Cloning of hemoglobin-α1 from half-smooth tongue sole (Cynoglossus semilaevis) and its expression under short-term hypoxia

This study cloned the hemoglobin a1 from the marine teleost, the half-smooth tongue sole （Cynoglossus semilaevis）, and then examined its expression under hypoxia exposure. The full-length of CsHb-a1 （594 bp） cDNA contains an open reading frame encoding 144 amino acids. Sequence analysis shows that the predicted CsHb-a1 amino acids shares high identities with that of other species. Real-time PCR showed that CsHb-a1 was highly expressed in the heart, liver, spleen, kidney and blood. Five to 120 min esposure and long-term （36 h） exposure to hypoxia （1.0 mg/L） significantly increased CsHb-a1 mRNA expression in most tissues compared to those fish held in normoxic conditions （dissolved oxygen （DO）： 6.2 mg/L）. These results suggested that the up-regulation of Hb-a1 is an important component for adaptation of half-smooth tongue sole to short-term hypoxia.

Molecular Cloning and Expression Analysis of FTZ-F1 in the Half-smooth Tongue-sole, Cynoglossus semilaevis

To investigate the expression characteristics of sex related gene of FTZ-F 1 in the half-smooth tongue-sole （Cynoglossus semilaevis）, the homologue FTZ-F1 （hsFTZ-F1） full-length cDNA was isolated from the testis by homologous cloning, and the cDNA included the open reading frame and a 66bp 5＇-UTR, along with a 1619bp 3＇-UTR, encoding a predicted 485 amino acid protein. Sequence, tissue distribution and phylogenic analyses of the FTZ-F1 showed that the hsFTZ-F1 belonged to SF-1/Ad4BP group. The hsFTZ-F1 transcripts were highly abundant in the gonads, kidneys, brain and head-kidneys, but weakly in other tissues. However, the expression level in the brain and head-kidney of female was highly abundant than in the male. The hsFTZ-F1 expression was highly abundant in the embryo than in the larvae, which suggested that the hsFTZ-F1 may be involved in the organogenesis in the tongue sole.

Isolation and Identification of the Pathogen Causing Skin Ulcer Disease in Cynoglossus semilaevis Günther and Its Sensitivity to Chinese Herbal Medicine

In this study, the pathogen causing skin ulcer disease in Cynoglossus semilaevis Gunther was isolated for morphological observation, physiological and bio- chemical identification. According to the result, the isolated pathogen was identified as Vibrio harveyi. The results of recurrent infection of C. semilaevis Gonther showed that the pathogen was strongly pathogenic to C. semilaevis Gunther. In or- der to explore the pathogenesis, outer membrane protein （OMP） gene of C. semi- laevis Gunther was detected by PCR. The results showed that all the three repre- sentative strains harbored OMP gene. According to the results of sensitivity test of the pathogen to Chinese herbal medicine, Galla Chinensis, Fructus Mume, Fructus Hippophae and Lignum Sappan exerted strong antibacterial effects against V. harveyr, Pericarpium Granati exhibited slight antibacterial effect against V. harveyi; Pericarpium Citri Reticulatae, Rhizoma Acori GramineL Herba Houttuyniae, Herba Portulacae, Herba Andrographis, Eucalyptus globulus Labill. and Herba Menthae Heplocalycis had little effect on V. harveyi. Galla Chinensis, Fructus Mume, Fructus Hippophae and Lignum Sappan were prepared into three prescriptions, among which prescription 1 （Galla Chinensis ＋ Fructus Mume） exhibited the strongest antibacterial effect.

Histopathological Observation of Cynoglossus semilaevis Günther Infected with Red Abdominal Shell Disease

The histopathological changes in various organs of Cynoglossus semilaevis Günther infected with red abdominal shell disease were observed by dissection and HE staining. In the ill C. semilaevis, the body surface was congestive and bleeding;the skin in the blind side ulcerated with extravasated blood; the gill filaments ulcerated with a lot of mucus; the liver, pancreas and spleen were swelling and bleeding with extravasated blood; the small intestine was congestive and bleeding. Degeneration and necrosis, and submucosal bleeding occurred in the skin of C. semilaevis infected with red abdominal shell disease. There was red blood cell infiltration and deposition in the space among muscle fibers. Epithelial degeneration and necrosis occurred in the parotid gland. The efferent branchial artery was congestive. The myocardial fibers had a loose structure, and a large area of fracture appeared. The liver cells were swelling with degeneration and necrosis and concentrated nucleus. A large area of the renal interstitial was broken down and necrotic. The submucosa of the small intestine had necrosis and hemorrhage.

Isolation and Characterization of Pathogenic Listonella anguillarum of Diseased Half-Smooth Tongue Sole(Cynoglossus semilaevis Günther)

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among 14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 〈1.0×10^5 colony forming units （CFU）mL^-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillantm with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay （IFA） was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.

Effects of Starving and Re-feeding Strategies on the Growth Performance and Physiological Characteristics of the Juvenile Tongue Sole(Cynoglossus semilaevis)

Starving and re-feeding trials were conducted to evaluate the effects of starvation duration and recovery times on the growth performance and physiological characteristics of juvenile tongue sole Cynoglossus semilaevis, which included growth, body composition, intestinal morphology and digestive enzymes activities. The fish starved for one(D1), two(D2) and four(D4) days, respectively, were recovered for three(R3, R6, R12), seven(R7, R14, R28) and eleven(R11, R22, R44) folds of starvation days while the control fish were fed constantly. The experiment was conducted for 96 days. The fish individuals in D1R11, D2R14 and D2R22 caught up in weight with the control, indicating that complete compensatory growth existed in these fish individuals. The highest body weight gain and total specific growth rates were observed in D2R22. Food intake increased significantly in most treatments in comparison with that of the control except for D1R7 and D2R22(P < 0.05). Food conversion efficiency and apparent digest coefficient in D2R22 were significantly promoted, being higher than that of the control(P < 0.05). The activities of trypsin and lipase were found to be closely related to the growth performance. The activities of liver and hindgut trypsin, also midgut and hindgut lipase in D2R22 were significantly higher than those of the control(P < 0.05). The fold height(HF) of foregut and midgut increased significantly in D2R22, and HF decreased significantly in D1R3, D2R6 and D4R12(P < 0.05). On our findings, we may conclude that the optimum starving and re-feeding strategy is starving for 2 days and re-feeding for 22 days. And in this starving and re-feeding strategy, the compensatory growth could be mostly attributed to the promotion of food conversion efficiency and digestibility coefficiency.

Cloning,characterization of two female-specific AFLP markers and development of PCR-based sex identification method for the half-smooth tongue sole Cynoglossus semilaevis

Two female-specific AFLP(amplified fragment length polymorphism)markers(named CseF464 and CseF136)were isolated by using one selective primer combination(E-AGC/M-CTG)from the genomic DNA of 20 females and 20 males of the half-smooth tongue sole Cynoglossus semilaevis.Both the markers were re-amplified,recovered from the agarose gels,cloned and sequenced.Bioinformatics analysis indicated that the length of the two markers were 468 bp and 134 bp,respectively,and the sequences showed no similarity to each other,as well as to the known sequences deposited in the GenBank database using BLASTn.Two pairs of SCAR(sequence characterized amplified regions)primers were designed based on the sequences of the two female-specific markers.Furthermore,PCR-based genetic sex identification method was developed in Cynoglossus semilaevis.A specific fragment was amplified in all females but not in any males by using these SCAR primers on the initial 20 female and 20 male individuals of Cynoglossus semilaevis.The feasibility of the two SCAR primer pairs was confirmed in additional 100 individuals(50 females and 50 males).This allowed for reliable,rapid molecular identification of genetic sex of the species,genetic mapping on the sex chromosomes and better understanding of the sex determination and sex differentiation in the half-smooth tongue sole.

Analysis of SSRs Derived from an EST Library of Half-Smooth Tongue Sole Cynoglossus semilaevis

A complementary DNA(cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag(EST) database was generated,containing 3100 cDNA clones,of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats(SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats(51.4%) are the most abundant class of SSR in the dataset. The dimeric,trimeric,tetrameric and hexameric re-peats are represented in decreasing proportions of 27.2%,16.0%,2.8% and 1.9%,respectively. The frequency of pentameric repeats was observed the least(only 0.7%) . Most of the monomeric and dimeric repeats are distributed in 3' and 5' un-translation region. If translation regions are considered merely,trimeric repeats are the highest,accounting for 57% of the total microsatellites.

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole(Cynoglossus semilaevis)

Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization during Cynoglossus semilaevis early developmental process, eight candidate reference genes （ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S） were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the mature C. semilaevis. EFIA was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages （from oosphere to 90 days old） in this study. However, when divided the Ct value data of the above mentioned early developmental stages into two separate periods （embryo and post-hatching periods）, TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

Phenotypic and genetic parameter estimation of juvenile growth and bottom color traits in half-smooth tongue sole,Cynoglossus semilaevis

Half-smooth tongue sole（Cynoglossus semilaevis） is a commercially important fish species. There were no reports about accurate genetic parameters of growth traits in C. semilaevis, despite efforts aimed at the culture of this species. Therefore, the main goal of this study was to estimate the parameters for the early growth traits（total length, body depth and body weight） of C. semilaevis based on the data obtained from 80 families. The trait of fish with pure white bottom or not（named as the trait of bottom color） was also analyzed. All the heritabilities estimated for the traits were medium, which varied from 0.210 to 0.362. Genetic correlations among growth traits were highly positive, varying from 0.913 to 0.959, indicating that selection of one of the three traits would result in correlated increase in the others. Also, genetic correlations between bottom color and growth traits were positive and varying from 0.241 to 0.353, suggesting that selection for the bottom color can be utilized to enhance the selection of growth traits. In addition, fish from the top performing 16 full-sib families were selected as broodstock, based on the above analysis and the comparison of family breeding value. This research provides an important basic material to implement selective breeding in C. semilaevis.

Effects of the Amplitude and Frequency of Salinity Fluctuation on the Body Composition and Energy Budget of Juvenile Tongue Sole(Cynoglossus semilaevis)

Effects of the amplitude(± 2, ± 4, ± 6, and ± 8) and frequency(2, 4, and 8 d) of salinity fluctuation on the body composition and energy budget of juvenile tongue sole(Cynoglossus semilaevis) were investigated in a 64-d experiment. Results showed that the amplitude and frequency of salinity fluctuation had significant interaction and both substantially affected the final weight and specific growth rate of juvenile tongue sole. The tongue sole exhibited better growth in treatments with moderate amplitude and frequency of salinity fluctuation(amplitude ± 4–6; frequency 4–8 d) than in other treatments and the control. In terms of energy budget, salinity fluctuation strongly affected the proportions of energy components, including those deposited for growth and lost in respiration, feces, and excretion. Moderately amplitude and frequency of salinity fluctuationg that favored the growth of tongue sole partitioned more energy for growth and less energy for metabolism than the constant and other amplitude and frequency of salinity fluctuation. Average energy budget for tongue sole at moderately fluctuating salinity was determined to be 100C(food) = 30.92G(growth) + 10.30F(feces) + 6.77U(excretion) + 52.01R(respiration). Energetic advantage at moderately fluctuating salinity, including increased energy intake, high assimilation efficiency, reduced metabolism expenditure, and more energy partitioned into growth, might account for the enhancement of tongue sole growth. Commercial farmers are recommended to rear juvenile tongue sole with moderate salinity fluctuations for better growth performance of this species.

Functional morphology of the olfactory organ of the tongue sole, Cynoglossus semilaevis

The morphology and structure of the olfactory organ of Cynoglossus semilaevis Gunther are described. The oval olfactory sacs on both sides differ in size and in the number of lamellae, With those on the abocular side having smaller sacs and fewer lamellae than those on the ocular side. On the ocular side, the average ratio of sac length to eye diameter is 2.1 （i.e.〉1） with an average of 91 lamellae, while on the abocular side, the values were 1.7 （i.e.〉1） and 69, respectively. In addition, the surface morphology varies in different parts of the lamella. The frontal part, near the anterior nostril, is a non-sensory margin with cilia-free epidermal cells. Within this is an internal ciliated sensory area, which is intercalated with ciliated receptor cells and a few ciliated non-sensory cells. Additionally, some dense ciliated non-sensory cells make up a non-sensory area, which also contains cilia-free epidermal cells distributed in patches. In the rear of the olfactory sac near the posterior nostril, the lamellae differ in morphology from those of the frontal olfactory sac but are similar in having few ciliated receptor cells. In other words, the surface of the lamellae in the rear part of the olfactory sac is mainly non-sensory. At present, four types of lamellae （~ E IlIand IV） have been recognized in relation to the pattern of the sensory epithelium. In this study, the frontal and rear lamellae resembled types I and IV, respectively, but are referred to as types r and IV because they are slightly less developed. Data on the ratio of length of lamellae to eye diameter, number of lamellae and the type of surface pattern of the lamellae show that the development of the olfactory system of C. semilaevis facilitates prey capture.

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.

Analysis of phenotypic and genetic parameters for growth-related traits in the half smooth tongue sole,Cynoglossus semilaevis

Phenotypic and genetic parameters for growth-related traits in the half-smooth tongue sole, Cynoglossus semilaevis, were estimated in 22 full-sib families produced by normal and neo-male breeding stocks. As phenotypic males with female genotypes, neo-males are harmful in C. semilaevis aquaculture because they reduce overall production. The present study evaluated the difference in the growth-related traits： total length （TL）, body weight （BW） and square root of body weight （SQ_BW） at the age of 570 days between normal and neo-male offspring （neo-males used as male parents）. The difference in the proportion of females between normal and neo-male offspring was also assessed. Based on the linear mixed model, restricted maximum likelihood （REML） and best linear unbiased prediction （BLUP） were used to estimate various （co）variance components and estimated breeding values （EBVs） of growth-related traits. As a result, all the mean values of the three studied traits were significantly larger in normal offspring than in neo-male offspring. Additionally, the female proportion was significantly larger in normal offspring than in neo-male offspring. Heritability was 0.128＋0.066 2 for TL, 0.128-4-0.065 5 for BW and 0.132~0.062 9 for SQBW, all of which were low level heritabilities. The correlation coefficients of EBVs and phenotypic values of the target traits were 0.516 for TL, 0.524 for BW and 0.506 for SQ_BW, all of which were highly significant （P〈0.01）. Genetic correlations among TL, BW and SQ_BW were positive high （0.921-0.969） and higher than those of phenotype （0.711-0.748）, both of which had low standard errors （0.063-0.123 for genotype, and 0.010-0.018 for phenotype）. Compared with normal offspring, neo-male offspring have lower breeding values for each studied trait through EBVs comparison. Therefore, neo-male offspring should not be used as broodstock in a C. semilaevis breeding programs.

Effects of Na^+/K^+ Ratio of Groundwaters on the Gill Ion-Transport Enzyme Activity,Plasma Osmolality and Growth of Cynoglossus semilaevis Juveniles

The effects of environmental Na+/K+ ratio on the gill ion-transport enzyme activity,plasma osmolality and growth of Cynoglossus semilaevis juveniles were investigated. The results showed that,plasma osmolality was similar among flounder adapted to different Na+/K+ ratios of saline groundwaters (P>0.05),while the growth,gill Na+,K+-ATPase and HCO3--ATPase activities were affected by Na+/K+ ratio significantly (P<0.05). The gill Na+,K+-ATPase activity reached its maximum on day 3,then decreased gradually from day 3 to day 9 and remained constant from day 9 to day 15. The peak values of gill Na+,K+-ATPase activity were detected on day 3 for all Na+/K+ ratios of saline groundwaters,then the enzyme activities descended,and on day 9 the enzyme activi-ties achieved steady state,and the gill HCO3--ATPase activity increased rapidly and achieved steady state after one day. During steady state,the gill Na+,K+-ATPase and HCO3--ATPase activity of Na+/K+ ratios 20 and 40 treatments were significantly higher than those in the control group (Na+/K+ ratio 27.5),while there were no significant differences between the Na+/K+ ratio 30 treatment and the control group; the gill Na+,K+-ATPase activity of Na+/K+ ratio 20 and 40 treatments were significantly higher than that for ratio 30 treatment,but there were no significant differences of gill HCO3--ATPase activity among these treatments. At the end of the 15-day experiment,the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higher than those in groundwaters; significant differences also occurred among the treatments; Na+/K+ ratio 30 treatment had the highest values (33.7% and 1.94 respectively),which were significantly higher than those under Na+/K+ ratios 20 and 40 treatments. There-fore,for the saline groundwater used in this experiment,it is suggested that the Na+/K+ ratio be adjusted to approximately 30,i.e.,as close to that of natural seawater as possible in the culture of flounder.

Large-Scale Screening of Growth-Related Variants in Chinese Tongue Sole(Cynoglossus semilaevis)

Growth is the most valuable economic trait for improving aquaculture fish species,since fast growth can reduce labor cost and make more economic benefits.However,the knowledge about how many and which genes are related to the growth of Chi-nese tongue sole is limited.High-throughput sequencing screening of variants is a fast,economical and accurate assay to identify genes related to growth in crops,livestock and other aquaculture products.In the present study,genome-wide resequencing of 30 female Chinese tongue sole individuals from large and small groups to screen growth trait-related variations(SNPs,InDels)was carried out.In total,6545735 SNPs and 1016745 InDels were detected,while 31 genes related to growth traits were identified.Their functions were mainly involved in muscle architecture,post-embryonic development,neurosensory development and hormone regulation.Fur-thermore,18 of the 31 genes related to growth trait are located on W chromosome,indicating that W chromosome plays an important role in regulating the body size of female tongue soles.The markers and genes identified in our study can be applied to selective breeding of tongue sole and thus will promote the aquaculture industry and increase economic efficiency.

DNA Methylation of pacap Gene Is Involved in Growth Superiority of Female Half Smooth Tongue Sole(Cynoglossus semilaevis)in Different Salinities

Pituitary adenylate cyclase activating polypeptide(PACAP)and growth hormone-releasing hormone(GHRH)play important roles in the GH/IGF growth axis in fishes.To determine whether epigenetic change is involved in the regulation of pacap and ghrh responses to low salinity stress in Cynoglossus semilaevis,the correlation between growth traits,DNA methylation status and gene expression level in low salinity(15,S15)and optimal salinity(30,S30)at day 7(D7)and day 60(D60)were analyzed.Results showed that exposure to low salinity for 60 days attenuated C.semilaevis growth rate.Under low salinity,DNA methylation level of pacap promoter increased in females and decreased in males at day 7,but inverted at day 60.Additionally,pacap expression was up-regulated in both males and females.The pacap promoter methylation level was higher and expression level was lower in female than in male.The results suggest that pacap promoter methylation level is negatively correlated to mRNA level and positively correlated to body weight,while gene expression level is negatively related with body weight.With the decrease of salinity,DNA methylation level of ghrh promoter and exon1,as well as its gene expression displayed minor changes.Overall,pacap gene seems to play an important role in fish growth,contributing to female growth superiority,while ghrh gene seems not pertinent under salinity stress.The results indicate that low salinity potentially affects fish growth through regulating DNA methylation in pacap promoter.This study expands the understanding of the molecular mechanism of how salinity modulates fish growth from the epigenetic perspective.

Cloning and expression of follistatin gene in half-smooth tongue sole Cynoglossus semilaevis during the reproduction cycle

Follistatin(FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole,Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction(RTPCR). FST m RNA was expressed in all 13 tissues analyzed,and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis,the transcript level of FST was the highest in the perinucleolus stage,decreased in the primary yolk stage,slightly increased in the tertiary yolk stage,and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone,which stimulated oocyte development. However,no significant variation was observed across all stages of testis development,although the expression level in the spermatogenesis stage was relatively low,which may result from the regulation of FST by aromatase.

Changes in Plasma Osmolality, Cortisol and Amino Acid Levels of Tongue Sole(Cynoglossus semilaevis) at Different Salinities

A serial of salinity transferring treatments were performed to investigate the osmoregulation of tongue sole(Cynoglossus semilaevis). Juvenile tongue sole were directly transferred from a salinity of 30 to 0, 10, 20, 30, 40 and 50. Blood sampling was performed for each treatment after 0, 1, 6 and 12 h, as well as after 1, 2, 4, 8, 16 and 32 d. The plasma osmolality, cortisol and free amino acids were assessed. Under the experimental conditions, no fish died after acute salinity transfer. The plasma cortisol level increased 1 h after the abrupt transfer from a salinity of 30 to that of 0, 40 and 50, and decreased from 6 h to 8 d after transfer. Similar trends were observed in the changes of plasma osmolality. The plasma free amino acids concentration showed a ‘U-shaped' relationship with salinity after being transferred to different salinities for 4 days. More obvious changes of plasma free amino acid concentration occurred under hyper-osmotic conditions than under hypo-osmotic conditions. The concentrations of valine, isoleucine, lysine, glutamic acid, glycine, proline and taurine increased with rising salinity. The plasma levels of threonine, leucine, arginine, serine, and alanine showed a ‘U-shaped' relationship with salinity. The results of this study suggested that free amino acids might have important effects on osmotic acclimation in tongue sole.

Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole(Cynoglossus semilaevis)

Chinese tongue sole(Cynoglossus semilaevis)is an economically important marine fish species with a ZZ/ZW sex determination mechanism,which can be influenced by temperature.Alternative splicing(AS)is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation,but has rarely been reported in fish.In this study,to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation,we combined isoform and RNA sequencing(Iso-Seq and RNA-Seq)to perform transcriptome profiling of male and female gonads in C.semilaevis.In total,81883 and 32341 full-length transcripts were obtained in males and females,respectively.A total of 8279 AS genes were identified,including 2639 genes showing differential AS(DAS)between males and females.Many intersecting DAS genes and differentially expressed genes(DEGs)were enriched in the meiotic cell cycle pathway,and genes related to gonadal differentiation,such as esrrb and wt1a,were found to have sex-specific isoforms.Thus,this study revealed AS events in the gonadal transcriptomes of male and female C.semilaevis,described the characteristics of active transcription in the testes,and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.