Studies on hybridization effects of different geographic populations of Chlamys farreri Ⅱ.The medium-term growth and development of Chlamys farreri populations from China and Russia and their reciprocal crosses

Based on the research of juvenile (2, 3, 4 months) growth and survival of three populations of two different geographic areas in Chlamys farreri from Russian and China and their F1 hybrids derived from Chinese cultural population (CC) (?) × Russian population (RW) (?) , Chinese wild population (CW) (?) × Russian population (RW) (?), Russian population (RW) (?) × Chinese wild population (CW) (?) , the study of the medium-term (6, 8, 10, 12 months) growth and development of Chlamys farreri was carried out. The four determined results indicated that there existed different extent heterosis (3% -52%) for the growth in three types of F1 hybrids, and the offspring derived from CC(?) ×R(?) had a stronger heterosis among the crosses at the medium-term; the uptrend among traits are wet weight >shell width>shell length> shell height, Chinese cultural population could be recognized as excellent parent, and seasonal variations influence very much on the daily increment and growth rate of each trait of Chlamys farreri and it is only able to survive and could barely grow in winter (6-8 months), but grows fast in temperate season (10-12 months).

IMPACT OF STARVATION ON SURVIVAL, MEAT CONDITION AND METABOLISM OF CHLAMYS FARRERI

The effects of 60 day starvation on survival rate, condition index (CI), changes of nutrient composition of different tissues, respiration and excretion of scallop Chlamys farreri were studied in laboratory from Oct. 17 to Dec. 15,1997. Two groups (control and starvation with 200 individuals each) were cultured in two 2 m 3 tanks, with 31 to 32 salinity water at 17℃. Starvation effects were measured after 10, 20, 40 and 60 days. There was no mass mortality of scallops of the two tanks and survival rates of the control and starvation groups were 93.5% and 92.0%, respectively. Starvation had strong effect on the meat condition of the scallops, especially after 10 days; when relative lipid percentage dropped sharply while relative protein percentage increased. The impact of starvation on the oxygen consumption rate (OCR) and the ammonia N excretion rate (AER) was obvious. The OCR increased rapidly after 10 days but decreased after 20 days. The AER increased after 10 days and 20 days, but decreased obviously from 20 to 40 days. The O:N ratios varied to different degrees, and minimized after 20 days. The low O:N ratios implied that the protein was the main material for the metabolism of C. farreri.

Characterization, Expression and Function Analysis of DAX1 Gene of Scallop(Chlamys farreri Jones and Preston 1904) During Its Gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop(Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at m RNA and protein levels. The c DNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame(ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher(P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.

Protective Effect of Topically Applied Polypeptide from Chlamys farreri Against Ultraviolet Radiation-Induced Chronic Skin Damage in Guinea Pig

Polypeptide from Chlamys farreri (PCF) , a topical polypeptide isolated from Chlamys farreri , was used in this experiment aimed to investigate the photoprotective effect of PCF against chronic skin damage induced by ultraviolet A (UVA) and ultraviolet B (UVB) radiation. The chronic ultraviolet irradiated guinea pig model was established, and visible changes in the skin including wrinkling, sagging and erythema were observed. Malondialdehyde (MDA) and antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH px) in the dorsal skin were determined using biochemical methods. The results showed:(1)PCF (5 % and 20%) could greatly protect the dorsal skin of guinea pig against wrinkling, sagging and erythema induced by UV radiation in a concentration dependent manner.(2)PCF could reduce MDA formation in the dorsal skin caused by UV irradiation, while increasing the activities of SOD and GSH px.(3)The differences among the PCF groups and UV model group were significant ( P <0.05, P <0.01). These results indicated that topical application of PCF provided broad solar UV spectrum photoprotection; and that the antioxidant property of PCF might play a role in photoprotection.

Association of myostatin Variants with Growth Traits of Zhikong Scallop(Chlamys farreri)

Scallop is a popular sea food and an important aquaculture shellfish.Identification of genes and genetic variants relating to scallop growth could benefit high-yielding scallop breeding.Myostatin(MSTN) is a conservative regulator of muscle growth,and has become one of the most important target genes for genetic improvement of the production of farmed animals.In this study,four single nucleotide polymorphisms(SNPs) were identified in the 5' flanking region of MSTN gene(Cf MSTN) in Zhikong scallop(Chlamys farreri).The association of these SNPs with scallop growth traits,including shell length,shell height,body weight and striated muscle weight was analyzed.The SNP g-1162G>T was found to associate with shell length,shell height,and striated muscle weight.The TT type scallops showed significantly higher trait values than those of GT type,and the GG type individuals exhibited median values.On the contrary,significantly more Cf MSTN transcripts were detected in the striated muscle of GT type scallops than in those of TT and GG type ones.Our results suggested that Cf MSTN might regulate the scallop muscle growth negatively,and SNP g-1162G>T can be used as a candidate marker for the selective breeding of high-yielding scallop.

Free radical scavenging abilities of polypeptide from Chlamys farreri

We investigated the radical scavenging effect and antioxidation property of polypeptide ex- tracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions ( O?2 ), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions ( O2? ) (IC50 =0.3 mg/ml), hydroxyl radicals (OH·) (IC50 = 0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50 = 0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibi- tion ratio on lipid peroxidation of linoleic acid was 43 %. The scavenging effect of PCF on O?2 , OH· and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an anti- oxidant from marine products and potential for industrial production in future.

Size fraction of phytoplankton and the contribution of natural plankton to the carbon source of Zhikong scallop Chlamys farreri in mariculture ecosystem of the Sanggou Bay

The biomass and size fraction of phytoplankton in terms of chlorophyll a（Chl a） was measured during four cruises conducted in April, July, October 2013 and January 2014 in mariculture area, the Sanggou Bay, China.Results show that total Chl a levels in the surface seawater of the Sanggou Bay generally range from 0.10 to 20.46μg/L, with an average value of 2.13 μg/L. Nano-phytoplankton was the most important size-fraction and accounted for about 65.1% of total Chl a. In order to evaluate the importance of the ＂protozoan trophic link＂ for energy transfer from the microbial loop to filter-feeding feeders, Zhikong scallop Chlamys farreri was then offered a natural planktonic community as potential prey. Results show that scallops obtained carbon source from natural plankton with the rate of 11 033.05 μg/（g·d）. Protists（nanoflagellates and ciliates） were the dominant source of carbon retained by scallop（48.78%）. The microbial loop provided 58.45% of the carbon source for farmed scallops. These results indicate that the microbial loop represent a valuable trophic resource in mariculture system of the Sanggou Bay.

The Polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydro-genase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glu-tathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25%-l%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant pro-erty.

Application of the first internal transcribed spacer(ITS-1)of ribosomal DNA as a molecular marker to population analysis in farrer's scallop Chlamys farreri

Sequence variation of the first internal transcribed spacer of ribosomal DNA （ ITS - 1 ） was examined and its application to the study of genetic variation was explored in four populations of farter＇ s scallop Chlamys farreri. ITS - 1 fragments, with a length of about 300 bp,of 78 individuals collected from Dalian, Qingdao, Yantai in China and Korea respectively were amplified via PCR, cloned and sequenced. Intra-genomic variation was examined by sequencing several clones of single individuals. Alignment and polymorphism analysis detected 44 haplotypes and 50 polymorphic sites which consist of 30 substitutions and 20 indels, indicating a high level of polymorphisms. Sequence analysis also showed a very low level of intra-individual variation. All these features validated the feasibility of application of ITS - 1 fragment to population analysis. Polymorphism analysis showed that the Korea sample has the richest genetic variation, followed by Yantai and Qingdao samples. AMOVA （analysis of molecular variance） showed that the majority （96.26%） of genetic variation was distributed within populations and 3.74% resulted from among populations, but with P 〈 0.05 （ = 0.042）, indicating that the populations in this study have significant divergence. This output was basically concordant with the result arising from RAPD data and different from that from mitochondrial 16S rDNA sequence data. Discussion on this inconsistency was made accordingly.

Effects of polypeptide from Chlamys farreri on amino acid con-tent in guinea pig skin irradiated by chronic ultraviolet A and B

We examined the effects of polypeptide from Chlamys farreri (PCF) on the amount of hy-droxyproline in guinea pig skin irradiated by chronic ultraviolet A (UVA) and ultraviolet B (UVB) radiation. PCF was applied locally before repeated exposure of guinea pig to UVA and UVB. The contents of hy-droxyproline and other amino acids in guinea pig skin were determined by automatic amino acid analyzer. Our results showed that: (1) long-time UVA and UVB radiation can reduce dramatically the amounts of hy-droxyproline, aspartic acid, threonine, glycine, phenylalanine and lysine in guinea pig skin in comparison with the control group (P < 0.05); (2) Compared with model group, pre-treatment with 5 % and 20 % PCF prior to UVA and UVB radiation can inhibit the decline of amino acids content in guinea pig skin in a dose-dependent manner (P < 0.05). As the decrease of hydroxyproline, glycine and lysine contents in the skin directly reflexes type I collagen degeneration, our results indicated that the chronic application of PCF can protect skin type I collagen against UV radiation, and thus protect skin from photoaging.

Removal of Arsenic from Chlamys farreri with Different Methods

Arsenic is commonly found in seafood,and the toxicity of different speciation varies widely.Therefore,there is an urgent need to reduce the content and analyze the speciation of arsenic to improve the safety of seafood.In this study,arsenic was removed from Chlamys farreri by adding ferric ions and sodium citrate or reducing the salinity of seawater.Then,the speciation of arsenic was analyzed.The results showed that the removal rates of arsenic were 20%,23%,24%and 18%when C.farreri individuals were treated with 0.1 g L^(−1)Fe^(3+),0.4 g L^(−1),0.6 g L^(−1)sodium citrate or decreasing seawater salinity to 17 within 48 h,respectively.Furthermore,it was detected that the arsenic speciation in the C.farreri was mainly polar arsenic,and among the polar arsenic was mainly arsenic betaine.These results indicated that the removal of arsenic can be achieved by adding the removal agent or changing the seawater salinity under the experimental conditions.The speciation of arsenic did not change during the removal process and it was mainly excreted in the speciation of arsenic betaine.

Sperm Nuclear Expansion and Meiotic Maturation in Normal and Gynogenetic Eggs of the Scallop,Chlamys farreri

Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamys farreri eggs were observed under a fluorescence microscope. The effects of ultraviolet (UV) irradiation on the fer- tilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases (phase A, meta- phase I; phase B, polar body formation; and phase C, female pronuclear development and expansion) that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but con- densed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transforma- tion of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body (DCB). At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.

CYTOLOGICAL COMPARISON OF GAMETOGENESIS OF SCALLOPS,ARGOPECTEN IRRADIANS AND CHLAMYS FARRERI

Histological characteristics of gametogenesis of two kinds of scallops, gonochoric Chinese scallop, Chlamys farreri and hermaphroditic bay scallop, Argopecten irradians were investigated in this study. Spermatogenesis in C. farreri has different developmental stages: spermatogonia, primary spermatocyte, second spermatocyte, spermatid and spermatozoon. A large number of same developmental stage spermatic cells converge at a definite area of the testis. Premeiotic, previtellogenic and vitellogenic oocytes can be found during oogenesis in C. farreri , where oocyte distribution is obviously irregular. The A. irradians gonad consists of two different parts in one individual: one part functions as testis, the other as ovary. Between these two parts is a special appearance area, where a large number of spermatic cells are bound with two layers of acellular substance with many oocytes in it.

Selection of anticoagulant solution to the hemolymph of Chlamys farreri by transmission electron microscropy and flow cytometry

Mixtures of hemolymph from Chlamys farreri with three different anticoagulant solutions were incu-bated for an hour in vitro, then the ultrastructural alterations of hemocytes were observed, and the aggre-gation rate was analyzed by using transmission electron microscropy and flow cytometry respectively. The results showed that Formula 3 （glucose 20.8 g L^-1; EDTA 20mM; sodium chloride 20 g L^-1; Tris-HCI 0.05M;pH 7.4）was the desirable anticoagulant solution for C. farreri hemocytes. Further phagocytosis assay showed that no obvious negative effect was given to the hemocyte phagocytic activity when using Formula 3 as the anticoagulant solution.

Cross-breeding of Different Geological Populations of Scallop, Chlamys farreri

Wild populations of Chlamys farreri derived from Japan and China are used as parents in a cross breeding experiment conducted in Zhangzi Island of Liaoning Province in the early May of 2000. Four different first filial generations of the crossbreeding, namely JC, CJ, CC and JJ, which represent different cross combinations of the parents, are differentiated from each other in some observed quantitative traits including survival rate, hatchery rate and attachment rate, etc. By ANOVA analysis, significant differences in growth rate are found among the four populations of the offspring after one year of culturing in Shandong Province. JJ population shows the fastest growth rate and has shell height of 46.1±0.71(SD)mm in average, whereas the other three populations, CC, CJ and JC, has the shell height of 43.1±0.42mm, 41.5±0.48mm and 37.2 ±0.31mm, respectively. No heterosis is detected in the experiment.

Comparative transcriptomics reveals genes involved in metabolic and immune pathways in the digestive gland of scallop Chlamysfarreri following cadmium exposure

Chlamys farreri is an economically important mollusk that can accumulate excessive amounts of cadmium(Cd). Studying the molecular mechanism of Cd accumulation in bivalves is difficult because of the lack of genome background. Transcriptomic analysis based on high-throughput RNA sequencing has been shown to be an efficient and powerful method for the discovery of relevant genes in non-model and genome reference-free organisms. Here, we constructed two c DNA libraries(control and Cd exposure groups) from the digestive gland of C. farreri and compared the transcriptomic data between them. A total of 227 673 transcripts were assembled into 105 071 unigenes, most of which shared high similarity with sequences in the NCBI non-redundant protein database. For functional classification, 24 493 unigenes were assigned to Gene Ontology terms. Additionally, Eu Karyotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes analyses assigned 12 028 unigenes to 26 categories and 7 849 unigenes to five pathways, respectively. Comparative transcriptomics analysis identified 3 800 unigenes that were differentially expressed in the Cd-treated group compared with the control group. Among them, genes associated with heavy metal accumulation were screened, including metallothionein, divalent metal transporter, and metal tolerance protein. The functional genes and predicted pathways identified in our study will contribute to a better understanding of the metabolic and immune system in the digestive gland of C. farreri. In addition, the transcriptomic data will provide a comprehensive resource that may contribute to the understanding of molecular mechanisms that respond to marine pollutants in bivalves.

Variations in retention efficiency of bivalves to different concentrations and organic content of suspended particles

Retention efficiencies (RE) of scallop (Chlamys farreri),oyster (Crassostrea gigas) and mussel (Mytilus edulis) in a flow-through system were measured to understand the short-term response to various particle and organic matter concentrations.By comparing the RE of C.farreri with that of C.gigas and M.edulis,we gained further knowledge on the feeding physiological characteristics of C.farreri and ascertained the possible cause of high summer mortalities of this species.The experimental feeding conditions included natural differences in the abundance and composition of suspended seston,as well as conditions in which seston abundance and composition were manipulated by adding natural silt or cultured microalgae.The results show that in natural sea water,the minimum particle size for maximal retention in M.edulis,C.gigas and C.farreri was approximately 4,6,and 8 μm,respectively;the RE of 2-μm (equivalent spherical diameter) particles was 17%,19%,and 8%,respectively;and the relative standardized RE was 58%,49%,and 18%,respectively.In C.gigas and M.edulis,the minimal particle size for maximal retention did not change with food quality (organic content).C.farreri was more sensitive to fluctuations in particle concentration and organic content.With particle concentration increase,the minimal particle size for maximal retention in scallop shifted to large particles and the RE for 2-μm particle decreased from 8% in natural seawater to 1.6%-6% in silt-enriched groups.With organic content increase,the minimal particle size for maximal retention shifted from 8 to 5 μm in natural seawater.Variation in RE of C.farreri with food conditions and the relationship between lower RE and smaller particle size may hamper C.farreri from food taking due to the decrease in the size distribution of phytoplankton in Sungo Bay.

Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

Phenoloxidases(POs)are a group of copper proteins including tyrosinase,catecholase and laccase,which play crucial roles in the innate immune response of mollusks.In this research,POs were studied in cultured mollusk species,including scallop Chla-mys farreri,abalone Haliotis discus hannai and clam Scapharca subcrenata.The POs were isolated from hemocytes using linear-gradient native-PAGE combined with catechol staining.The PO activities and their characters were investigated.The molecular mass of PO in C.farreri was 576 kDa,and it was 228 kDa in H.discus hannai.In S.subcrenata,four POs were detected and their mole-cular masses were 391 kDa,206 kDa,174 kDa and<67 kDa,which were named as 391-PO,206-PO,174-PO and s-PO,respectively.Ki-netic analyses indicated that all of the POs,except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine.However,all of the POs failed to oxidize tyrosine.The effects of divalent metal ions on POs’activities were assayed,in-cluding Fe^(2+),Mg^(2+),Zn^(2+),Mn^(2+),Cu^(2+)and Ca^(2+)from FeCl_(2),MgSO_(4),ZnSO_(4),MnCl_(2),CuSO_(4)and CaCl_(2).The POs were inhibited by Fe^(2+)at all determined concentrations.Additionally,the inhibition assay showed that all of the POs were inhibited by cysteine,ascorbic acid,sodium sulfite,citric acid,ethylenediaminetetraacetic acid disodium(EDTA)and sodium diethyldithiocarbamate(DETC).The inhibition effects of critric acid and EDTA are dose-dependent.H.discus hannai PO and 391-PO were slightly inhibited by sodium azide,and H.discus hannai PO,391-PO and 174-PO were slightly inhibited by thiourea.In conclusion,the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.

Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

In situ loop-mediated isothermal amplification （in situ LAMP） combines in situ hybridization and loop-mediated isothermal amplification （LAMP） techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization （FISH）, primed in situ labeling （PRINS）, and cycling primed in situ labeling （C-PRINS）. Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop （ Chlarnys farreri）.