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.

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.

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.

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%.

Insights into the genetic covariation between harvest survival and growth rate in olive flounder(Paralichthys olivaceus)under commercial production environment

In aquaculture,selective breeding for survival till harvest have become an alternative strategy for improving disease resistance and production.However,knowledge of genetic parameters of harvest survival,e.g.,heritability and genetic correlations between survival and growth rate traits,is still scarce.The aims of this study were to estimate genetic parameters for harvest survival and growth rate traits under commercial farming conditions in olive flounder(Paralichthys olivaceus).Harvest survival was defined as a binary trait;growth traits were measured as average daily gain(ADG),specific growth rate(SGR),daily growth coefficient(DGC)and body weight(BW).Data from a population of 241 full-sib families(involving 39,904 individuals,four generations)were used.Heritabilities of survival were low but significant,which were 0.15±0.04 and 0.22±0.01 on observed and underlying scale,respectively.Heritability estimates for ADG,SGR and DGC were medium to high,which were 0.33±0.06,0.83±0.07,0.58±0.07,respectively.While the heritability of BW is of low magnitude(0.17±0.08).The genetic correlations between harvest survival and three growth rate traits(i.e.,ADG,SGR and DGC)were very strong(ranging from 0.66 to 0.79),which is an exciting result.However,the genetic correlation between harvest survival and BW was much lower(0.17±0.08).These results suggest that selection for harvest survival would consequentially result in concomitant increase of growth rate,and vice versa.Our findings revealed novel insights into the genetic improvement of growth rate and harvest survival through genetic selection in olive flounder.

Effects of dietary supplementation of Lactobacillus plantarum and Bacillus subtilis on growth performance,survival,immune response,antioxidant capacity and digestive enzyme activity in olive flounder(Paralichthys olivaceus)

In present study,a 10-month feeding trial was carried out to investigate effects of dietary Lactobacillus plantarum and Bacillus subtilis supplementation singly on growth performance,innate immune response,antioxidant capacity and digestive enzyme activity of olive flounder under a commercial production environment.Two experimental groups fed basal diet with supplementation of L.plantarum(5.0×107 CFU/g diet)and B.subtilis(1.0×108 CFU/g diet)respectively,and control group fed with basal diet without probiotics supplement.At harvest,serum immune parameters,liver antioxidant indices and digestive enzyme activities in liver,stomach and intestines were measured.Fish fed with B.subtilis supplemented diet showed the highest growth rate which was significantly higher than L.plantarum supplemented group(P<0.05)which was also significantly higher than control group(P<0.05).Survival rate were significantly elevated by feeding diet with L.plantarum supplementation(P<0.05),while the improvement was not significant for B.subtilis supplemented diet compared to control group.All the studied immune parameters in serum were substantially enhanced in both experimental groups.Similar results were observed for the antioxidant capacity in liver.Besides,the activities of digestive enzymes increased substantially in both experimental groups compared to control group and the detected improvement in dietary B.subtilis supplemented group were all higher than in L.plantarum supplemented group,though not all the improvements were significant.In summary,the separate supplementation of B.subtilis and L.plantarum in diet is more beneficial for growth performance and healthy status respectively.Hence,in olive flounder aquaculture,favorable effects of compounded supplementation of L.plantarum and B.subtilis are expected.

Interactive effect of dietary vitamin E and inorganic mercury on growth performance and bioaccumulation of mercury in juvenile olive flounder, Paralichthys olivaceus treated with mercuric chloride

A 6-week feeding trial was carried out to evaluate the effects of dietary vitamin E(DL-a-tocopheryl acetate, TA) on growth and mercury(Hg) accumulation in juvenile olive flounder(Paralichthys olivaceus)treated with mercuric chloride(HgCl_2). Vitamin E and HgCl_2 were added to the semi-purified basal diet.Six semi-purified diets in a 2 × 3 factorial design were formulated to contain 2 levels of Hg(0 or 20 mg HgCl_2/kg diet) and 3 levels of vitamin E(0, 100, or 200 mg TA/kg diet). Experimental fish(n = 360,9.99 ± 0.15 g) were randomly allocated into 30-L tanks at a density of 20 fish per tank with 3 replicates in each treatment and were fed twice a day. At the end of the feeding trial, dietary Hg depressed the growth performances in terms of weight gain(WG), specific growth rate(SGR), feed efficiency(FE) and protein efficiency ratio(PER) in fish, while fish fed the diets supplemented with vitamin E showed significant growth improvement in both presence and absence of HgCl_2 in the diets(P < 0.05). Survival rate was not affected in fish fed the experimental diets. Whole body compositions of fish such as lipid and moisture contents were influenced by dietary vitamin E supplementation. Total Hg contents of muscle, liver and kidney tissues were significantly reduced in fish fed diets supplemented with vitamin E(P< 0.05), while the two-way ANOVA showed that increasing Hg concentration has resulted in a reduction in vitamin E.Whole body fatty acids of fish like eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA) contents were decreased by dietary Hg. However, supplementation of dietary vitamin E improved the a-linolenic acid(ALA) and EPA contents in fish. Our results suggest that dietary supplementation of vitamin E has potential effects on growth improvement and ameliorating inorganic Hg bioaccumulation in juvenile olive flounder.

Comparison of juvenile growth of wild and hatchery-raised olive flounders Paralichthys olivaceus as examined by otolith microstructure

Fish stocking is a common practice for restocking wild populations of fishes,and the growth conditions of hatchery-raised fishes are a major key for successful recruitment.Yet,there are few studies that compare the growth of hatchery-reared juvenile fishes with wild populations.We compared the growth conditions of hatchery flounders,Paralichthys olivaceus,at two fish farms in Korea with that of natural stocks.All of the relationships of growth parameters including total length-weight,otolith-total length,and weight/length ratio relative to otolith length indicated that the growth was similar between the wild and the reared fishes at the two fish farms.The results suggest that the fishes grown at farming facilities should have a similar probability of recruitment as wild juveniles.

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 Paralichtbys 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.

Estimationof genetic parameters for juvenile growth performance traits in oliveflounder(Paralichthys olivaceus)

The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder(Paralichthys olivaceus).A total of 82 full-sib families from 2 year-classes with complete pedigree information were used to estimate heritabilities and genetic/phenotypic correlations for 4 growth performance traits(body weight BW,total length TL,body height BH and condition factor K)based on uni-trait and multi-trait linear mixed models,respectively.Data were analyzed by the restricted maximum likelihood(REML)method.The heritabilities of BW,TL and BH were 0.81,0.70 and 0.61,which are relatively high,however,heritability for K is low(0.11).For all growth traits,the common full-sib effects(c^(2))were at a low to moderate level(0.14–0.20);the maternal effects(m^(2))of the traits(except K)were also detected(0.15–0.19).The phenotypic and genetic correlations among BW,TL and BH were highly positive(0.70–0.91),however,the correlations between K and the other 3 traits was low or negligible(−0.22–0.26).According to our results,these traits(except K)could be selected and enhanced simultaneously.In addition,direct selection based on TL or BH,which is an easily measurable trait,will be more favorable than selection on BW.Moreover,our results indicated that growth parameters estimated in the juvenile stage could be used as a predictor of growth performance at later growth stages.