Half-smooth Tongue Sole Cynoglossus Semilaevis Research Articles

Focal adhesion signaling pathway involved in skin immune response of tongue sole Cynoglossus semilaevis to Vibrio vulnificus infection

Focal adhesion (FA) plays a key role in cell adhesion, migration and antibacterial immune, but it remained unclear in fish. In this study, half-smooth tongue sole Cynoglossus semilaevis were infected with Vibrio vulnificus, and then immune-related protein in the skin, especially for FA signaling pathway were screened and identified by iTRAQ analysis. Results showed that the differentially expressed proteins (DEPs) in skin immune response (eg., ITGA6, FN, COCH, AMBP, COL6A1, COL6A3, COL6A6, LAMB1, LAMC1, FLMNA) were firstly found in FA signaling pathway. Furthermore, the validation analysis of FA-related genes were basically consistent with the iTRAQ data at 36 hpi (r = 0.678, p < 0.01), and their spatio-temporal expressions were confirmed by qPCR analysis. The molecular characterization of vinculin of C. semilaevis was described. This study will provide a new perspective for understanding the molecular mechanism of FA signaling pathway in the skin immune response in marine fish.

A saposin domain-containing protein of tongue sole Cynoglossus semilaevis: Antimicrobial activity and mechanism

Prosaposin is a precursor that can be processed into four different saposins, designated as A, B, C, and D, which have multiple functions in mammals, including neuroprotection and immune modulation. The immune function of saposin in teleost remains largely unknown. In the present study, a saposin (SAP) domain-containing protein was identified in half-smooth tongue sole Cynoglossus semilaevis and named CsSDP. CsSDP harbors one SAP A domain and two SAP B domains. When expressed in HEK293T cells, CsSDP was specifically localized in the lysosome. When overexpressed in Escherichia coli, CsSDP markedly inhibited bacterial growth, and the inhibitory effect depended on two specific regions in the SAP A and SAP B domains. Two polypeptides (P32 and P30) derived from the above SAP A and B domains could bind to and inhibit the growth of both Gram-negative and Gram-positive bacteria. The ultrastructural analysis revealed that P32 and P30 killed target bacteria by disrupting the bacterial cell wall and inducing substantial release of cytoplasmic contents. These results shed new lights on the immune function of saposin domain-containing protein in teleost.

Brain and Pituitary Transcriptome Analyses Reveal the Differential Regulation of Reproduction-Related LncRNAs and mRNAs in Cynoglossus semilaevis.

Long noncoding RNAs (lncRNAs) have been identified to be involved in half-smooth tongue sole (Cynoglossus semilaevis) reproduction. However, studies of their roles in reproduction have focused mainly on the ovary, and their expression patterns and potential roles in the brain and pituitary are unclear. Thus, to explore the mRNAs and lncRNAs that are closely associated with reproduction in the brain and pituitary, we collected tongue sole brain and pituitary tissues at three stages for RNA sequencing (RNA-seq), the 5,135 and 5,630 differentially expressed (DE) mRNAs and 378 and 532 DE lncRNAs were identified in the brain and pituitary, respectively. The RNA-seq results were verified by RT-qPCR. Moreover, enrichment analyses were performed to analyze the functions of DE mRNAs and lncRNAs. Interestingly, their involvement in pathways related to metabolism, signal transduction and endocrine signaling was revealed. LncRNA-target gene interaction networks were constructed based on antisense, cis and trans regulatory mechanisms. Moreover, we constructed competing endogenous RNA (ceRNA) networks. In summary, this study provides mRNA and lncRNA expression profiles in the brain and pituitary to understand the molecular mechanisms regulating tongue sole reproduction.

Complete genome analysis of a virulent Vibrio scophthalmi strain VSc190401 isolated from diseased marine fish half-smooth tongue sole, Cynoglossus semilaevis

BackgroundVibrio scophthalmi is an opportunistic bacterial pathogen, which is widely distributed in the marine environment. Earlier studies have suggested that it is a normal microorganism in the turbot gut. However, recent studies have confirmed that this bacterial strain can cause diseases in many different marine animals. Therefore, it is necessary to investigate its whole genome for better understanding its physiological and pathogenic mechanisms.ResultsIn the present study, we obtained a pathogenic strain of V. scophthalmi from diseased half-smooth tongue sole (Cynoglossus semilaevis) and sequenced its whole genome. Its genome contained two circular chromosomes and two plasmids with a total size of 3,541,838 bp, which harbored 3185 coding genes. Among these genes, 2648, 2298, and 1915 genes could be found through annotation information in COG, Blast2GO, and KEGG databases, respectively. Moreover, 10 genomic islands were predicted to exist in the chromosome I through IslandViewer online system. Comparison analysis in VFDB and PHI databases showed that this strain had 334 potential virulence-related genes and 518 pathogen-host interaction-related genes. Although it contained genes related to four secretion systems of T1SS, T2SS, T4SS, and T6SS, there was only one complete T2SS secretion system. Based on CARD database blast results, 180 drug resistance genes belonging to 27 antibiotic resistance categories were found in the whole genome of such strain. However, there were many differences between the phenotype and genotype of drug resistance.ConclusionsBased on the whole genome analysis, the pathogenic V. scophthalmi strain contained many types of genes related to pathogenicity and drug resistance. Moreover, it showed inconsistency between phenotype and genotype on drug resistance. These results suggested that the physiological mechanism seemed to be complex.

Effect of Halomonas storage poly‐β‐hydroxybutyrates on survival, growth and vibriosis resistance of half‐smooth tongue sole Cynoglossus semilaevis juveniles

Half-smooth tongue sole Cynoglossus semilaevis is a high-value marine fish species. The intensive farming of this flatfish species has been suffered from disease outbreak. Poly-β-hydroxybutyrate (PHB) is a carbon storage compound accumulated by a wide variety of prokaryotes and is considered biocontrol agent applied in aquaculture. In this study, the dietary effect of PHB-accumulating Halomonas (HM.PHB) was investigated on survival, growth, resistance on Vibrio anguillarum and intestine microbial composition of C. semilaevis juveniles. Halomonas was fermented, which contained 76% PHB on cell dry weight. The fish juveniles were fed formulated feeds containing 1% HM.PHB, 3% HM.PHB and 5% HM.PHB, and 3% particle PHB (Par.PHB) and 3% Halomonas containing no PHB (HM) for 6 weeks. The basal feed feeding was considered as the control. The results revealed that both Halomonas-PHB and particle PHB significantly improved the survival and growth of the juveniles than the control (p < .05), while HM was in between. When challenged with V. anguillarum, the survival rate in 1% HM.PHB, 3% HM.PHB and 5% HM.PHB was significantly improved than the control. Moreover, the increased intestine fold, epithelium height and surface were also observed in 3% HM.PHB. PCoA based on high-throughput sequencing of 16S rRNA gene fragment indicated that dietary Halomonas-PHB and particle PHB modified and stabilized the intestine microbial community of the fish. Our study demonstrated that Halomonas-PHB is an effective feed additive for C. semilaevis juvenile culture. The outcome of this study facilitates the application of Halomonas-PHB in C. semilaevis and other marine fish aquaculture.

Establishment and characterization of a new cell line derived from half-smooth tongue sole Cynoglossus semilaevis kidney

In this study, we established a new cell line (Cynoglossus semilaevis kidney cells, CSK) from kidney of the half-smooth tongue sole (Cynoglossus semilaevis). The cells were subcultured over 1 000 days and passaged for more than 100 times. Additionally, CSK cells were optimally maintained in Dulbecco’s modified Eagle medium nutrient mixture F-12 supplemented with HEPES, antibiotics, fetal bovine serum, 2-mercaptoethanol, and basic fibroblast growth factor. The optimum growth temperature for CSK cells was 25°C, and the cells showed a fibroblast-like phenotype. Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2n=42. CSK cells were susceptible to Grouper nervous necrosis virus (NNV), and cytopathic effects were observed at 3–5 days postinfection. The NNV sensitivity of CSK cells was related to the high abundance of virions in the cytoplasm, as observed by electron microscopy. Additionally, CSK cells could be successfully transfected with a green fluorescent protein reporter plasmid, and fluorescent signals were easily observed. Finally, immunocytochemistry analysis showed that CSK cells were supporting cells. Overall, we established this new cell line, which may have potential applications in the identification of viral pathogens affecting the half-smooth tongue sole.

Temporal expression profiles of leptin and its receptor genes during early development and ovarian maturation of Cynoglossus semilaevis.

Leptin (Lep) plays a key role in the regulation of food intake and energy homeostasis in vertebrates. Our previous studies have provided evidence for the existence of two leptin genes (lepa and lepb) and one leptin receptor (lepr) gene in a flatfish, the half-smooth tongue sole (Cynoglossus semilaevis). However, the spatial-temporal expression patterns and possible roles of the leptin system during early development and ovarian maturation are still poorly understood in teleosts. In the current study, we evaluated dynamic expression profiles of lepa, lepb, and lepr mRNAs during various developmental stages in this species. Quantitative RT-PCR analysis indicated that both ligand (lepa and lepb) and receptor (lepr) genes were detected in unfertilized eggs and during embryogenesis but with different expression profiles. In addition, lepa, lepb, and lepr transcripts levels increased significantly during larval development, reaching the peak at 10, 25, and 30 days post-hatching (dph), respectively. On the other hand, changes in mRNA expression of these three genes at the brain-pituitary-gonad (BPG) axis were also investigated during ovarian maturation, and lepa, lepb, and lepr mRNAs varied greatly. Taken together, our results encompass the first study reporting the dynamic expression patterns of leptin and its receptor mRNAs in the order Pleuronectiformes, providing evidence that the leptin system could be functional and play important roles during early development and ovarian maturation in tongue sole.

Isolation, characterization and expression analysis of TRPV4 in half-smooth tongue sole Cynoglossus semilaevis

The transient receptor potential vanilloid 4 (TRPV4), another Ca2+ entry channel, belongs to the vanilloid subfamily and responds to a number of different physical and chemical stimuli and exists widely in mammals. However, our understanding of the TRPV4 in fish remains poor. Therefore, we studied the TRPV4 gene from Cynoglossus semilaevis, named CsTRPV4 that encodes a putative protein of 870 amino acids common in structure and characteristic of mammalian TRPV4, including the domains of ANK repeats, six TM, TRP domain, and CaMBD. The CsTRPV4 was expressed ubiquitously in examined tissues: higher expression in the heart, spleen, testis, and eye, but lower expression in kidney and liver. Surprisingly, the expression of CsTRPV4 in lateral line was significantly higher than in many other tissues as the CsTRPV4 was expressed significantly in the free neuromasts. In addition, CsTRPV4 in the free neuromast from the larval fish was significantly expressed in the hair cells of the free neuromasts. Therefore, the free neuromasts can act as a mechano-sensor to the mechanical stimulation in molecular level in C. semilaevis, which lays a foundation for further study of the functions of the free neuromasts.

Extraction Optimization and Characterization of Gelatin from Half-Smooth Tongue Sole (Cynoglossus semilaevis Gunther) Skin

ABSTRACTHalf-smooth tongue sole is a popular warm-water fish, and its skin may be an available resource for gelatin extraction. Response surface method was used to optimize the extraction of gelatin from the fish skin. The most suitable conditions for maximum gelatin yield (24.2%) were pretreatment with 1.9 g/L NaOH, followed by hot water extraction at 63.54ºC for 4.81 h. The obtained gelatin had a high imino acid content (185 residues/1,000 residues), with a high content of crude protein and low levels of ash, fat, and moisture. The predominant sodium dodecyl sulfate–polyacrylamide gel electrophoresis bands revealed that the gelatin contained low-molecular-weight peptides, α-, β-, and γ chains. As compared with bovine gelatin, the fish skin gelatin solution had lower values of strength and clarity and higher levels of L* and viscosity. Additionally, Fourier-transform infrared spectra showed that gel from the gelatin had four amide bands (amide A, amide I, amide II, and amide III) similar to commercial gelatin. The results suggest that half-smooth tongue sole skin could be a promising source of gelatin in view of the comparability between its gelatin and commercial gelatin in physicochemical properties.

Genome-wide identification and prediction of long non-coding RNAs in half-smooth tongue sole Cynoglossus semilaevis

At present, the function and profile of long noncoding RNAs (IncRNAs) in half-smooth tongue soles Cynoglossus semilaevis remain poorly understood. Lherefore, we identified IncRNAs in the species using large-scale deep sequencing approaches. A total of 96 726 222 clean reads and 2497 IncRNAs were obtained from the compound samples. In total, 1 694 known IncRNAs and 803 novel IncRNAs were identified. Most of the novel IncRNAs distributed mainly in a range of 200–3 000 nt and contained 2–6 exons. Six novel IncRNAs were selected for expression analysis by quantitative reverse transcription-polymerase chain reaction, of which lnc_770 and lnc_150 were expressed mainly in the ovary. Lhis study provides a valuable resource for IncRNA studies of half-smooth tongue sole and improves our understanding of the function of non-coding RNA in fish.

Developmental expression of LPXRFa, kisspeptin, and their receptor mRNAs in the half-smooth tongue sole Cynoglossus semilaevis

Both gonadotropin-inhibitory hormone (GnIH) and kisspeptin play a critical role in the control of reproduction in vertebrates. However, the spatial–temporal expression patterns and possible roles of these two peptides during development are poorly understood. To this end, we evaluated dynamic expression profiles of lpxrfa, the piscine ortholog of gnih, kisspeptin (kiss2) and their receptor mRNAs during embryogenesis and early larval development in a flatfish, Cynoglossus semilaevis. Quantitative RT-PCR analysis indicated that both ligand (lpxrfa and kiss2) and receptor (lpxrfa-r and kiss2r) mRNAs were detected in unfertilized eggs and during embryogenesis, but with different expression profiles. In addition, lpxrfa-r, kiss2 and kiss2r transcripts increased significantly at 6 days post-hatching (dph), while lpxrfa mRNAs did not vary significantly during all larval development. Taken together, these results of the present study suggest that both LPXRFa and kisspeptin systems could be functional and play differential roles during embryogenesis and early larval development in tongue sole.

CDNA cloning and expression analysis of glutaredoxin (Grx) 2 in the Pacific white shrimp Litopenaeus vannamei

Glutaredoxin (Grx) is a class molecule oxidoreductase, which can regulate the redox state of proteins and plays a key role in antioxidant defense. However, the informations of Grx cDNA sequences and their functions are lack in decapod crustacea. In the present study, the cDNA of LvGrx 2 was cloned from the Pacific white shrimp, Litopenaeus vannamei. The open reading frame (ORF) of LvGrx 2 was 360 bp, which encoded a polypeptide of 119 amino acids. The molecular mass of the predicted protein is 12.87 kDa with an estimated pI of 8.22. Sequence alignment showed that the amino acid sequence of LvGrx 2 shares 59%, 59% and 58% identity with that of the coelacanth Latimeria chalumnae, the plateau frog Nanorana parkeri and the half-smooth tongue sole Cynoglossus semilaevis, respectively. Quantitative real-time PCR analysis revealed that LvGrx 2 were detected in a wide range of tissues, with highest expression in gill, hepatopancrea and intestine, and weakest expression in muscle. The expression responses of LvGrx 2 were analyzed in hepatopancrea and gill after ammonia-N stress or lipopolysaccharide (LPS) injection. During ammonia-N exposure, the LvGrx 2 transcriptions in hepatopancrea and gill significantly up-regulated, and the peak value appeared after 12 h and 24 h exposure respectively. After LPS injection, expression levels of LvGrx 2 in hepatopancrea obviously increased in the early and late stages, while LvGrx 2 transcription in gill sharply up-regulated in the middle period. These results suggest that LvGrx 2 may play a vital role in shrimp defense system against environmental stress and pathogen infection. RNA interference experiment was designed to further probe roles of LvGrx 2 during ammonia-N exposure. Ammonia-N induced obvious improvement in expression levels of LvGrx 2, LvGrx 3, GPx, GST and Trx, accompanied by increases of protein carbonyl and malondialdehyde (MDA) contents. However, transcription of GPx and GST were much weaker in LvGrx 2 interfered-shrimp, and oxidative damage in both lipid and protein were more serious. These results further suggest that LvGrx 2 in shrimp participates in oxidative defence and regulation of antioxidant system.

Structural and functional conservation of half-smooth tongue sole Cynoglossus semilaevis RIP3 in cell death signalling

Receptor interacting protein kinase 3 (RIP3) has emerged as an essential molecular regulator in human inflammation with accumulating evidence in vertebrates. However, the structure and functions of RIP3 in teleosts remains elusive. Here we identified one RIP3 homologue from half-smooth tongue sole Cynoglossus semilaevis, designated CsRIP3. The open reading frame (ORF) of CsRIP3 contained 1491 nucleotides and encoded a protein with 496 amino acids (Mw = 55.44 kDa). CsRIP3 expression was detected in various tissues in half-smooth tongue soles. CsRIP3 expression was up-regulated after pathogens i.e. Vibrio and poly (i:c) treatment in vivo, indicating its possible role in teleost immune response. Further analysis using human cells demonstrated that CsRIP3 exhibited highly conserved pro-apoptotic and pro-necroptotic functions in comparison with human RIP3. In conclusion, for the first time we reported the homologous structure and functions of CsRIP3 in inflammatory cell death, which provides novel perspectives on fish immunity studies in future.

Molecular Characterization and Expression and DNA Methylation Analyses of a Galectin-Related Protein Gene from Cynoglossus semilaevis

Galectins, a family of β-galactoside-binding proteins, participate in both innate immunity and adaptive immunity. This study identified one novel galectin-related protein from half-smooth tongue sole Cynoglossus semilaevis, which was designated as CsGRP. The full-length cDNA of its encoding gene was 785 bp in length with a 528 bp open reading frame encoding a putative protein of 176 amino acids. The deduced CsGRP contained a putative 131-aa galactoside-binding lectin domain and 3 critical residues responsible for carbohydrate binding (R93, W109 and R114). Genomic structural analysis revealed that CsGRP consisted of five exons and four introns. CsGRP showed 68% similarity with Poecilia latipinna GRP and 67% similarity with Stegastes partitus GRP. CsGRP showed the highest expression level in liver, although its expression was detected in all tested tissues. When challenged with Vibrio harveyi, the expression of CsGRP was significantly down-regulated in liver (P < 0.05). In addition, we found that in spleen and kidney of C. semilaevis, the CpG island of CsGRP showed significantly higher (P < 0.05) methylation level in disease-resistant family of C. semilaevis (DR-Cs) than in disease-susceptible ones (DS-Cs). Our results suggested that CsGRP may play important roles in the immune response of C. semilaevis. Moreover, DNA methylation patterns provided valuable data for understanding the relationship between epigenetic regulation and immunity, which would assist the animal genetic research and improve the animal breeding in the future.

Quality improvement of half-smooth tongue sole (Cynoglossus Semilaevis) fillets by chitosan coatings containing rosmarinic acid during storage

ABSTRACTThe effects of chitosan edible coating incorporated with rosmarinic acid on the quality of half-smooth tongue sole fillets were examined during 18 days of refrigerated storage (4 ± 0.5°C). The samples were analyzed by physicochemical, microbiological, and sensory characteristics. Results showed that samples coated with 1% chitosan containing 30 or 40 mg/L rosmarinic acid significantly lower the total volatile basic nitrogen, K value, free fatty acids, trimethylamine, total viable counts, and H2S-producing bacteria. Low field NMR analysis (LF-NMR) results indicated that chitosan-rosmarinic acid edible coating had positive effects on maintaining the water content and water migration. These results indicated that 1% chitosan coating with 30 mg/L rosmarinic acid was a potential alternative method to improve the quality of half-smooth tongue sole fillets during refrigerated storage.

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half-smooth tongue sole Cynoglossus semilaevis.

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD) and mucous-cell types was evaluated in the gastrointestinal tract of the half-smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH2·5) periodic acid Schiff (AB-PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB-) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS-AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.

Identification and characterization of a progestin and adipoQ receptor (PAQR) structurally related to Paqr7 in the ovary of Cynoglossus semilaevis and its potential role in regulating oocyte maturation

Membrane progestin receptors (mPRs) play an important role in the regulation of oocyte meiotic maturation in fish. However, details of the molecular endocrine mechanism regulating oocyte maturation in multiple spawning fish with asynchronous ovarian development remain unclear. The cDNA encoding a novel progestin and adipoQ receptor with structural similarity to mPRα (Paqr7), herein called Paqr7b, was cloned and sequenced from the ovary of half-smooth tongue sole Cynoglossus semilaevis. Phylogenetic analysis showed that Paqr7b represents an evolutionary intermediate between mPRα and mPRβ and shares high homology with other similar Paqr proteins in other teleost species. However, the tongue sole Paqr7b protein showed much greater homology to teleost mPRαs (average 52%) than mPRβs (average 40%), suggesting it may have arisen from gene duplication of mPRα. paqr7b and paqr7 mRNA exhibited similar patterns of tissue expression. The mRNA and protein of Paqr7b were ubiquitously detected in all tissues analyzed, including the ovary. Moreover, in situ hybridization results revealed that paqr7b was expressed in stage V oocytes, as well as in scattered cells in the pituitary. The expression of paqr7b mRNA in brain and ovary significantly increased from ovarian development stage II to stage V (P<0.05), and was maximal at stage V, and then sharply decreased at stage VI. The transcript level of paqr7b mRNA in the pituitary also peaked at stage V (P<0.05). Treatment of tongue sole ovarian follicles with gonadotropin consistently increased the expression level of Paqr7b protein and mRNA in both a dose- and stage-dependent manner. Microinjection of tongue sole oocytes with a morpholino antisense oligonucleotide to Paqr7b blocked the progestin induction of oocyte maturation. Our findings demonstrate an important role of Paqr7b in the regulation of oocyte maturation in tongue sole and suggest the receptor may also influence other aspects of reproduction, such as pituitary function.

Molecular characterization and expression analysis of Patched 1 gene in the half-smooth tongue sole (Cynoglossus semilaevis)

The signaling molecule hedgehog (Hh) is essential for cellular signaling required for body pattern formation in embryonic and individual development in vertebrate and invertebrates. Patched 1 (Ptc1), a receptor of Hh, mediates the activity of Hh by binging the Hh ligands. In this study, the Ptc1 of half-smooth tongue sole Cynoglossus semilaevis (CsPtc1) was cloned and characterized. The full-length of CsPtc1 cDNA is 5 212 nucleotides (nt), and encodes a protein with 1 543 amino acid residues. CsPtc1 shared many features with Ptc1 of other telesot species. Real-time quantitative PCR revealed CsPtc1 mRNA expressed in brain, liver, heart, gill, intestines, spleen, gonad and kidney, with expression level in testis significantly higher than in ovary. In testis, hybridization signals were mainly detected in primary spermatocytes, secondary spermatocytes, and sertoli cells, while weak signals were found in oocytes. Correspondingly, the degree of methylation is higher in female than in male and pseudomale. Results indicate that CsPtc1 may be involved in Desert Hedgehog (DHH) maintenance of the male and pseudo-male germ line and spermatogenesis.

Morphological variation and distribution of free neuromasts during half-smooth tongue sole Cynoglossus semilaevis ontogeny

This study was conducted to clarify the distribution and morphology of free neuromasts during the development of half-smooth tongue sole (Cynoglossus semilaevis) using scanning electron microscopy. During development, (1) the apical surface of free neuromasts changed in shape from a circle to a four-point star; (2) the external structure changed from being level with the epidermis to papilla-like above the level of the epidermis; (3) the neuromast cupula changed from cylindrical to blade-shaped; (4) the free neuromasts went from occurring singly to being in clusters of a few single organs; (5) the arrangement changed from a linear array to no discernable pattern; (6) there was a significant increase in the number of free neuromasts after metamorphosis. In adult C. semilaevis, free neuromasts were only observed on the abocular side of the head. Thus, there were more free neuromasts located on the abocular side of the head with a higher concentration around the anterior nostril and mouth, which may have a mechanical sensory function to help locate food as an adaptation to a benthic mode of life.

Location of Vibrio anguillarum resistance-associated trait loci in half-smooth tongue sole Cynoglossus semilaevis at its microsatellite linkage map

A cultured female half-smooth tongue sole (Cynoglossus semilaevis) was crossed with a wild male, yielding the first filial generation of pseudo-testcrossing from which 200 fish were randomly selected to locate the Vibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 cM in length with an average spacing of 7.68 cM, covering 72.07% of that expected (1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection (SHAVI) and Immunity of V. Anguillarum Infection (IVAI). Above a logarithm of the odds (LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identified. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci (QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.