Abstract:The key to sustain the development of marine finfish aquaculture is to improve the understanding of reproductive physiology, which will pave the way for the successful breeding programs and larviculture. Recently, in China, northern coastal research institutes have made significant advances in the field of reproductive physiology in flatfish with focus on the hypothalamus- pituitary-gonadal axis (HPG axis), including histological characteristics, gonadal development, plasma sex steroids, and reproductive endocrine and regulatory mechanisms. This paper summarized the mechanisms of important functional genes in the HPG axis using Cynoglossus semilaevis and Paralichthys olivaceus as examples. Furthermore, we provided here a synoptic contents of some of these important advances related to the HPG axis, including the histology and endocrinology of secretory tissue cells, physiological characters of gonads, environmental factors, hormone-induced spawning, temperature, photoperiod and sex steroid levels, with emphasis on C. semilaevis, Verasper moseri and Kareius bicoloratus. This paper aims to provide the guidance for new breeding programs and laviculture in flatfish.

Abstract:This research focuses on the expression characteristics and mechanism of the novel membrane progestin receptor-like (mPR-Like, mPRL) gene of half-smooth tongue sole (Cynoglossus semilaevis) to improve the technique of artificial breeding. In the present study, using the quantitative real-time PCR (qRT-PCR) assays, the mPRL mRNA expression levels were measured in the isolated oocytes during different stages of oogenesis. With the oocyte development, the mPRL transcript levels of oocytes significantly increased from stage Ⅱ to stage Ⅴ and reached the peak at stage Ⅴ. The spatial expression of mPRL mRNA in the ovary, pituitary and brain of C. semilaevis was demonstrated using in situ hybridization. Results revealed that the mPRL expression was observed on the membrane of oocytes, and the positive signals were also observed in the scattered cells throughout the pituitary and in the brain neurons. Western blotting analysis identified the immunoactive protein that bands in the ovary, brain, pituitary, liver, head kidney and kidney of C. semilaevis. However, the expression levels of the protein in the ovary, brain and pituitary were higher than other tissues. Immunohistochemistry detection revealed that the cellular localization of mPRL protein was similar with that of mRNA in C. semilaevis. The signals were observed in the scattered cells of pituitary and the brain neurons. The intensity of the positive signals was found in the membrane of oocyte. After incubation in vitro in the presence or absence of hCG, the mPRL mRNA and protein levels were measured in the oocytes at different developmental stages using qRT-PCR and western blotting analysis. In response to the hCG treatment, the mPRL mRNA and protein expression increased in a step-wise manner during follicle development, with the highest level detected in the stage V of oocytes. The mPRL expression characteristics at both transcript and protein levels implied that mPRL was involved in regulating the reproduction of female C. semilaevis through the brain-pituitary-ovary axis endocrine system. Moreover, the evidence supports that mPRL has a functional role in the oocyte maturation in C. semilaevis by acting as a mediator of progesterone.

Abstract:This research examined the expression characteristics and mechanism of the novel membrane progestin receptor-like (mPRL) in Japanese flounder Paralichthys olivaceus. In the present study, the temporal changes of mPRL mRNA expression level in isolated oocytes at various oogenesis stages were measured using quantitative real-time PCR (qRT-PCR). The mPRL transcript levels significantly increased with the oocyte development, with the peak level at stage Ⅴ. However, the transcript level decreased significantly at stage Ⅵ. These expression characteristics of mPRL showed that mPRL is a good marker for studying the oocyte maturation. The spatial expression of P. olivaceus mPRL mRNA in the ovary and brain was demonstrated using in situ hybridization. Results revealed that mPRL transcript was localized at the membrane of oocytes, and the positive signals were also observed in the brain neurons of P. olivaceus. Specifically, high-titer rabbit polyclonal antibody was generated against synthetic peptides derived from the C-terminal domain of P. olivaceus mPRL amino acid. Immunobloting analysis identified the immunoactive protein that bands in the ovary, brain, head kidney, kidney and liver of P. olivaceus. However, the expression levels of protein in ovary and brain were higher than other tissues. The protein was localized in the major reproductive associated organs including the ovary and brain using immunohistochemistry. Moreover, the cellular localization of mPRL protein was similar with that of mRNA in P. olivaceus. The intensive signals were observed at the edge of pituitary and brain, and the intensity of positive signals was found on the membrane of oocytes. This is to date the first study that examined the tissue specific localization of mPRL protein and transcript in the reproductive organs of P. olivaceus. The mPRL expression characteristics implied that mPRL regulates the reproduction of P. olivaceus through the endocrine system. Furthermore, the evidence supports a functional role of mPRL in the oocyte maturation of P. olivaceus, possibly, by acting as a mediator of progesterone.

Abstract:In this study we investigated the expression patterns of the mRNA and proteins of membrane progestin receptor alpha (mPRα) in different tissues and oocyte phases using quantitative real-time PCR (qRT-PCR), in situ hybridization, immunohistochemistry, and western blotting analysis. Mature tissue samples were collected from female half smooth tongue sole Cynoglossus semilaevis. After in vitro incubation with or without human chorionic gonadotropin (HCG), the levels of mPRα mRNA and proteins in the oocytes at different developmental stages were measured with qRT-PCR and western blotting analysis. It was shown that the highest level of mPRα mRNA appeared in Phase V oocyte of mature C. semilaevis. 20 IU/ml HCG promoted the mRNA and protein expression more effectively than 10 IU/ml HCG. This further implied that mPRα was involved in the regulation of oocyte maturation through guiding progesterone. The western blotting analysis confirmed that the expression of mPRα proteins was higher in the brain, pituitary, and ovary than in other tissues. These results indicated that mPRα could play certain roles in different tissues, especially the endocrine-related ones. Results of in situ hybridization and immunohistochemistry clearly showed that the mPRα mRNA and proteins were expressed on the oocyte membrane and the peripheral or bureaucratic structures of other organs. Our study enriched the knowledge about mechanisms of membrane receptor signal transduction.

Abstract:In this study we collected the mature tissue samples of female Japanese Flounder Paralichthys olivaceus and investigated the expression patterns of mRNA and proteins of membrane progestin receptor alpha (mPRα) in different tissues and different oocyte phases, using techniques such as quantitative real-time PCR, in situ hybridization, immunohistochemistry, and western blotting. It was shown that the highest level of mPRα mRNA appeared in the Ⅴ phase of oocyte maturation. This further implied that mPRα was involved in the regulation of oocyte maturation through guiding progesterone. The western blotting analysis confirmed the expression of mPRα proteins in the ovary, brain, head kidney, kidney, and liver. Moreover, the expression of mPRα mRNA and proteins was higher in the brain and ovary than in other tissues. These results indicated that mPRα could play certain roles in different tissues, especially the endocrine-related ones. Results of in situ hybridization and immunohistochemistry clearly showed that the mPRα mRNA and proteins were expressed on the oocyte membrane and the bureaucratic structures of other organs. Our study enriched the knowledge about mechanisms of membrane receptor signal transduction.

Abstract:In this study we investigated the characteristics and mechanisms of the expression of progesterone receptor membrane component 1 (PGRMC1) during oocyte maturation of the half-smooth tongue sole (Cynoglossus semilaevis). Expression of PGRMC1 mRNA in the oogenesis of the ovarian development stages Ⅳ and Ⅴ were monitored with qRT-PCR technique. The results showed that the highest level of PGRMC1 mRNA appeared in Phase Ⅴ oocytes of the mature C. semilaevis. In general, the transcription of PGRMC1 peaked in different developmental phases of oocyte. This suggested that PGRMC1 participated in ovarian development and maturation of C. semilaevis. Next we tested whether HCG treatment affected PGRMC1 expression in different stages of oogenesis using qRT-PCR and western blotting analysis. It was shown that HCG could up-regulate the expression of PGRMC1 mRNA and protein, and that 20 IU/ml HCG was more powerful than 10 IU/ml (P<0.05). The effect of HCG varied in different phases of oocyte and was the most obvious in Phase Ⅴ, indicating that PGRMC1 played an important role in the mature stage. The results above suggested that PGRMC1 could regulate the maturation of C. semilaevis oocytes. Our findings may shed light on future exploration of the role of PGRMC1 in the reproductive function of C. semilaevis.

Abstract:Progesterone receptor membrane component 1 (PGRMC1) may play an important role in oocyte maturation of the female half-smooth tongue sole (Cynoglossus semilaevis). To better understand the underlying mechanisms, here we measured the mRNA expression of PGRMC1 in different tissues with in situ RNA hybridization and immunohistochemistry, and analyzed the protein expression with western blotting. According to the results of in situ RNA hybridization, the mRNA of PGRMC1 was detected in the membrane of oocytes, in scattered cells of the entire pituitary, and in the neurons of the brain. Immunohistochemistry showed strong signals of PGRMC1 in the oocyte membrane of the mature ovary, and signals were also observed in the liver, kidney, and head kidney of C. semilaevis. In the liver, PGRMC1 was mainly expressed in the bile duct and hepatic veins that were connected to the outer tissue. In the kidney, the PGRMC1 was expressed in the vicinity of the renal tubules. The results above demonstrated that PGRMC1 was a membrane receptor that functioned on the cell membrane. We also analyzed the protein sequence of PGRMC1, selected epitope of the sequence, and synthesized the corresponding immune polypeptide. We obtained the polyclonal antibody of PGRMC1 by immunizing New Zealand rabbit with the recombinant protein. The antibody was applied to measure the expression level of PGRMC1. Western blotting results showed that the expression of PGRMC1 protein was higher in the ovary, liver, and brain, and was relatively low in the kidney, head kidney, and pituitary. Our results may provide valuable information on the physiological function of PGRMC1 in the oocyte maturation and reproductive endocrinal regulation of C. semilaevis

Abstract:The neuroendocrine regulation of reproduction in vertebrates, including fish, is primarily controlled by the hypothalamo-pituitary-gonadal (HPG) axis, with each component secreting specific neuropeptides or hormones. A classic example of such regulation is the gonadotropin-releasing hormone (GnRH) system. It was believed that GnRH was the only hypothalamic stimulator of the release of pituitary gonadotropins, and that there was no inhibitory neuropeptide in the reproductive axis. However, this notion has been challenged by the recent discovery of a vertebrate hypothalamic neuropeptide that suppresses pituitary gonadotropins release. Gonadotropin-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide which was identified in 2000 from the brain of Japanese quail (Coturnix japonica). To date, GnIH orthologs have been isolated from fish to mammals and GnIH is the only reported inhibitor of reproduction in any vertebrate. GnIH acts on the pituitary and on GnRH neurons in the hypothalamus to decrease gonadotropin synthesis and release, inhibiting gonadal development and maintenance via a novel G-protein-coupled receptor (GPR147). It is well accepted that GnIH acts as an inhibitory factor in the control of reproduction in birds and mammals. However, the role of GnIH in the control of gonadotropin synthesis and release has been debatable in fish. In this study, we evaluated the effects of GnIH peptides on the reproduction-related gene expression in the hypothalamus of half-smooth tongue sole using a primary hypothalamus culture system for the first time. Our results showed that tsGnIH-1 increased the expression of gnrh2 and gnih mRNAs, but had no effects on gnrh3 or kiss2 mRNA expression. On the other hand, tsGnIH2 significantly inhibited gnrh3 mRNA expression. However, tsGnIH2 altered neither gnrh2, kiss2 nor gnih mRNA levels. Our findings suggested that GnIH peptides derived from the same precursor played different roles in the reproduction-related gene expression in tongue sole, and provided information for the future studies on the regulation of reproduction by GnIH peptides in fish.

Abstract:In order to clarify the physiological roles of gonadotropin-releasing hormone 2 (GnRH2) during ovarian maturation in half-smooth tongue sole (Cynoglossus semilaevis), we first cloned the full-length cDNA of GnRH2 by RT-PCR coupled with RACE. Then, the tissue distribution and changes in gnrh2 mRNA levels during ovarian maturation were evaluated by real-time quantitative PCR. Our data showed that the full-length cDNA of GnRH2 was 538 bp in size, excluding the poly-A tail. It consisted of a 5¢ untranslated region (UTR) of 154 bp, a 3¢ UTR of 126 bp and an open reading frame (ORF) of 258 bp which encoded an 85-amino acids preprohormone with a deduced molecular mass and isoelectric point of 9.69 kDa and 8.55, respectively. GnRH2 precursor contained a signal peptide, a mature decapeptide, a processing site, and a GnRH-associated peptide. Multiple sequence alignments indicated that GnRH2 preprohormones were highly conserved among teleosts, especially the decapeptide (QHWSHGWYPG) motif. Tongue sole GnRH2 precursor displayed the highest sequence identities with the order Perciformes (89.41%–90.59%), followed by Pleuronectiformes, Salmoniformes, and Tetraodontiformes (78.82%– 85.88%), and the lowest sequence identities with Cypriniformes (61.18%–71.76%). The tissue distribution showed that gnrh2 transcripts could be detected at the highest levels in the brain and at lower levels in the pituitary and other peripheral tissues. The developmental stages of ovaries were divided into five stages (Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ) by histological analysis and the expression patterns of gnrh2 mRNA during ovarian maturation were also investigated. In the brain, the mRNA expression of gnrh2 peaked at stage Ⅲ, declined sharply at stage Ⅳ and reached a minimum at stage Ⅴ. However, the brain gnrh2 mRNA levels ascended again at stage Ⅵ. The pituitary gnrh2 mRNA levels declined gradually, except for a temporary increase at stage Ⅴ. These data indicate that brain GnRH2 appears to be involved in ovarian maturation in the half-smooth tongue sole, and this study extends our knowledge of the roles of GnRH2 on the regulation of reproduction in fish.

Abstract:In order to deeply understand underlying mechanisms for regulating reproductive trait of Cynoglossus semilaevis, the physiological role of GH/IGF-Ⅰaxis in ovarian development was explored. The levels of growth hormone (GH) mRNA of pituitary and IGF-Ⅰ mRNA of liver, brain, and ovary at different ovarian development stages were determined by quantitative real-time PCR (qRT-PCR) analysis. Meanwhile, the expression levels of serum GH, IGF-Ⅰ, estradiol (E2) and testosterone (T) were detected by radioimmunoassay (RIA) and ELISA methods, respectively. Furthermore, the correlation between the mRNA and peptides expression levels of GH, IGF-Ⅰand gonadosomatic index (GSI), and the one between the expression levels of serum E2 and serum T during ovarian development was calculated with statistical methods. The results showed that, the levels of pituitary GH mRNA significantly increased at stage Ⅳ ovary, and peaked at stage Ⅴ ovary, but decreased significantly post spawning (stage Ⅵ). It has significantly positive relationship with the expression levels of GSI, serum GH and serum IGF-Ⅰ during ovarian development. The serum GH levels showed similar trend with pituitary GH mRNA expression levels. Liver IGF-Ⅰ mRNA achieved the minimum levels at stage Ⅳ and stage Ⅴ ovaries, but with a significant increase at stage Ⅵ ovary. It was significantly positive correlated with brain IGF-Ⅰ mRNA levels and serum IGF-Ⅰ levels, but significantly negative correlated with serum E2 and serum T levels during ovarian development. Brain IGF-Ⅰ mRNA levels significantly increased since stage Ⅱ ovary and peaked at stage Ⅳ ovary, and showed a significant negative relationship with serum IGF-Ⅰ levels. Ovary IGF-Ⅰ mRNA levels significantly increased at stage Ⅱ ovary and peaked at stage Ⅳ ovary, then significantly declined at stage Ⅴ ovary until stage Ⅵ ovary. It was significantly correlated with serum E2 levels but negatively correlated with serum IGF-Ⅰlevels. Serum IGF-Ⅰ levels showed similar trend with liver IGF-Ⅰ mRNA levels, which significantly decreased at stage Ⅳ ovary, but then significantly ascended to peak at stage Ⅵ ovary. These results revealed that GH and IGF-Ⅰ synergistically or antagonistically play physiological role in ovarian development of C. semilaevis in endocrine or paracrine ways, and their regulation functions are related with synthesis/secretion during ovary development regulation. The present results could provide a new clue for better understanding the possible mechanism for reproductive regulation of C. semilaevis.

Abstract:Hypermelanosis is an abnormal coloration that commonly occurs on the blind-side of half-smooth tongue sole Cynoglossus semilaevis in captivity, resulting in deterioration in flesh quality and reduced market price. This problem has become the bottleneck for sustainable development of flatfish farming industry. Melanin-concentrating hormone (MCH) is produced in pituitary acting as an inhibitor for hypermelanosis in fish. The present study aims to identify the role of MCH in regulating the blind-side hypermelanosis in C. semilaevis. cDNAs encoding two MCH alleles (pMCH1 and pMCH2) were cloned using RACE method and their structures were analyzed. The spatial and temporal expression patterns of MCH mRNA were measured using the quantitative real-time PCR. Results showed that C. semilaevis pMCH1 cDNA sequence was 476 bp in length and encoded 134 amino acids, which shares high identity (66.9%) with Paralichthys olivaceus. C. semilaevis pMCH1 was clustered with the Pleuronectiformes, Tetraodontiformes and Perciformes species based on the phylogenetic analysis. By contrast, C. semilaevis pMCH2 cDNA sequence was 626 bp in length and encoded 147 amino acids. The transcript levels of both MCH1 and MCH2 had the highest expression level in pituitary compared to other tissues. MCH1 mRNA was also detected among other tissues. Furthermore, MCH2 mRNA was highly expressed in the brain, eye-side skin, blind-side skin, gonad and gill, whereas the peripheral tissue had relatively low expression level. Correlating the MCH mRNA expression levels and the degree of the blind-side hypermelanosis showed that, MCH1 mRNA expression levels in the pituitary and skin had similar trends, which peaked when fish had about 10% blind-side hyperpigmentation, and then significantly reduced as the blind-side hypermelanosis level increased. For fish with normal blind-side coloration and 50% blind-side hyperpigmentation, their pituitary MCH2 mRNA levels were significantly higher than those from fish with 10% and 80% blind-side hyperpigmentation. The skin MCH2 mRNA level significantly increased with the increased hyperpigmentation degree on the blind-side of fish. The present study provides new insights into the mechanisms underlying the regulation of blind-side hypermelanosis in C. semilaevis.

Abstract:The full-length cDNA of melanin concentration hormone receptor (MCHR) was isolated from the brain of Cynoglossus semilaevis using RACE (rapid amplification of cDNA ends) method. The spatial and temporal expression patterns of MCHR mRNA in different tissues were analyzed to link with the degree of pigmentation on the blind-side of fish by the quantitative real-time PCR. Results showed that C. semilaevis MCHR1 cDNA sequence was 1685 bp in length and contained a 642 bp of open reading frame encoding 359 amino acids. It shared 83.3% identity with Paralichthys olivaceus. Based on the phylogenetic analysis, C. semilaevis MCHR1 was clustered with the Pleuronectiformes, Cyprinodontiformes and Perciformes species. In addition, C. semilaevis MCHR2 cDNA sequence was 1626 bp in length and contained a 1044 bp of open reading frame encoding 347 amino acids. The identity was more than 90% homology to pleuronectiformes species. C. semilaevis MCHR2 was clustered with the pleuronectiformes and perciformes species based on the phylogenetic analysis. MCHR1 mRNA was mainly expressed in gill and MCHR2 mRNA was primarily expressed in the eye-side skin, followed by gonad. The spatial expression patterns of MCHR mRNA implied that MCHR regulates the physiology of C. semilaevis through endocrine, paracrine and autocrine pathways. Pituitary MCHR1 mRNA level significantly increased during the initial stage of blind-side pigmentation and peaked when fish had 50% pigmentation on the blind-side. By comparison, skin MCHR1 mRNA maintained at high levels during the pigmentation occurring on the blind-side of fish. Both pituitary and skin MCHR2 mRNA levels peaked when fish had about 10% blind-side pigmentation, then significantly decreased as the degree of pigmentation reduced. The results showed that the MCHR is directly or indirectly involved in the regulation of blind-side hypermelanosis in half-smooth tongue sole.

Abstract:Blind-side hypermelanosis is a serious problem among intensively cultured Paralichthys olivaceus, resulting in significant economic losses. To date, no treatment is available to prevent this problem due to lack of knowledge. However, the degeneration of blind-side hypermelanosis was observed among pond-cultured P. olivaceus recently. To investigate the possible mechanisms, three types of P. olivaceus were identified based on the pigmentation on the blind side: normal blind-side type (NBS), blind-side hypermelanosis type (BSH), and blind-side degeneration type (BSD), and used for the comparison of morphological and associated endocrine changes at hormonal and mRNA levels. For BSD type, the number of melanocytes in the hypermelanosis-degenerated parts of blind side was reduced significantly comparing to those in the eye-side parts and in the blind-side parts in the BSD type. Moreover, the scales in the hypermelanosis-degenerated parts experienced the transformation from ctenoid to weak ctenoid, and cycloid shape. Meanwhile, the number of ctenoid scale spines on the blind- side decreased in hypermelanosis-degenerated parts among BSD fish. The plasma melanocyte-stimulating hormone (MSH) and melanin-concentrating hormone (MCH) levels were determined and compared between the three types of P. olivaceus. Our results showed that the BSD plasma MCH level was significantly higher than that of NBS and BSH, whereas the BSH had the highest MSH level among all three types of fish. Gene expression analysis showed that the BSD pituitary MCH mRNA level was remarkably higher than that of BSH, but for POMC mRNA level, the BSD was significantly low. Results from the present study significantly improved the understanding of the hyperpigmentation on the blind- side of P. olivaceus and help to develop the protocol to control this problem in aquaculture production.

Abstract:A healthy microbial community in digestive tract is critically important during early life stages of fish. To examine the microbial diversity in the gastrointestinal tract of Japanese flounder (Paralichthys olivaceus), we surveyed the intestinal bacteria of P. olivaceus during the larvae and juvenile stages. In this study, Illumina MiSeq of 16S rRNA and biological information analysis method were used to explore the intestinal bacterial community composition in indoor tank-culture system. Sampling was carried out throughout the larvae and juvenile stages for six time points. The obtained 7462 operational taxonomic units (OTUs) were classified into 42 bacterial species and 972 genera. The results indicated that larval fish (1 day-post-hatch; dph) displayed a distinct and diversified gut microflora, with Proteobacteria, Bacteroidete and Firmicutes dominating the community structure. During the age of 9 and 21 dph, Proteobacteria population increased significantly and remained to be the main species. By comparison, Firmicutes had fast turnover rate and increased sharply at 45 dph after feeding formulated food. The change of intestinal dominant microflora genus was also significant. The relative abundance of Vibrio was the highest during the period of live feed (9 and 21 dph sampling), and was the lowest level after feeding formulated feed (115 dph). By contrast, the Bacteroides and Prevotella became the predominant intestinal bacteria at 80 dph, whereas Firmicutes became a core microbiota at 80 dph. Blautia and other related genus colonized in the intestinal tract of P. olivaceus and became the dominant microbiota between 80 and 115 dph. The results revealed the change and establishment of microbiota during the transition stages from larvae to juvenile in response to live and formulated feed. Our results provided a database for analyzing the role of the intestinal micro ecological system of P. olivaceus at larvae and juvenile stages and have important implication for larval production of P. olivaceus.

Abstract:Good water quality is essential for the healthy growth of pond-cultured marine fish. Balanced microecological environment plays an important role in the maintenance of water quality and control of fish disease. To identify the mechanisms regulating the balance in microecological environment and to find specific and high-efficiency probiotics for pond culture of marine fish, here we tested two strains of bacteria isolated from sewage sludge of the estuary area of the Futuan River in Rizhao. They were identified as Lactobacillus plantarum and a Bacillus subtilis-like species according to the analysis such as morphological characteristics, biochemical properties, and 16S rRNA sequencing. The optimum growth conditions for L. plantarum and B. subtilis were pH 6.5/temperature 30℃ and pH 6.0/temperature 36℃ respectively. In the laboratory, the degrading effects of the mixed bacteria strains A and B (1:1) on ammonia nitrogen, nitrite, and phosphate in the seawater were 73.2%, 58.0%, and 52.4% respectively. Next, we examined the degrading effects of the bacteria in two outdoor marine ponds rearing Japanese flounder. One was designated as the experimental pond added with the probiotics (mixture of strain A and strain B), and the other one was the control. The applications of the probiotics (1:1, bacteria density 5×104–10×104 cell/ml) caused significant decrease in the concentrations of ammonia nitrogen, nitrite, and phosphate, and the degrading rates were 86.3%, 88.9%, and 68.3% respectively. Our results provided useful information for large-scale production of the two probiotics and for their application in aquaculture in the future.

Abstract:Seriola aureovittata, yellowtail kingfish, is a globally-distributed marine pelagic fish species. This fish has high market value because of its excellent taste and nutrition. Many countries including China have launched artificial culture programs of S. aureovittata. In order to better evaluate its nutritional value and edibility, we analyzed and compared the nutritional composition of the muscles of farmed and wild fish using national standard methods. It showed that S. aureovittata had high levels of crude protein, essential amino acids, and delicious amino acids in the muscle, which well complied with the ideal protein standard suggested by Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). This demonstrated that S. aureovittata was a good protein source for human. According to the amino acid score (AAS) and chemical score (CS) values, the first and second restriction amino acids of the fish were Met and Val respectively. We also found the crude fat content in the muscle of S. aureovittata was higher than that of other fish species such as salmon, tuna, and groupers. Moreover, the contents of unsaturated fatty acid, especially the DHA-EPA combination in the muscle were high, which guaranteed good tasty and high nutritional value. Furthermore, the high level of minerals in the muscle was greatly beneficial for human metabolism and health. It was also shown that there was no significant difference in nutritional composition between farmed and wild S. aureovittata, although the former had moderately higher crude fat content and lower water content. The EP values for farmed and wild S. aureovittata were 13.44 kJ/g and 8.68 kJ/g respectively. In conclusion, S. aureovittata is enriched in proteins and high quality fat, therefore should be a promising candidate fish species for large-scale aquaculture in the near future.

Abstract:Karyological study is a useful tool in exploring evolutionary mechanisms of fish because it provided basic information on the number, size and morphology of chromosomes. In this study we characterized the karyotype and chromosomal characteristics of the yellowtail kingfish Seriola aureovittata by examining metaphase spreads from head kidney cells of one-year-old fish artificially bred from wild broodstock in the Dalian coast of China. Chromosome samples were prepared through in vivo injection of phytohemagglutinin (PHA), olchicine-air drying technique, and Giemsa staining. A total of 80 mitotic metaphases from 21 individuals were analyzed, and 65 spreads showed 2n=48 chromosomes, which represented 81.25% of the observed metaphases. The relative size of the chromosomes in this subspecies varied from (2.50±0.23)% to (5.16±0.56)%. The long arm, short arm, and total length of the first submetacentric chromosomes were (1.81±0.09) μm, (0.87±0.03) μm, and (2.68±0.09) μm respectively. Two pairs of subtelocentric chromosomes were found in this subspecies. The length of their short arms was (0.55±0.04) μm and (0.39±0.06) μm, and that of the long arms was (2.10±0.06) μm and (1.87±0.07) μm. The diploid consisted of 4 submetacentric, 6 subtelocentric, and 38 telocentric chromosomes, and the fundamental number of chromosome arms was 54. The first pair of chromosomes had secondary constriction and satellite. The karyotype formula for this species was determined to be 2n=48 = 6sm + 4st + 38t. The total haploid chromosome length was approximately 51.97 μm. The fact that S. aureovittata has 3 pairs of submetacentric and 2 pairs of subtelocentric chromosomes distinguishes this species from other previously reported Seriola species. The comparison of karyotypes suggested that S. aureovittata might belong to an advanced and specific evolutionary group. Our study promoted the understanding of the specific karyotype evolution of the genus Seriola, and provided information on the polyploidy manipulation, hybridization, and sex control.

Abstract:为了全面认识黄条魳(Seriola aureovittata)生物学特性，利用传统测量方式、框架测度法、几何形态测量法和解剖学方法，观察和测度了其外部形态特征、可量与可数性状及内部结构特征，并模拟构建了黄条魳形态性状度量框架图。观察了黄条魳不同部位鳞片和耳石形态特征，比较了各形态度量性状的比值关系，发现全长与体长比值、下颌长与上颌长比值、尾柄长与尾柄高比值变异较小，表明这些性状关联密切。利用统计分析方法建立了黄条全长(TL)与体重(BW)之间的关系模型：BW=2.1652TL2– 140.35TL+2479.9 (R²=0.9812)，体高(BH)与体重间的关系模型：BW=0.7575BH3.0059(R²=0.9816)。研究分析表明，在黄条的12个可量形态性状中，除眼径外的其他11个形态性状间均存在显著相关关系。通径分析进一步揭示了体高和体长性状是影响体重的2个关键因素，其对体重的决定程度分别达41.34%和13.11%，它们对体重的共同决定程度达42.88%。本研究观察描述了黄条魳内部结构特征，其比肠长为0.62–0.69，脊椎骨数量为23–25，总出肉率可达75%。本研究结果可为黄条魳种质判别、系统分类及人工繁育与养殖技术开发提供形态认知依据。

Abstract:This study was designed to improve the understanding of the alimentary canal of Cynoglossus semilaevis. The alimentary canal of C. semilaevis can be divided into five sections, including oropharyngeal cavity, esophagus, esophagus and intestinal junction, foregut and hindgut. The structure and ultrastructure of alimentary canal of C. semilaevis were investigated using anatomical, histological and electronic microscopy methods. The number of mucosal folds, the height of mucosal folds, the thickness of muscle layers (longitudinal muscle and circular muscle), the density of mucus cells were measured and analyzed. The results showed that there was no obvious morphological stomach in C. semilaevis, evident by the lack of typical histological structure. However, a cystic structure, namly oesogaster, was formed in posterior part of esophagus following food intake, and oesogaster shrinked significantry during no food period. The principal structures of intestine canal include mucosal layer, submucosal layer, muscle layer and serosal layer. Oesogaster and intestine canal had little morphological difference based on electronic microscopic analysis. Each part of mucosal epithelial cell had mucus cells. Microvilli on the intestinal surface had no morphological difference. Microvilli of different sections, however, had differernces in length, with the longest on the foregut. The size and shape of mucus cells were also different. Top cytoplasm contained significant amount of mucous granules, while surface microvilli were relatively short, inward concaving and compact together. The goblet cells were found in the front segment of esophagus. In terms of mucous cell pattern, type Ⅲ mucous cell was the dominant cell type throughout the whole esophagus. Specifically, the mucous cells in oesogaster belonged to type Ⅲ, whereas foregut and hindgut segments mainly contained type Ⅳ and type Ⅱ mucous cells, respectively. The present study provides basic knowledge of digestive structure and mechanism of C. semilaevis and potentially can help to manufacture commercial diet for C. semilaevis.

Abstract:Pacific cod (Gadus macrocephalus) is a commercial potential species for marine fish aquaculture. The present study used a wild population of Gadus macrocephalus that was captured from the open sea of Weihai (Yellow Sea region). Under captivity, 49 wild individuals survived and acclimated to broodstock rearing conditions. Shortly, 15 batches of fertilized eggs were collected. Embryogenesis and early larval development of G. macrocephalus, including morphological features and development time, were described. G. macrocephalus spawned demersal and slightly adhesive eggs. Eggs were almost spherical and had no oil globules with diameter about 0.9–1.1 mm. The embryonic development of G. macrocephalus was divided into five stages, namely cleavage stage, blastula stage, gastrula stage, neurula stage as well as organogenesis stage. Embryos hatched out after 312 h 30 min in the sea water when the temperature was 9–10℃. The total length of newly hatched larva was (3.85±0.12) mm．Larva opened mouth at 6 days post hatching (dph) and started exogenous nutrition (first-feeding) stage. The yolk was completely absorbed at 8 dph. Primordial swimming bladder appeared at 6 dph and was filled with air at 16 dph. The first and second intestine physiological curvature formed at 12 dph and at 22 dph, respectively. This study provides the important baseline reference for broodstock management and larviculture of G. macrocephalus.

Abstract:In this study we tested the feasibility of coded wire tags (CWTs) as a novel high-efficiency tagging method for Japanese flounder (Paralichthys olivaceus). A total of three size groups were tested. The total lengths for small, medium, and large size groups were (5.92±0.41) cm, (8.92±0.36) cm, and (12.06±0.62) cm respectively. In a short-term experiment, we marked hatchery-reared juveniles with CWTs in the dorsal muscle and examined whether the fish size affected the tag retention and tagging-induced mortality. Tag retention was above 96% in all the three size groups and appeared to be unaffected by the fish size. Tag loss was only observed within 4 days post tagging. Tagging-induced mortality for small juveniles (13%) was significantly higher than that for the medium (4%) and the large (1%) juveniles. The tag shedding rates for the small, medium, and large juveniles were 3.3%, 2.4%, and 0.7% respectively. Linear dependence models were established to describe the relationships of tagging-induced mortality, the total length, and the body thickness. Death of small marked juveniles usually occurred within 4 days post tagging. In order to reduce the post-tagging mortality and biases in stocking assessment, we recommended CWT tagging on juveniles at least 6 cm in body length. The CWT tagging may have no effects on the growth and swimming behavior of the fish, therefore could be an ideal tagging method for Japanese founder juveniles. It is potentially a good tool in large-scale tagging and evaluation of Japanese flounder in the future.