Histological Assessment and Transcriptome Analysis Provide Insights into the Toxic Effects of Perfluorooctanoic Acid to Juvenile Half Smooth Tongue Sole Cynoglossus semilaevis

Perfluorooctanoic acid(PFOA)is a widespread synthetic persistent organic pollutant that may enrich along the food chain and affect the growth,development,reproduction,and lipid metabolism of aquatic organisms,particularly the benthic organisms.How-ever,the toxic effects of PFOA on the half-smooth tongue sole Cynoglossus semilaevis,a commercial benthic fish in China,have rarely been reported.Because juvenile fish are sensitive to environmental pollutants,in the present study,histological assessment and tran-scriptome sequencing were performed to determine the short-term impact of PFOA on juvenile half-smooth tongue soles.Histologi-cal analysis showed that PFOA exposure caused hepatocyte rupture,intestinal villi breakage,increased goblet cell count,and brain ab-normal.Transcriptome results showed that some interesting signaling pathways,such as glycolysis/gluconeogenesis,PPAR signaling pathway and GABAergic synapse signaling pathway,were enriched after PFOA exposure.In addition,some metabolic,immune and neural genes were differentially expressed,which including ependymin,hbb1-like and gad 1,and they were up-regulated after 14 days of exposure.Transcriptome results also indicated that half-smooth tongue sole might improve energy metabolism in response to PFOA toxicity after 7 days of exposure.These findings provide a basis for studying the ecological effects of PFOA on marine benthic fishes.

基于低压静电场技术(LVEF)对半滑舌鳎(Cynoglossus semilaevis)的保鲜效果及微生物菌落影响分析

为研究低压静电场对半滑舌鳎(Cynoglossus semilaevis)在贮藏过程中微生物群落特征,采用高通量测序技术对贮藏至30 d半滑舌鳎的微生物群落碱基信息测序,产生的碱基序列通过OTU聚类、多样性分析、相关性分析等手段,探讨影响半滑舌鳎保鲜效果的微生物群落组成和发育信息。研究不同低压静电场(2000、2500 V/m)对半滑舌鳎贮藏期间微生物群落组成的影响。结果表明:半滑舌鳎有效序列范围为76735~103583,平均长度为428.39~429.17 bp,OTU数目在45~218。贮藏期间样品的微生物多样性和相对丰度均有所下降。在门水平上共鉴定出四种微生物菌门,分别为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)和其他。在属水平上鉴定出11种微生物菌属,分别为不动杆菌属(Acinetobacter)、嗜冷杆菌属(Psychrobacter)、希瓦氏菌属(Shewanella)、环丝菌属(Brochothrix)、漫游菌属(Vagococcus)、发光杆菌属(Photobacterium)、Myroides和肉食杆菌属(Carnobacterium)、假单胞菌属(Pseudomonas)、无色杆菌属(Achromobacter)和其他。贮藏至30 d时,CK组优势腐败菌为环丝菌属,LVEF-1(2000 V/m)和LVEF-2(2500 V/m)组样品中环丝菌属相对丰度显著上升,但是低于CK组,且嗜冷杆菌属和希瓦氏菌属相对丰度仍然占据一定比例。由此推测LVEF能够抑制半滑舌鳎优势腐败菌属相对丰度的增加。研究结果为低压静电场保鲜贮藏半滑舌鳎微生物多样性变化提供了理论基础。

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.

Molecular cloning,characterization and expression analysis of Sox9a and Foxl2 genes in half-smooth tongue sole(Cynoglossussemilaevis)

Two transcription factors, Sox9a and Foxl2 were cloned from half-smooth tongue sole （Cynoglossus semilaevis）. Sox9a is a new duplication of C.semilaevis Sox9 gene. The complete cDNA of Sox9a gene was 1 842 bp long coding for 487 amino acids and Foxl2 gene was 1 817 bp coding for 308 amino acids. Sox9a was expressed higher in male brain, pituitary and gonad and Foxl2 were higher in female brain, pituitary and gonad. The expression of Sox9a gene in gonads of neo-males was higher than that of normal females. Sox9a and Foxl2 were expressed higher in gastrula stage than in other stages. In the period of sex differentiation, the expression of Sox9a was first going up and then going down and Foxl2 was higher expressed at 37 dph. The highest expressions of Sox9a and Foxl2 genes occurred in nine-month and 12-month old gonad tissues, respectively. Sox9a gene was considered to have inevitable links with sex reversal, sex differentiation and cell differentiation of embryos and formation of spermatogenic ceils. Foxl2 was considered to play a role in sex differentiation, cell differentiation of embryos but not to be necessary for sex determination and sex reversal.

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.

Antimicrobial and hemolytic activity of fish epidermal mucus Cynoglossus arel and Arius caelatus

Objective:To study the antimicrobial,hemolytic activity and immunomodulatory activity of fish epidermal mucus and their chemical constituents from Cynoglossus arel(C.arel) and Alius caelatus(A.caelatus).Mucus plays an important role in the prevention of colonization by parasites,bacteria and fungi.Methods:Epidermal mucus was obtained from two marine fishes,lyophilized and the chemical composition of epidermal mucus was analysed by FT-IR analysis.The in vitro antimicrobial activity against human pathogens(fungi,gram positive and gram-negative bacteria) and also the hemolytic activity and immunomodulatory activity were determined.Results:Totally ten human pathogens were tested against the fish mucus.Out of the ten pathogens,five pathogens have proved to be sensitive to the mucus.Maximum zone of inhibition was observed against Vibrio cholera(V.cholera)(9 mm and 2 mm in diameter),followed by Staphylococcus aureus(S.aureus) with a inhibition zone of(6 mm and 3 mm),Streptococcus areus(S.areus)(5 mm and 4 mm),Vibrio parahemolyticus(V.parahemolyticus)(4 mm and 5 mm) respectively.Conclusions:The present investigation has revealed that positive progresses in the fish mucus extracts against human pathogens and hemolytic activity.But further efforts are required for the purification and isolation of the active antimicrobial compounds in order to establish their possible applications.

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.

Long duplication of 18S ribosomal DNA in Cynoglossus lineolatus(Pleuronectiformes： Cynoglossidae)： novel molecular evidencefor unequal crossing over model

Although 18 S r DNA sequence is extremely conservative, the polymorphism still has been found in few species.In the present study, three types(Type A, B and C) of 18 S r DNA sequence coexisted in Cynoglossus lineolatus genome, suggesting a non-concerted evolution process, rather than a strictly concerted evolution fashion.Based on the differences of sequence variation, GC content, secondary structure and minimum free energy,Types A and B were speculated as the potential pseudogenes. Additionally, a fascinating finding was a 189-bp duplication of 18 S r DNA in Type A sequence. To our knowledge, this is the first report on such a long duplication in teleostean ribosomal DNA. Compared with several theories accounting for the formation of tandem repeats, the unequal crossing over model was thought to be the most likely mechanism to generate the189-bp duplication of 18 S r DNA. These results not only provide a novel molecular evidence for the unequal crossing over model, but also benefit for the further study on 18 S r DNA in fishes.

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.

Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole,Cynoglossus semilaevis

The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sex-determining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1;functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1-Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.

FOOD HABITS AND SEASONAL VARIATION OF STOMACH CONTENTS OF TONGUE SOLE CYNOGLOSSUS SEMILAEVIS (GUNTHER) IN THE BOHAI SEA

Examination of the food habits and seasonal variation of the stomach contents of adult tongue sole, Cynoglossus semilaevisGunther) taken in July 1982-July 1983 from Laizhou Bay and the Huanghe River estuary of the Bohai Sea showed Crustacea, bivalvia and small fishes comprised the main prey. Invertebrates such as polychaeta, cephalopoda, gastropoda, echinodermata and actiniaria were also intermittently found in them. They intensively fed all the year round (monthly feeding rate of over 80%). The main food items were Alpheus japonica, Alpheus distinguendus, Oratosquilla oratorio, Eucrate crenata and Carcinoplax vestitus, etc. In summer and autumn, the portion of bivalvia such as Cultellus attenuatus and Musculus senhousei increased steadily. From summer to winter, a stable proportion of small fishes such as Rhinogobius pflaumi and Setipinna taty was in the diet.

QTL Detection for Albinism-Related Loci in Chinese Tongue Sole(Cynoglossus semilaevis)

The Chinese tongue sole(Cynoglossus semilaevis) is widely cultured in the coastal region of East Asia and has excellent economic value. However, the high albino rate of the breeding population has caused a significant loss to the aquaculture industry. To study the molecular mechanism of albinism, the present study used an albino Chinese tongue sole family to construct three simple sequence repeat(SSR) linkage groups, and draft a preliminary linkage map related to albinism. After albinism-related loci mapping, 18 albinism-related loci were detected under two models(containing 2407 genes) compared to the Chinese tongue sole genome. One of these loci, the tyrosinase related protein(tyrp2), which has been reported previously as an important gene regulating both eumelanin and phaeomelanin levels, was indicated to be the possible cause of albinism. Thirty-five Gene Ontology(GO) terms and 14 Kyoto Encyclopedia of Genes and Genome pathways were annotated via bioinformatic analyses. One GO term with protein tyrosine kinase activity, which contained 10 genes, was previously suggested to affect fish albinism. These results establish a foundation for further in-depth study of albinism in Chinese tongue sole.

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.

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.

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.