Verasper Variegatus Research Articles

Identification of two female-specific DNA markers in Verasper variegatus

Sex-specific DNA markers are powerful tools for genetic sex identification and sex control breeding of fish. Spotted halibut (Verasper variegatus) is a kind of marine flatfish with important nutritional and economic value, which exhibits significant sex-related growth dimorphism that the female individuals grow faster than the male individuals. However, there is no rapid, effective and universal sex-specific DNA marker used for genetic sex identification and sex control breeding in spotted halibut up to date. In the present study, whole genome resequencing was performed on 17 male and 17 female spotted halibut individuals to identify sex-specific markers. A total of 325.36 Gb and 328.99 Gb data were obtained from females and males, respectively. After reads mapping, sequencing depth and coverage analysis, 359 potential female-specific DNA sequences were identified. Then, after two rounds of verification, two female-specific 66 bp and 68 bp insertions were identified as female-specific markers which can accurately identify the genetic sex of spotted halibut through PCR amplification and 2% agarose gel electrophoresis. Subsequently, these two female-specific DNA markers and the PCR method were further validated using 117 spotted halibut individuals from another breeding population to confirm their genetic sex, which was in accordance with their phenotypic sex determined by histological section of the gonads. This may be the first two rapid, accurate and effective female-specific DNA markers used for genetic sex identification in spotted halibut. The identification of these two female-specific DNA markers will not only provide important genetic evidence to elucidate the sex determination mechanism of spotted halibut, but also help to advance the process of sex control breeding in spotted halibut.

Assembly of the poorly differentiated Verasper variegatus W chromosome by different sequencing technologies

The assembly of W and Y chromosomes poses significant challenges in vertebrate genome sequencing and assembly. Here, we successfully assembled the W chromosome of Verasper variegatus with a length of 20.48 Mb by combining population and PacBio HiFi sequencing data. It was identified as a young sex chromosome and showed signs of expansion in repetitive sequences. The major component of the expansion was Ty3/Gypsy. The ancestral Osteichthyes karyotype consists of 24 protochromosomes. The sex chromosomes in four Pleuronectiformes species derived from a pair of homologous protochromosomes resulting from a whole-genome duplication event in teleost fish, yet with different sex-determination systems. V. variegatus and Cynoglossus semilaevis adhere to the ZZ/ZW system, while Hippoglossus stenolepis and H. hippoglossus follow the XX/XY system. Interestingly, V. variegatus and H. hippoglossus derived from one protochromosome, while C. semilaevis and H. stenolepis derived from another protochromosome. Our study provides valuable insights into the evolution of sex chromosomes in flatfish and sheds light on the important role of whole-genome duplication in shaping the evolution of sex chromosomes.

Radial polarity in the first cranial neuromast of selected teleost fishes.

The neuromast is a sensory structure of the lateral line system in aquatic vertebrates, which consists of hair cells and supporting cells. Hair cells are mechanosensory cells, generally arranged with bidirectional polarity. Here, we describe a neuromast with hair cells arranged radially instead of bidirectionally in the first cranial neuromast of four teleost species: red seabream (Pagrus major), spotted halibut (Verasper variegatus), brown sole (Pseudopleuronectes herzensteini), and marbled sole (Pseudopleuronectes yokohamae). In these four species, this polarity was identified only in the first cranial neuromast, where it appeared at the rostral edge of the otic vesicle before hatching. We investigated the initial appearance and fate of this unique neuromast using scanning electron microscopy. We also assessed characteristics of radial neuromast pertaining to morphogenesis, development, and innervation using a vital fluorescent marker and immunohistochemistry in V. variegatus. The kinocilium initially appears at the center of each hair cell, then moves to its outer perimeter to form radial polarity by around 7 days postfertilization. However, hair cells arranged radially disappear about 15 days after hatching. This is followed by the appearance of bidirectionally arranged hair cells, indicating that polarity replacement from radial to bidirectional has occurred. In P. herzensteini, both afferent and efferent synapses between the nerve fibers and hair cells were observed by transmission electron microscopy, suggesting that radial neuromast is functional. Our discovery suggests that neuromasts with radial polarity could enable larval fish to assimilate multiaxial stimuli during this life stage, potentially assisting them in detecting small water vibrations or water pressure changes.

Intermediate-Term Storage of Spotted Halibut (Verasper variegatus) Sperm: Effects of Storage Methods, Extenders Supplemented with Antibiotics and Antioxidants on Sperm Quality.

Intermediate-term preservation of sperm assists the reproductive management of fish spermatozoa; however, no information is available on sperm of the spotted halibut, Verasper variegatus. We aimed to identify the optimum diluents, temperatures, dilution ratios, antibiotics, and antioxidants for sperm motility and cell viability. The diluents evaluated were marine fish Ringer’s solution (MFRS), Stein’s solution, 300 mM sucrose, and 300 mM glucose (diluted 1:1 [sperm: diluent], 1:2, 1:4, and 1:10 and stored at 0, 2, 4, and 6 °C). Neomycin and gentamycin (100, 200, 400, and 800 mg/L) and antioxidants (Mito-TEMPO [0, 25, 50, 75, 100, 125, 150, 175, and 200 µM], reduced glutathione [0, 2, 4, 6, 8, and 10 mM], and trehalose [0, 50, 100, 150, 200, and 250 mM]) were assessed in terms of sperm preservation. The most effective condition for cold storage of spotted halibut sperm was Stein’s solution at a dilution ratio of 1:4 at 2 °C, with a combination of neomycin 800 mg/L and 250 mM trehalose that showed spermatozoa motility of > 43% after 60 days. These storage conditions will be valuable for spotted halibut hatcheries.

Chromosome-level genome assembly of the Verasper variegatus provides insights into left eye migration

Chromosome-level genome assembly of the Verasper variegatus provides insights into left eye migration

A low body-color regulating ability of spotted halibut Verasper variegatus: Evaluation of the roles of melanin-concentrating hormone and proopiomelanocortin systems

A low body-color regulating ability of spotted halibut Verasper variegatus: Evaluation of the roles of melanin-concentrating hormone and proopiomelanocortin systems

An evaluation of the growth-promoting effects of green light on spotted halibut for its practical application in aquaculture

We examined the effects of tank color and stocking density on the growth of spotted halibut (Verasper variegatus) under a green light-emitting diode (LED). Fish reared for 40 days under a green LED (10 μmol m–2 s–1 at the water surface) grew faster than those reared under a white LED, irrespective of tank color (white or black). Green light promoted higher growth than indoor room light when fish were kept for 32 days under both the tested stocking densities, 4.3 kg m–2 and 13 kg m–2, but stocking density alone did not affect fish growth. These results suggest that a growth-promoting effect of green light can be expected within the range of stocking densities applied here for fish housed in both black and white tanks.

Complete mitochondrial genomes of two Pleuronectid species: Clidoderma asperrimum and Verasper variegatus (Teleostei: Pleuronectiformes: Pleuronectidae)

Complete mitochondrial genomes of two Pleuronectid species, Clidoderma asperrimum and Verasper variegatus (Teleostei: Pleuronectiformes: Pleuronectidae) were analysed using the primer walking method. Their mitogenomes were 17,632 and 17,273 bp in total length, respectively and comprised 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Their gene contents and orders were similar to those of typical vertebrates. All Pleuronectid species were subdivided into three clades in the phylogenetic tree, and the two Pleuronectid species analysed in this study formed a strong monophyletic group comprising species belonging to three genera, Hippoglossus, Reinhardtius, and Verasper.

Tumor necrosis factor-alpha (TNF-α) in spotted halibut Verasper variegatus at the embryonic and metamorphic stages

As an aquatic fish, the spotted halibut Verasper variegatus is highly susceptible to bacterial and virus infections. Tumor necrosis factor-alpha (TNF-α) as a cytokine could control the inflammatory responses. The functions of TNF-α in many species have been widely studied, particularly in mammals. However, little is known about the TNF-α functions in V. variegatus. We first cloned and sequenced the TNF-α gene in V. variegatus (VvTNF-α). The two conserved cysteine residues, transmembrane sequence, Thr-Leu motif, and TNF family signature, as well as the TA-rich motifs of its proteins related to inflammatory responses had high similarity to those of the other teleost and mammalian TNF-α. The phylogenetic analysis showed that VvTNF-α was consistent with TNF-α genes of other vertebrates. The VvTNF-α transcripts were extensively distributed in the peripheral blood leukocytes (PBLs), spleen, and gill, indicating that the VvTNF-α had a role in immune function. Furthermore, treatment with pathogen-associated molecular patterns (PAMPs) could induce a rapid and significant increase of VvTNF-α in the PBLs, which reveals that VvTNF-α does participate in the host immune responses against bacterial and viral pathogens. We found that VvTNF-α had an interesting expression pattern during metamorphosis, showing that the flatfish TNF-α may have some novel functions during specific developmental stages. In addition, the 3D structure prediction of VvTNF-α provided an indication of how it is likely to interact with other proteins. Therefore, VvTNF-α has multiple functions, and provides valuable information to explore novel functions of TNF-α.

Teleost Type 2 Interleukin-1 Receptor (IL-1R2) from the Spotted Halibut (Verasper variegatus): 3D Structure and a Role in Immune Response

Teleost Type 2 Interleukin-1 Receptor (IL-1R2) from the Spotted Halibut (Verasper variegatus): 3D Structure and a Role in Immune Response

Pharmacokinetic profiles of florfenicol in spotted halibut, Verasper variegatus, at two water temperatures.

The pharmacokinetic profiles of florfenicol in the spotted halibut (Verasper variegatus) were investigated at 15 and 20°C water temperatures, respectively. Florfenicol content in plasma samples was analyzed using an HPLC method. Drug concentration versus time data were best fitted to a three-compartment model after a single intravenous administration (15mg/kg BW), and fitted to a two-compartment model after an oral administration (30mg/kg BW) at 15 and 20°C. The florfenicol concentration in the blood increased slowly during the 12hr following an oral administration at 15°C, with a peak concentration (Cmax ) of 9.1mg/L, and then declined gradually. The half-lives of absorption, distribution, and elimination phase were 2.18, 5.66 and 14.25hr, respectively. The bioavailability (F) was calculated to be 24.14%. After an oral administration at 20°C, shorter half-lives of absorption (1.33hr), distribution (2.51hr) and elimination (9.71hr), a higher Cmax (12.2mg/L), and a similar F (23.98%) were found. Based on the pharmacokinetics and pharmacodynamics, an oral dose of 30mg/kg BW was suggested to be efficacious for bacterial disease control in spotted halibut farming.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ± 36 mg) were about 12 and 630 μg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 μg/L and a no-observed-effect concentration of 2.1 μg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 μg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

Novel Functions of Interleukin-1 Beta in Spotted Halibut (Verasper variegatus) at Its Embryonic and Metamorphic Stages

Interleukin-1beta (IL-1β) is a cytokine involved in the control of inflammatory responses. The functions of IL-1β have been extensively studied in many vertebrates. However, few studies focused on the functions of IL-1β in the spotted halibut (Verasper variegatus), an endangered aquatic fish that easily suffers from bacterial and viral infections. We first cloned and sequenced the IL-1β gene from V. variegatus (VvIL-1β). Its conservative interleukin-1 propeptide and interleukin-1/18 domains corresponding regions, characteristics of the IL-1 protein family and TA-rich motifs relating to inflammatory responses were similar to those of the IL-1β genes from other teleost and mammals. The phylogenetic analysis showed that VvIL-1β was homologous to the IL-1β gene of other vertebrates. Treatment with pathogen-associated molecular patterns (PAMPs) induced a significant and rapid up-regulation of VvIL-1β expression in the peripheral blood leukocytes (PBLs), revealing that VvIL-1β is actively involved in the host immune responses against bacterial and viral pathogens. The expression level of VvIL-1β was relatively high in the PBLs, gill, and intestine, indicating the immunological function of VvIL-1β. VvIL-1β also had interesting expression patterns at specific developmental stages, implying that the IL-1β of flatfishes may have some novel functions during embryonic development and metamorphosis. The 3D structure prediction also provided information about how VvIL-1β interacts with its receptor protein. These results suggested that VvIL-1β may have multiple functions in V. variegatus and provided us valuable information for understanding the novel functions of IL-1β.

Green-shifting of SWS2A opsin sensitivity and loss of function of RH2-A opsin in flounders, genus Verasper.

We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2‐a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2‐a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λmax) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue‐sensitive, whereas in V. variegatus (485.4 nm), it was green‐sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green‐sensitive, while its overall structure remains conserved as a blue‐sensitive opsin. Further, the rh2‐a function seems to have been reduced during the evolution of flounders. λmax values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue‐sensitive. Thus, the green‐sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2‐A.

Effect of Dietary Antarctic Krill Meal on Growth Performance, Muscle Proximate Composition, and Antioxidative Capacity of Juvenile Spotted Halibut, Verasper variegatus

AbstractA 7‐wk feeding trial was conducted to evaluate the effects of dietary Antarctic krill meal (AKM) on the growth performance, proximate composition of muscles, and antioxidative capacity of juvenile spotted halibut. Six diets were formulated to contain about 50% protein and 8% lipid. A control diet (R0) without AKM and the other five diets with 8.1, 16.2, 24.3, 32.4, and 42.5% AKM supplementation (R10–50) to replace 10, 20, 30, 40, and 50% fishmeal protein were used to feed to juvenile spotted halibut. The juveniles were fed with each diet using three replicates and cultivated in the indoor culture system. Results showed that the specific growth rate, feed intake, and protein efficiency ratio in the R30 and R40 groups were significantly higher than that in other groups (P < 0.05). Survival rate in the R50 group was significantly lower compared with the R0 group and the other four AKM supplementation groups. Moreover, the rising AKM levels in diets had significant effects on the chemical composition of juvenile spotted halibut, showing significantly decreased contents of crude protein, but increased lipid and ash contents (P < 0.05). The total superoxide dismutase activity and catalase activity of serum and liver in AKM groups were significantly higher than those in the control group (P < 0.05). In contrast, the contents of malondialdehyde in serum and liver were significantly lower compared with the control group. These findings illustrate that a moderate AKM level in diets can significantly improve the growth performance, feed utilization, and antioxidative capacity in juvenile spotted halibut, which support the finding that AKM may be used as a good protein source for halibut in the aquafeed industry.

SNP discovery in spotted halibut (Verasper variegatus) using restriction site-associated DNA sequencing(RAD-seq)

The spotted halibut (Verasper variegatus) is an important economic marine flatfish species in China and Japan. However, the wild population of V. variegatus has suffered a drastic decline in recent years. We implemented restriction site-associated DNA sequencing(RAD-seq) to develop SNP markers from 36 individuals. In total, 100 high quality SNPs were obtained after a stringent SNP filter process. The minor allele frequency of these SNPs ranged from 0.0455 to 0.5000. The range of observed heterozygosity and expected heterozygosity were from 0.0909 to 1.0000 and from 0.0881 to 0.5077, respectively. The inbreeding coefficient varied from −1.000 to 0.220. Among these SNPs, 16 loci deviated significantly from Hardy–Weinberg equilibrium after Bonferroni correction. These novel SNP markers will serve as a valuable tool for future population and conservation genetics studies for this species.

Horizontal movement and emigration of juvenile spotted halibut Verasper variegatus released in a shallow brackish lagoon: Matsukawa-ura, northeastern Japan, revealed by acoustic telemetry

To elucidate the post-release movement and emigration of juvenile spotted halibut Verasper variegatus, ten hatchery-reared age-0 individuals (10 months old, 17.0–19.7 cm total length) surgically implanted with acoustic transmitters were released near the center of Matsukawa-ura, a shallow brackish lagoon, on 25 November 2009 (water temperature 13.8 °C). They were monitored by ten acoustic receivers for 5 months. Of ten individuals, eight left the release site during December–March. Three of these emigrated to the outer ocean on 17 and 29 January and 30 March 2010, respectively. Juveniles probably started wintering migration in December and January when water temperatures decreased considerably in the lagoon (mean 9.5 and 6.0 °C, respectively). They stopped their migration in the coldest month, February (mean 5.7 °C), and restarted it in March when water temperatures frequently exceeded a plausible threshold for movement (≥6 °C). Statistical analyses revealed that the fish started migration significantly more frequently at nighttime. The migration track of an individual recorded from 11 to 30 March showed gradual nocturnal movements and a slow migration speed (estimated maximum speed 2.2 m/min). Our results revealed that water temperature primarily governed the seasonal timing of nocturnal migratory movements of juvenile spotted halibut.

Path analysis of effects of morphometric traits on body weight in spotted halibut Verasper variegatus at different growth stages

PDF HTML阅读 XML下载 导出引用 引用提醒 不同生长期圆斑星鲽形态性状对体重影响的通径分析 DOI: 作者: 作者单位: 1. 中国水产科学研究院 黄海水产研究所, 山东 青岛 266071;2. 上海海洋大学 水产与生命学院, 上海 201306;3. 天津渤海水产研究所, 天津 300457 作者简介: 边力(1988-),男,助理研究员,博士,研究方向为鱼类繁育与遗传育种.E-mail:bianli@ysfri.ac.cn 通讯作者: 中图分类号: S96 基金项目: 天津市农业科技成果转化与推广项目（201604100）；中国水产科学研究院黄海水产研究所基本科研业务费（20603022016005）. Path analysis of effects of morphometric traits on body weight in spotted halibut Verasper variegatus at different growth stages Author: Affiliation: 1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;3. Tianjin Bohai Sea Fisheries Research Institute, Tianjin 300457, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:圆斑星鲽（）作为中国鲽形目（Pleuronectiformes）养殖鱼类的重要代表种类，具有生长速度快，可食用部分多，肉质细嫩等特点。为了探究圆斑星鲽形态性状与体重的关系，本研究分别测定11月龄、14月龄和17月龄3个不同生长期圆斑星鲽的体重及全长（FL）、体长（BL）、体高（BH）、头长（HL）、头高（HH）、尾柄长（CL）、尾柄高（CH）、吻长（SL）共计8个形态性状，分别进行相关分析和通径分析，剖析形态性状对于体重的直接作用和间接作用；利用回归分析的方法，建立各个时期以形态指标为自变量，体重为因变量的最优线性回归方程。结果显示，1）不同生长阶段，影响体重的重点形态指标存在差异，影响11月龄圆斑星鲽体重的主要形态性状为体高和体长，14月龄阶段为全长、体高和头长，17月龄阶段为全长和体高；2）11月龄、14月龄和17月龄形态性状对体重（BL，XHL，；3）除本研究所包括的形态指标外，体厚可能为另外一个影响圆斑星鲽体重的重要指标。本研究阐明了体重与形态指标间的相关关系，为圆斑星鲽的良种选育提供了生物学依据。 Abstract:is an important representative species of the Pleuronectiformes. It has a fast growth rate, most parts of its body are edible, and the meat is tender and delicious; all these features make a good aquaculture candidate. In order to explore the relationship between morphometric traits and body weight in , full length (FL), body length (BL), body height (BH), head length (HL), head height (HH), caudal peduncle length (CL), caudal peduncle height (CH), snout length (SL), and body weight (BW) of at 11, 14, and 17 months of age were measured. Correlation and path analysis were conducted, and the effects of each morphometric trait on body weight were separated into direct and indirect effects. Regression analyses were conducted with morphological traits as independent variables and body weight as the dependent variable at each growth stage. The results showed that (1) different morphometric traits had significant path coefficients at each growth stage; at the 11 month stage, the morphometric traits that significantly affected body weight were BH and BL, at the 14 month stage, the significant morphometric traits were FL, BH, and HL, at the 17 month stage, the significant traits were FL and BH. (2) The regression equations at 11, 14, and 17 months of age were =-362.484+13.145, and , respectively. (3) Besides the morphometric traits included in this study, body thickness is another potentially important trait that can affect body weight. This study demonstrated the relationship between morphometric traits and body weight. The results provide valuable information and theoretical guidance for breeding programs. 参考文献 相似文献 引证文献

A hepatic metabolomics-based diagnostic approach to assess lethal toxicity of dithiocarbamate fungicide polycarbamate in three marine fish species

The present study was performed to evaluate the toxic effect of the dithiocarbamate fungicide polycarbamate (PC) on the hepatic metabolic profiles of three marine fish species, red sea bream (Pagrus major), spotted halibut (Verasper variegatus), and marbled flounder (Pleuronectes yokohamae). First, juvenile fish were exposed to graded concentrations of PC for 96h; the 96-h LC50 values obtained were 22–29, 239–553, and 301–364µgL−1 for red sea bream, spotted halibut, and marbled flounder, respectively, indicating that red sea bream possessed higher sensitivity to PC than the two benthic species. Second, the fish were exposed to lethal-equivalent concentration (H group) or sub-lethal (one-tenth of the H group concentrations; L group) for 24 and 96h and gas-chromatography based metabolomics approach was employed to explore the crucial biomarker metabolite associated with lethal toxicity. Of the 53 metabolites identified, only reduced glutathione (GSH) was consistently elevated in the H group for the three fish species at 96h. The calculated cut-off value of GSH (mM) based on receiver operating curve analysis between H group and the other treatment groups (control, solvent control, and L group) was obtained at 0.56mM, which allowed to distinguish between the groups with high confidence for the three fish species. These results are the first to demonstrate the potential of using GSH as a possible biomarker metabolite and its usefulness of threshold cut-off value for diagnosing life-threatening health conditions of fish.