Effects of salinity on growth and feeding of juvenile Starry flounder (Platichthys stellatus)

The survival rate, feeding, growth of juvenile Starry flounder were studied at different salinities. The results showed that when the fish was directly translated from salinity of 32 to 0 and more than 0, the survival rate was 100% in 96 hours. If the fish was acclimatized, it was still 100% in 42 days. The fish at the salinity of 0 - 16 grew faster than others; feeding rate varied as salinity changed, and the highest one was at salinity of 32 and the lowest one was at the salinity of 16. As to feed conversion efficiency, the highest one was at the salinity of 16, and the lowest one was at the salinity of 24. There were no significant differences among the treatments （P＆gt;0.05）. It was found that low salinity benefited for recovering albinism.

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis.A new Sertoli cell line(POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages.Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P.olivaceus.Cells were optimally maintained at 25℃ in DMEM/F12 medium supplemented with fetal bovine serum,basic fibroblast growth factor,and epidermal growth factor.The growth curve of POSC showed a typical "S" shape.Chromosome analysis revealed that the cell line possessed the normal P.olivaceus diploid karyotype of 2n=48t.POSC expressed dmrt1 but not vasa,which was detected using RT-PCR and sequencing.Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL.Therefore,POSC appeared to consist of testicular Sertoli cells.Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid,with the transfection efficiency reaching 10%.This research not only offers an ideal model for further gene expression and regulation studies on P.olivaceus,but also serves as valuable material in studying fish spermatogenesis,Sertoli cell-germ cell interactions,and the mechanism of growth and development of testis.

Molecular Cloning and Expression Analysis of Toll-like Receptor 1 cDNA in Japanese Flounder,Paralichthys olivaceus

[Objective] The paper aimed to clone the full length gene of Toll-like recep- tors （TLRs） in Japanese flounder （Paralichthys olivaceus）, and analyze their structural features and expression regularity. [Method] The full length cDNA sequence of Toll like receptor 1（TLR1） gene was identified from Japanese flounder head kidney by ho- mologous cloning and rapid amplification cDNA ends （RACE）. The bioinformatics and expression model of this gene was analyzed. [Result] The TLR1 cDNA was 2 947 bp, a 2 418 bp open reading frame （ORF）, encoding 805 amino acid （aa） residues, including signal peptide, six leucine-rich repeat（LRR） motifs, two transmembrane zones and one TolI/IL 1 receptor （TIR） domain. The molecular weight of the deduced protein was 91.15 KDa, and the isoelectric point was 6.49. The amino acid sequence of Japanese flounder TLR1 possessed 69%-35% identity with the TLRls of other verte- brates, further analysis showed that the TIR domain of Japanese flounder TLR1 shared 84%-62% identities with TIR domains in other vertebrates. Japanese flounder TLR1 protein firstly clustered with TLRls in Epinephelus coioides in the phylogenetic analysis. The transcription of Japanese flounder TLR1 was examined by real-time quantitative PCR, and its mRNA was mainly detected in liver, heart and spleen. [Conclusion] The results lay a foundation for further studying the functions of TLR1 and developing immune potentiator in Japanese flounder.

Effects of Dietary Soy Isoflavones on Feed Intake,Growth Performance and Digestibility in Juvenile Japanese Flounder(Paralichthys olivaceus)

An 8-week feeding trial was conducted to investigate the effects of dietary soy isoflavones on feeding intake,growth performance,and digestion of juvenile Japanese flounder(Paralichthys olivaceus).Four isonitrogenous(49% crude protein) and isoenergetic(20.1 MJ kg-1) diets were formulated to contain four graded levels of soy isoflavones,namely,0,1,4 and 8 g soy isoflavones in 1 kg of diet.Each diet was randomly fed to triplicate tanks of fish(Initial average weight:2.58 g ± 0.01 g),and each tank was stocked with 35 fish.No significant difference was observed among diets with levels of 0,1 and 4 g kg-1 soy isoflavones in feed intake,weight gain,feed efficiency ratio(FER),proximate composition of fish whole body and apparent digestibility coefficients(ADC) of nutrients and energy(P>0.05).However,high dietary soy isoflavones level(8 g kg-1) significantly depressed weight gain,FER,whole-body crude lipid content of fish and ADC of nutrients(P<0.05).These results indicate that high level of dietary soy isoflavones(above 4 g kg-1) significantly depresses growth responses and FER of Japanese flounder.However,as the content of soy isoflavones in soybean meal is around 1 to 3 g kg-1,the adverse effects might be neglected when soybean products are used as a fish feed ingredient.

Single Nucleotide Polymorphism of FSHβ Gene Associated with Reproductive Traits in Japanese Flounder (Paralichthys olivaceus)

Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P<0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P<0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.

Effects of Calanoid Copepod Schmackeria poplesia as a Live Food on the Growth, Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder, Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.

Analysis of New Microsatellite Markers Developed From Reported Sequences of Japanese Flounder Paralichthys olivaceus

The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs,including 440 dinucleotide,254 trinucleotide,53 tetranueleotide,95 pentanucleotide and 40 hexanucleotide microsatellites respectively.The CA/TG and GA/TC repeats were the most abundant microsatellites.AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites.PCR primers were designed to amplify 10 identified microsatellites loci.The PCR results from eight pairs of primers showed polymorphisms in wild populations.In 30 wild individuals,the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8.These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.

Tissue Expression and Stock Variation of Isozymes of Stone Flounder(Kareius bicoloratus)

Tissue expression and stock variation of isozymes of stone flounder （Kareius bicoloratus） were analyzed with horizontal starch gel electrophoresis. For the fourteen enzymes assayed, 31 loci were recorded. The results indicated that all the isozymes examined were obviously tissue-specific. The expressions of SOD＊, GDH＊, G3PDH-2＊ and ADH-2＊ were detected only in liver, SDH-1＊, MDH-1＊ and ADH-1＊ only in muscle, and LDH-B＊ and LDH-C＊ only in eyes. In comparison, MDH-2＊, GPI-3＊ and SDH-2＊ were detected in all tissues examined. Other loci examined were detected in a variety of tissues. Muscle and liver were selected to detect the isozyme variation of the two geographic stocks of Qingdao and Weihai, Shandong Province, China. The percentages of polymorphic loci （P0.99） were 29.17% and 25.00%, the observed heterozygosities （H0） were 0.028 ±0.014 and 0.040 ± 0.019, and the expected heterozygosities （He） were 0.039±0.017 and 0.052±0.022 in Qingdao and Wethai stock, respectively. The coefficient of gene differentiation （Fst） and genetic distance （D） between the two stocks was 0.012 and 0.0011, respectively, indicating that the genetic differentiation is low between them. Compared with other species of Pleuronectiformes, both the percentage of polymorphic loci and the mean heterozygosity ofK. Bicoloratus were at a middle level.

Ontogeny of the Lymphoid Organs of Japanese Flounder,Paralichthys olivaceus

Histological study on the ontogeny of the lymphoid organs, kidney, thymus and spleen of Japanese flounder, Paralichthys olivaceus, from hatching to 40 d was carried out. The pronephric kidney duct appeared early in hatching although the primordial haemopoietic stem cells were observed within a week after hatching. The spleen was first seen after 8 d of hatching. The thymus appeared after 15 d, situated near the pronephric kidney. Small lymphoid cells appeared during the later phase of the post-larval stage in the sequence of thymus, kidney and spleen. During the 40 d of observations, there were no distinct inner or outer zones in thymus and no red or white pulp in spleen. These results suggest that the nonspecific defense immune system plays a very important role in the early larval stage of Japanese flounder.

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea （Qingdao population, QD） and East China Sea （Zhoushan population, ZS） using 10 polymorphic mierosatellite loci and cytochrome c oxidase subunit I （COI） sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data （Fst=0.048 7, P〈0.001） and mitochondrial DNA data （Fst=0.128, P〈0.001） both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.

Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid （IMI） to the gill cell line of flounder （FG） that collected in the gill ofParalichthys olivaceus, was examined by 3 widely used endpoint bioassays： NR （neutral red）, MTT （3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazoliurn bromide） and TCP （total cell protein）. The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The ICs0 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulurn （RER） remained normal. This would suggest that the mitochondria are probably the primary target of IMI.

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV （lymphocystis disease virus）-infected Japanese flounder （Paralichthys olivaceus）, serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry （LC-MS/MS）. Two immunityrelevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.

Dietary vitamin A,ascorbic acid and α-tocopherol affect the gonad development and reproductive performance of starry flounder Platichthys stellatus broodstock

The present trial was conducted with starry flounder Platichthys stellatus broodstock to evaluate the effects of dietary vitamin A, ascorbic acid, and a-tocopherol on the gonadal development and reproductive performance. 8 000 IU/kg diet vitaminA （VA group）, 500 mg/kg diet ascorbic acid （Vcpp group）, or 250 mg/kg diet a-tocopherol （a-TA group） was added into basal diet to create 3 vitamin experimental diets, respectively. Each diet was fed to 450 starry flounder broodstock for 104 days. Samples were collected weekly. The gonadosomatic index （GSI） of 3 vitamin experimental groups first decreased and then increased. Maximum GSI of Vcpp group was higher than that of ct-TA group but lower than that of VA group. The spawning periods of 3 vitamin experimental groups lasted 49, 56, and 45 days, respectively. No mature eggs were observed in the control group during the trial. The absolute fecundity （AF） and relative fecundity （RF） of a-TA group was higher than that of Vcpp group but lower than that of VA group. The results suggest that different vitamins play different roles in the fish reproductive process. Vitamin A stimulated the maturation of the ovary, ascorbic acid prolonged the spawning period, and a-tocopherol affected the development of the eggs.

Genome-wide identification and transcriptome-based expression analysis of sox gene family in the Japanese flounder Paralichthys olivaceus

Sox genes are transcription factors that ubiquitously exist in animal species, and share a conserved high mobility group(HMG) box. They play important biological roles in diverse developmental processes, such as sex determination and differentiation, chondrogenesis, neurogenesis, and early embryonic development. In this study, we identified 25 sox genes from genome and transcriptome of Japanese flounder Paralichthys olivaceus. These s ox genes could be mainly classified into seven subfamilies(B1, B2, C, D, E, F, and K), and each subfamily exhibited a relatively conserved gene structure. Besides, subfamilies A and G were found exclusively in human and mouse, and sox 32 in subfamily K only existed in teleosts. Compared with other mammals, some s ox genes in teleosts had two duplicates. The loss, duplication, and divergence of sox genes during evolution provided an evidence for whole-genome duplication that occurred in the radiation of teleosts. The expression of Japanese flounder sox genes was also analyzed by FPKM value. Our results showed that certain s ox genes exhibited obviously tissue-specific and spatio-temproal expression. Especially, gonal-basied expression analysis uncovered that s ox7 and s ox2 were ovary-biased, and s ox8 b was testis-biased. Moreover, sox10 a was expressed specifically in ovary, and sox8 a in testis. Therefore this study provide a solid foundation for future functional and evolutionary analysis of sox genes in Japanese flounder.

Characterization,tissue distribution,and expression of neuropeptide Yin olive flounder Paralichthys olivaceus

Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture.

DEVELOPMENT OF RETINAL STRUCTURE AND VISUAL ACUITY IN JAPANESE FLOUNDER(PARALICHTHYS OLIVACEUS)

The retinal structure and visual acuity in Japanese flounder Paralichthys olivaceus at different stages of development were examined by light microscopy. The resolving power of the retina, the visual axis and the best visual field were estimated based on the distribution of cone cells in the retina. The visual system of the larvae appears poorly developed at hatching. The larvae with total length (TL) of less than 10 mm, have single cones only and the eyes were well pigmented. At 10-11 mm TL, most single cones fused to form double cones, with the single and double cones forming a mosaic pattern. From larvae to early juvenile the retina stretches, the cones increase in diameter and rods increase in number. Based on the highest density of the cones in the ventro temporal region, the visual axis was orientated up forward. The resolving power of the retina in 40-530 mm TL Japanese flounder was found to range from 25.1 to 11.5 min. The results indicated continual improvements in the visual system of the growing fish towards higher resolving power, visual acuity and sensitivity.

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 （IL-2）-like factor was detected in the supernatant of plant haemoglutinin （PHA）-stimulated lymphocyte culture from peripheral blood,spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-1ike factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes （P〈0.01）. The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2 （rhIL-2） was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor （IL-2R） on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25（IL-2R） was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder(Paralichthys olivaceus)

Follistatin （Fst） is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns ofFst gene at five different development stages of Japanese flounder （stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months）. The muscle tissue of Japanese flounder was obtained at different development stages in dais experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Genome Editing in the Olive Flounder(Paralichthys olivaceus)Using CRISPR/Cas9 and a Simple Microinjection System

The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.