 976 bp, including a 5' non-coding region of 342 bp, a 3' non-coding region of 963 bp, and an open reading frame (ORF) of 1 671 bp encoding 557 amino acids. The predicted molecular weight of Aj-MMP-16 protein was 63.11 ku and isoelectric point was 4.79. Functional domain analysis revealed the typical MMPs family protein structure of Aj-MMP-16 including N-terminal propeptide region, hinge region, catalytic region, hemopexin-like domain and transmembrane region. Multiple sequence alignment and phylogenetic analysis showed that Aj-MMP-16 shared a certain degree of conservatism with MMPs of other species and had the highest identity with MMP-16 of Strongylocentrotus purpuratus. Quantitative real time PCR showed that the Aj-MMP-16 mRNA was expressed in all tissues of A. japonicus, and the expression levels were from high to low in the order of respiratory tree, intestine, coelomocytes, tube feet, muscle and body wall. At different stages of skin ulceration syndrome progression, the expression of Aj-MMP-16 mRNA in ulcerate body wall was significantly higher than that of the normal body wall. After the pathogenic bacteria challenge, Aj-MMP-16 mRNA expression increased significantly in coelomocytes. The results suggested that Aj-MMP-16 may play important roles in visceral regeneration, inflammation and immune response in sea cucumber.

Abstract:To analyze the influence of n-3/n-6 PUFAs levels on the growth performance and growth- related genes mRNA expression in common carp, five isonitrogenous and isoenergetic experimental diets based on fish oil and mixed vegetable oil were fed to the Cyprinus carpio haematopterus for 10 weeks. The control diet (T1) was only supplemented with fish oil as lipid source, n-3/n-6 ratio was 0.97. the second group (T2) was only supplemented with peanut oil, n-3/n-6 ratio was 0.02. n-3/n-6 ratio of the other three experiments groups were 0.46(T3), 1.09 (T4) and 1.53 (T5) with peanut oil and perilla seed oil as feed source. After 10 weeks cultivation experiments, we checked each group for fish growth performance, serum growth hormone and growth-related gene expression level in different parts of muscle tissue. The results indicated that there were no significant differences in the viscerosomatic index (VSI), feed coefficient (FCR), feed intake(FI)among the five tested groups. Weight gain rate of the fish (WGR) was raised first and subsequently declined with the increase of the n-3/n-6 ratio. WGR climbed the peak at T4 group. The serum GH levels of T2 group were significantly higher than those of others, and the level of GH in serum gradually reduced with the increase of of n-3/n-6 ratio. The expression levels of growth-related genes in the dorsal muscles showed that gh, ghr, igf-1 and myod were enhanced first and subsequently declined. However, igf-1r displayed the opposite expressive trend, while there were no significant differences in mstn level among the five treated groups. In red muscle, the expression levels of gh and myod were gradually decreased with the increase of n-3/n-6 ratio, and the expressions of ghr and mstn exhibited no significant differences in five groups. Besides, the expressive trends of igf-1 and igf-1r were similar to that of dorsal muscles. Interestingly, the expressions of gh, ghr and igf-1 in the abdominal muscles were consistent with the dorsal muscles. But, the levels of igf-1r and myod manifested no significant difference in five tested groups. There were significant differences in the number of muscle fibers in the T2 group and T4 group, and the muscle fibers in T4 group were thicker than those of T2. This study showed that vegetable oil contributed to improve the WGR of the fish and the n-3/n-6 ratio of the diets not only affected the serum GH level, but also influenced the growth related genes of GH/IGF axis in different parts of the muscle tissues.