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

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

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

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

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

Journal of Oceanology and Limnology - The transient receptor potential vanilloid 4 (TRPV4), another Ca2+ entry channel, belongs to the vanilloid subfamily and responds to a number of different...     

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

Journal of Oceanology and Limnology - At present, the function and profile of long noncoding RNAs (IncRNAs) in half-smooth tongue soles Cynoglossus semilaevis remain poorly understood. Lherefore,...     

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 discemable 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.     

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

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

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

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

Development of 57 novel polymorphic microsatellite markers in half-smooth tongue sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 ind...     

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℃,and the cells showed a fibroblast-like phenotype.Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2 n=42.CSK cells were susceptible to Grouper nervous necrosis virus(NNV),and cytopathic ef fects 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 af fecting the half-smooth tongue sole.     

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(C ynoglossus semilaevis) was crossed with a wild male, yielding the fi rst fi lial generation of pseudo-testcrossing from which 200 fi sh were randomly selected to locate the V ibrio 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 c M in length with an average spacing of 7.68 c M, 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. A nguillarum Infection(IVAI). Above a logarithm of the odds(LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identifi ed. 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.     

Effects of BPA and E2 on expression profiles of genes related to hypothalamic-pituitary-gonadal axis of half-smooth tongue sole Cynoglossus semilaevis

Endocrine disrupting chemicals (EDCs) are increasingly viewed as persistent pollutants, similar to natural hormones in function. This paper describes the expression profiles of 7 genes (DMRT, VTG, GnRHR, FSHR, CYP17A, CYP19A, and CYP19B) involved in sex steroid synthesis and action as well as sexual development in adult male and female Cynoglossus semilaevis, after exposure to different concentrations of Bisphenol A (BPA) and 17β-estradiol (E2). Both BPA (1, 10, 50, 125, and 250 mg/kg) and E2 (0.5, 5, and 10 mg/kg) induced changes in target gene expression, although the estrogenic effects of E2 as a model estrogen were stronger. Among the 7 genes, VTG, CYP17A and CYP19 responded strongly to BPA or E2 exposure and can thus serve as reference biomarkers for estrogenic EDCs exposure in marine teleosts. These data will provide a window to establish a hypothalamic-pituitary-gonadal model in C. semilaevis to better understand the effect pathways of EDCs.     

Identification of two major histocompatibility (MH) class II A genes and their association to Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex class II antigens are important in vertebrate immune system.In the present study,the full cDNA sequence of class II A gene was synthesized by RACE-PCR from half-smooth tongue sole(Cynoglossus semilaevis),and its open reading frame(ORF) polymorphism was studied.The whole cDNA sequence was 992 bp in length,including the ORF with 717 bp.Twenty-five alleles were identified and clustered into two distinct groups according to the specific nucleotides/amino acids in specific positions.Eleven alleles belonged to Cyse-DAA while fourteen alleles belonged to Cyse-DBA.Four Cyse-DAA alleles were observed in one individual,and three to five Cyse-DBA alleles were observed in each of the three detected individuals,which indicated that at least two loci existed in each gene.Moreover,in order to study the function of the alleles in resistance to infection,200 individuals were intraperitoneally injected with Vibrio anguillarum and the first 20 dead individuals and 20 surviving ones were selected for genotype analysis.Fifty-six alleles were identified among the 40 individuals.Twenty-nine alleles belonged to Cyse-DAA and the other 27 alleles belonged to Cyse-DBA.Eighteen alleles were selected for studying their function in resistance to infection.Alleles Cyse-DAA*0201,Cyse-DAA*1101,Cyse-DBA*0401,Cyse-DBA*1102,Cyse-DBA*1801 and Cyse-DBA*2201 were identi-fied only in surviving individuals,while alleles Cyse-DAA*0901,Cyse-DBA*1101 and Cyse-DBA*1401 occurred more frequently in dead individuals.This study confirmed the existence and polymorphism of two class II A genes as well as the relationship between alleles of class II A genes and disease susceptibility/resistance in half-smooth tongue sole.     

Sequence polymorphism of two major histocompatibility (MH) class II B genes and their association with Vibrio anguillarum infection in half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex (MHC) class Ⅱ B molecules play an important role in the adaptive immune response in fish. Previous study has reported that two highly polymorphic class ⅡB genes, Cyse-DAB and Cyse-DBB exist in half-smooth tongue sole (Cynoglossus semilaevis). In this study, the polymorphism within exon 2 of the class Ⅱ B genes following bacterial challenge was evaluated. Two hundred C. semilaevis individuals were injected intraperitoneally with Vibrio anguillarum. Muscle tissue from the first 20 dead and 20 of the survivors was collected for genotyping. Sixty alleles from the 40 individuals were isolated, of which 32 belonged to Cyse-DAB and 28 belonged to Cyse-DBB. The rate of dN (non-synonymous substitution) was higher than that of dS (synonymous substitution) in the PBRs (peptide binding residues) of both class Ⅱ B genes. Conversely, the rate of dS was higher than dN in the non-PBRs and the complete exon 2 sequence. Thus, the results suggest that positive selection has occurred in the PBRs and purifying selection in the non-PBRs and exon 2. Thirteen class Ⅱ B alleles were used to study the association between alleles and resistance to infection. Though not significant, alleles Cyse-DAB*0601, Cyse-DAB*0706, and Cyse-DBB*0101, Cyse-DBB*1301 were only found in surviving individuals and may represent alleles that have resistance against V. anguillarum infection. Alleles Cyse-DAB*0701 and Cyse-DAB*1301 were significantly more prevalent in dead individuals than in surviving ones and may represent alleles that are associated with increased susceptibility to V. anguillarum infection.     

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

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

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

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

Sensory bases and the mode of feeding behavior of tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

We conducted behavior experiments on the roles of vision, olfaction, and the lateral line in feeding in tongue sole Cynoglossus semilaevis Günter. Chemical cues from the prey alone could not elicit any searching or attacking behavior by the fish; only when moving foods were presented, the chemical nature of the foods affected feeding. In addition, we found that normal tongue sole did not accurately recognize moving artificial prey of different shapes. Neither chemosense nor vision plays an important role in prey detection. Reactions to moving objects and the response of the oropharyngeal cavity to different foods showed that the (eyeless) tongue sole use mainly the lateral line for prey detection and capture. Gustation in the oropharyngeal cavity is essential for the fish to swallow its prey.     

Analysis of phenotypic and genetic parameters for growthrelated traits in the half smooth tongue sole, <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

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

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

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

Analysis of SSRs derived from an EST library of half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

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