Search

Blind-side hypermelanosis has emerged as a major concern in flatfish aquaculture worldwide, including tongue sole (Cynoglossus semilaevis) in China. The causative gene and the molecular basis are still unclear. In this study, comparative transcriptome analyses were performed using different skin tissues of tongue sole: ocular-side normal (pigmented) skin, blind-side normal (non-pigmented) skin and blind-side hypermelanotic (pigmented) skin. Finally, 60 key hypermelanosis-related genes were mined, providing potential candidate gene resources involved in blind-side hypermelanosis. These genes were selected based on the log2(FoldChange) and false discovery rate (FDR) values (with corresponding P-Values

Background: In recent years, the disease outbreak caused by Vibrio harveyi upset the booming development of the Chinese tongue sole (Cynoglossus semilaevis) farming industry. Genomic selection (GS) is a powerful method to improve the traits of interest, which has been proved in livestock and some fishes. Besides, the single nucleotide polymorphism (SNP) array is an efficient genotyping platform that can be used for genetic studies. To improve V. harveyi resistance in C. semilaevis, we firstly constructed a reference group of 1,572 individuals and investigated accuracies of four genomic methods (genomic best linear unbiased prediction (GBLUP), weighted GBLUP, BayesB, and BayesC) at predicting the genomic estimated breeding value (GEBV) using five-fold cross-validation and SNPs varying from 0.5 k to 500 k. Then, an SNP array was developed using the Affymetrix Axiom technology, and its accuracy in genotyping was evaluated by comparing SNPs generated by the array and by the re-sequencing technology. Finally, we selected 44 candidates as the parents of 23 families of C. semilaevis to evaluate the feasibility of the SNP array for GS.Results: all genomic methods outperformed the pedigree-based BLUP (ABLUP) when at least 50 k SNPs used for prediction, of which GBLUP resulted in better estimation than ABLUP when more than 1 k SNPs used. A 38 k SNP array, “Solechip No.1”, was developed with an average of 10.5 kb inter-spacing between two adjacent SNPs. The SNPs generated by the array and by the re-sequencing reached an average consistency of 94.8 %, of which 79.3 % of loci had a more than 90 % of the consistency. The survival rates of these 23 offspring families had a correlation of 0.706 with the family GEBVs (mid-parental GEBVs), and the average survival rate of the top five families in GEBVs (79.1 %) is higher than the bottom five families (58.1 %).Conclusion: GS is an efficient method to improve the V. harveyi resistance in C. semilaevis, and the SNP array “Solechip No.1” is a convenient and reliable tool for the Chinses tongue sole selective breeding practice.

Single nucleotide polymorphism (SNP) arrays are a powerful genotyping tool used in genetic research and genomic breeding programs. Japanese flounder (Paralichthys olivaceus) is an economically-important aquaculture flatfish in many countries. However, the lack of high-efficient genotyping tools has impeded the genomic breeding programs for Japanese flounder. We developed a 50K Japanese flounder SNP array, “Yuxin No. 1”, and report its utility in genomic selection (GS) for disease resistance to bacterial pathogens. We screened more than 42.2 million SNPs from the whole-genome resequencing data of 1099 individuals and selected 48 697 SNPs that were evenly distributed across the genome to anchor the array with Affymetrix Axiom genotyping technology. Evaluation of the array performance with 168 fish showed that 74.7% of the loci were successfully genotyped with high call rates (> 98%) and that the polymorphic SNPs had good cluster separations. More than 85% of the SNPs were concordant with SNPs obtained from the whole-genome resequencing data. To validate “Yuxin No. 1” for genomic selection, the arrayed genotyping data of 27 individuals from a candidate population and 931 individuals from a reference population were used to calculate the genomic estimated breeding values (GEBVs) for disease resistance to Edwardsiella tarda. There was a 21.2% relative increase in the accuracy of GEBV using the weighted genomic best linear unbiased prediction (wGBLUP), compared to traditional pedigree-based best linear unbiased predictions (ABLUP), suggesting good performance of the “Yuxin No. 1” SNP array for genomic selection. In summary, we developed the “Yuxin No. 1” 50K SNP array, which provides a useful platform for high-quality genotyping that may be beneficial to the genomic selective breeding of Japanese flounder.

Chinese tongue sole (Cynoglossus semilaevis) is an important marine flatfish which is widely cultured in coastal areas in China. Infectious bacterial diseases such as Edwardsiella tarda imposed serious threats to this species. A potential strategy to prevent this disease is to select resistance strains. The aim of the present work was to estimate the genetic variation of disease resistance to E. tarda and to evaluate the genetic correlations between resistance traits and growth traits. Three types of models were fitted by using different trait definitions (binary, continuous and categorical). After a 9-day challenge test, the overall survival was 75.4% (ranging from 6.4% to 100% in families), and at test day 6, the overall survival was 50.7%. We set test day 6 and 9 as cut-off point times respectively. The heritabilities of survival traits were ranging from 0.10 to 0.36. Considerably higher heritability values were obtained at day 6 than at day 9, regardless of which model or trait definition used (except Trait 2 in LIN). The genetic correlations between disease resistance traits and growth traits (i.e. body weight and total length) were low and not significant from zero (−0.12–0.24). There is a substantial re-ranking of families when defined resistance as categorical and continuous traits compared to binary trait. These results confirm the existence of genetic variation for resistance against E. tarda and weak genetic correlations indicate that joint genetic improvement of E. tarda resistance and growth is scarcely available.

As one of the most valuable maricultured species, spotted knifejaw (Oplegnathus punctatus) has high popularity in eastern Asia. In recent years, diseases caused by Vibrio harveyi have brought huge economic losses in spotted knifejaw industry. To better understand the molecular mechanisms of immune response about V. harveyi resistance in spotted knifejaw, a comparative transcriptome analysis was performed on spleen tissues at five different time points post-infection (0, 12, 24, 48 and 72 hpi). A total of 4279 differentially expressed genes (DEGs) were identified. KEGG pathways analysis showed that multiple immune-related pathways were significant regulated, including Toll-like receptor signaling pathway, ECM-receptor interaction pathway, cytokine-cytokine receptor interaction pathway and hematopoietic cell lineage pathway. Weighted gene co-expression network analysis showed that several immune-related pathways of the highest correlation with 12 hpi (cor = 0.89, P = 7e-06) were significantly enriched. In addition, 12 hpi was a turning point for 7 gene clusters out of 9 that were divided according to gene expression patterns. Therefore, we speculated that 12 hpi might be a very critical time point for spotted knifejaw against V. harveyi infection. Additionally, qRT-PCR was carried out to validate the expressions of 12 DEGs. This study provided the first systematical transcriptome analysis of spotted knifejaw against V. harveyi. The results could help us better understand the dynamic immune responses of spotted knifejaw against bacterial infection, and provide useful information for antibacterial defense in spotted knifejaw industry as well.

Blind-side hypermelanosis has emerged as a major concern in commercial rearing environments of the flatfish aquaculture industry. To date, the underlying molecular mechanisms are not well understood. To fill this gap, in this study, whole transcriptomic sequencing and analyses were performed using normal skins and hypermelanic skins of the blind side of Chinese tongue sole (

The aim of this study was to evaluate genetic parameters of growth traits for improving selective breeding in olive flounder (Paralichthys olivaceus). A total of 82 full-sib families from 2 year-classes with complete pedigree information were used to estimate heritabilities and genetic/phenotypic correlations for 4 growth performance traits (body weight BW, total length TL, body height BH and condition factor K) based on uni-trait and multi-trait linear mixed models, respectively. Data were analyzed by the restricted maximum likelihood (REML) method. The heritabilities of BW, TL and BH were 0.81, 0.70 and 0.61, which are relatively high, however, heritability for K is low (0.11). For all growth traits, the common full-sib effects (c2) were at a low to moderate level (0.14–0.20); the maternal effects (m2) of the traits (except K) were also detected (0.15–0.19). The phenotypic and genetic correlations among BW, TL and BH were highly positive (0.70–0.91), however, the correlations between K and the other 3 traits was low or negligible (−0.22–0.26). According to our results, these traits (except K) could be selected and enhanced simultaneously. In addition, direct selection based on TL or BH, which is an easily measurable trait, will be more favorable than selection on BW. Moreover, our results indicated that growth parameters estimated in the juvenile stage could be used as a predictor of growth performance at later growth stages. Keywords: Genetic parameter, Growth trait, Heritability, Genetic correlation, Olive flounder, Paralichthys olivaceus

Recently, Vibrio harveyi has emerged as an important pathogenic disease with a high mortality rate (50%–70%) in Chinese tongue sole. The aim of this study was to estimate genetic parameters for resistance to V. harveyi in Chinese tongue sole and to evaluate the genetic correlation between resistance and growth traits (i.e. body weight and total length) (involving 50 full-sib families and 8547 individuals). Longitudinal linear models and cross-sectional threshold models were fitted by using different trait definitions (binary and categorical). After a 14-day test, the overall challenge test survival was 57.86% (ranging from 9.30% to 94.30% in families). The heritabilities of survival were ranging from 0.11 to 0.28, estimates obtained by linear models were higher than threshold models. The genetic correlations between resistance (binary and categorical traits) and two growth traits were moderately positive (0.27–0.51). Very high Pearson and Spearman correlations (0.99 to 1) of full-sib family EBVs between different models were all close to unity which might reveal the similar predictive ability of the four models. The favourable heritabilities and moderate positive genetic correlations indicate that joint genetic improvement of vibriosis resistance and growth performance would be feasible. Statement of relevance This paper offers guidelines to disease-resistance selective breeding strategies in Chinese tongue sole.

The phenomenon of sex dimorphism prevails among many fish species. It has attracted the general research interest due to its close relationship to fish growth and thus aquaculture productivity. In Chinese tongue sole (Cynoglossus semilaevis), 17β-estradiol (E2) can be used reportedly to induce the feminization of fish, although the detailed regulatory network remained elusive. We treated the tongue sole fry before sex differentiation with E2 (10 and 30 µg/L) to identify potential targets of E2 in Chinese tongue sole. The E2 treatment indeed induced genetic male fish sex-reversal to phenotypic female. Using an iTRAQ-based comparative proteomic analysis, 409 proteins that differentially expressed after E2 induction were identified, including 259 up-regulated and 150 down-regulated proteins. Furthermore, 19 differentially expressed proteins identified in the comparative proteomic analysis were selected to assess their transcription and eight of them exhibited the same tendency. Functions of the eight proteins included mainly nucleotide and protein binding. Interestingly, a far upstream element-binding protein 3-like isoform exhibited the significant upregulation both at transcription and translation levels after E2 treatment. This work identified a set of candidate proteins for E2 response and deepened our understanding of E2 regulatory mechanism.

The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.

The pore-forming protein perforin is one of the effectors of cell-mediated killing via the granule exocytosis pathway. In this study, a genome-wide association study was conducted in Vibrio harveyi disease-resistant and disease-susceptible families of half smooth tongue sole (Cynoglossus semilaevis) to determine the genes accounting for host resistance, and a perforin homologue was identified, designated perforin-1 like (CsPRF1l). The full-length cDNA of CsPRF1l is 1835 bp, and encodes 514 amino acids. The CsPRF1l gene consists of 10 exons and 9 introns, spanning approximately 7 kb. The amino acid sequence of CsPRF1l shows 60.35, 54.03, 41.92, and 34.17% identities to Morone saxatilis PRF1l, Oryzias melastigma PRF1l, Danio rerio PRF1.5 and Homo sapiens PRF, respectively. Sequence analysis revealed the presence of membrane attack complex/perforin (MACPF) and C2 domains in CsPRF1l. Quantitative real-time PCR showed that CsPRF1l presented a higher intestinal expression level in disease-resistant families than in susceptible families. Tissue expression pattern analysis showed that CsPRF1l is present in most of the tested tissues and highly expressed in the intestine, brain, stomach and gills. After challenge with V. harveyi, CsPRF1l mRNA was markedly upregulated in the liver, spleen, kidney, intestine, gills and skin. In addition, the recombinant CsPRF1l protein exhibited obvious antimicrobial activity against V. harveyi in vitro and in a zebrafish model. Collectively, these data indicate that CsPRF1l modulates host immune defense against V. harveyi invasion and provide clues about the efficacy of rCsPRF1l in fish that will give rise to useful therapeutic applications for V. harveyi infection in C. semilaevis.

Background Edwardsiella tarda causes acute symptoms with ascites in Japanese flounder (Paralichthys olivaceus) and is a major problem for China’s aquaculture sector. Genomic selection (GS) has been widely adopted in breeding industries because it shortens generation intervals and results in the selection of individuals that have great breeding potential with high accuracy. Based on an artificial challenge test and re-sequenced data of 1099 flounders, the aims of this study were to estimate the genetic parameters of resistance to E. tarda in Japanese flounder and to evaluate the accuracy of single-step GBLUP (ssGBLUP), weighted ssGBLUP (WssGBLUP), and BayesB for improving resistance to E. tarda by using three subsets of pre-selected single nucleotide polymorphisms (SNPs). In addition, SNPs that are associated with this trait were identified using a single-SNP genome-wide association study (GWAS) and WssGBLUP. Results We estimated a heritability of 0.13 ± 0.02 for resistance to E. tarda in Japanese flounder. One million SNPs at fixed intervals were selected from 4,978,724 SNPs that passed quality controls. GWAS identified significant SNPs on chromosomes 14 and 24. WssGBLUP revealed that the putative quantitative trait loci on chromosomes 1 and 14 contained SNPs that explained more than 1% of the genetic variance. Three 50 k-SNP subsets were pre-selected based on different criteria. Compared with pedigree-based prediction (ABLUP), the three genomic methods evaluated resulted in at least 7.7% greater accuracy of predictions. The accuracy of these genomic prediction methods was almost unchanged when pre-selected trait-related SNPs were used for prediction. Conclusions Resistance to E. tarda in Japanese flounder has a low heritability. GWAS and WssGBLUP revealed that the genetic architecture of this trait is polygenic. Genomic prediction of breeding values performed better than ABLUP. It is feasible to implement genomic selection to increase resistance to E. tarda in Japanese flounder with 50 k SNPs. Based on the criteria used here, pre-selection of SNPs was not beneficial and other criteria for pre-selection should be considered.

Tongue sole is an economically important marine flatfish with significant sexual growth dimorphism that female with superior growth performance, and phenotypic female proportion (FP) is rather low (10%–30%) under rearing conditions. Knowledge of whether genetic variation exists for FP is needed for determining the feasibility of including this desirable trait into breeding goal in tongue sole. The aims of this study were to estimate the heritability of FP and genetic correlations between FP and growth traits (body weight, BW; total length, TL) by using a population of 16,240 individuals from 157 full-sib families (involving four year-classes (YCs), three consecutive pedigreed generations). At juvenile stage, fish from each family were random sampled to measure BW and TL, and then sexed by examining gonad tissues under microscope. The overall FP was 43.8% across all year-classes, and family FP ranges from 25.4% to 69.9%. Across YCs, there was a low but significant genetic variation for FP with heritability estimates of 0.02 ± 0.00 (observed scale) and 0.03 ± 0.01 (underlying scale). Within YC, heritability estimates varied from 0.00 ± 0.00 to 0.08 ± 0.01 on observed scale, and from 0.00 ± 0.00 to 0.04 ± 0.01 on underlying scale. Genetic correlations between FP and BW, and between FP and TL were both of moderately positive magnitude, which were 0.46 ± 0.09 and 0.39 ± 0.10 respectively, and significantly different from zero (P

The Japanese flounder is one of the most widely farmed economic flatfish species throughout eastern Asia including China, Korea, and Japan. Edwardsiella tarda is a major species of pathogenic bacteria that causes ascites disease and, consequently, a huge economy loss for Japanese flounder farming. After generation selection, traditional breeding methods can hardly improve the E. tarda resistance effectively. Genomic selection is an effective way to predict the breeding potential of parents and has rarely been used in aquatic breeding. In this study, we chose 931 individuals from 90 families, challenged by E. tarda from 2013 to 2015 as a reference population and 71 parents of these families as selection candidates. 1,934,475 markers were detected via genome sequencing and applied in this study. Two different methods, BayesCπ and GBLUP, were used for genomic prediction. In the reference population, two methods led to the same accuracy (0.946) and Pearson's correlation results between phenotype and genomic estimated breeding value (GEBV) of BayesCπ and GBLUP were 0.912 and 0.761, respectively. In selection candidates, GEBVs from two methods were highly similar (0.980). A comparison of GEBV with the survival rate of families that were structured by selection candidates showed correlations of 0.662 and 0.665, respectively. This study established a genomic selection method for the Japanese flounder and for the first time applied this to E. tarda resistance breeding.

Despite Japanese flounder ( Paralichthys olivaceus ) is of importance in aquaculture of Northeast Asia, selective breeding programs for growth performance traits remain limited. The aim of this study was to estimate parameters of growth performance traits in this species. The heritabilities, genetic and phenotypic correlations, and breeding values were estimated for body weight (BW), total length (TL), condition factor (K), and average daily gain (ADG). The genetic analyses were performed on a total number of 186 full-sib families (108 sires and 146dams, 16,807 individuals with integrated pedigree information) from 4 year-classes based on three mixed animal models with tank and year-class as fixed effects, mean family trait values at tagging as a covariate, and additive genetic effect as a random effect using the restricted maximum likelihood method. For all growth traits the maternal and sir-dam interaction effects in proportion to phenotypic variance were very low (0.00–0.05). The heritability estimates for growth traits ranged from 0.12 to 0.39, which is in a low to moderate level. These results indicated that there is a considerable additive genetic variation in growth traits, and the ongoing selective breeding project will produce considerable genetic improvement in growth traits of Japanese flounder. The genetic and phenotypic correlations among BW, TL and ADG were high and positive (0.87–0.94, P Statement of relevance This paper offers guidelines to select breeding strategy in Japanese flounder.

Flatfish is an important group worldwide aquaculture species, and hypermelanosis on blind side is one of the prevalent and economically important problem which drops market price significantly. Knowledge of whether genetic variation exists for hypermelanosis is needed in order to determine the feasibility of including this trait into the breeding goal. Therefore, the objective of this study was to detect the magnitude of the heritable variation and estimate the heritability for blind-side hypermelanosis by using Chinese tongue sole (Cynoglossus semilaevis). Pigmentation on blind side was defined as three types and fitted in three models respectively, i.e., (i) continuous trait (proportion of non-pigmented area to total area of the blind side) fitted in a linear model (LM); (ii) binary trait (the melanistic scored 1 and the pure white scored 0 by eyes) fitted in cross-sectional threshold model (CTM); (iii) ordinal trait (four categories based on the extent of pigmentation) fitted in ordinal threshold model (OTM). The estimated heritability on observed scale was 0.34 (LM), and on underlying scale was 0.34 and 0.38 for CTM and OTM respectively. All the heritability estimates are associated with large standard errors (SEs) and not significantly different from zero. Anyway, these results point to the existence of genetic variation of blind-side hypermelanosis and the potential for future selective breeding. The greater than zero heritability estimates imply that the selection response is expected by selective breeding in flatfish. Further studies using bigger population data (large family numbers and sample size) are highly recommended.

Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9 cM corresponding to a 1.8 M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.

Flatfish have the most extreme asymmetric body morphology of vertebrates. During metamorphosis, one eye migrates to the contralateral side of the skull, and this migration is accompanied by extensive craniofacial transformations and simultaneous development of lopsided body pigmentation(1-5). The evolution of this developmental and physiological innovation remains enigmatic. Comparative genomics of two flatfish and transcriptomic analyses during metamorphosis point to a role for thyroid hormone and retinoic acid signaling, as well as phototransduction pathways. We demonstrate that retinoic acid is critical in establishing asymmetric pigmentation and, via cross-talk with thyroid hormones, in modulating eye migration. The unexpected expression of the visual opsins from the phototransduction pathway in the skin translates illumination differences and generates retinoic acid gradients that underlie the generation of asymmetry. Identifying the genetic underpinning of this unique developmental process answers long-standing questions about the evolutionary origin of asymmetry, but it also provides insight into the mechanisms that control body shape in vertebrates. National Natural Science Foundation of China [31130057, 31461163005, 31530078, 31472269, 31472262, 31472273]; State 863 High Technology R&D Project of China [2012AA092203, 2012AA10A408, 2012AA10A403-2]; Education and Research of Guangdong Province [2013B090800017]; Taishan Scholar Climb Project Fund of Shandong of China; Taishan Scholar Project Fund of Shandong of China for Young Scientists; Shanghai Universities First-class Disciplines Project of Fisheries; Program for Professor of Special Appointment (Eastern Scholar) at the Shanghai Institutions of Higher Learning; Shanghai Municipal Science, Special Project on the Integration of Industry info:eu-repo/semantics/publishedVersion

Dmrt2 is a member of the dmrt gene family with a conserved zinc finger-like DNA-binding motif (DM domain). In the present study, CS-dmrt2 was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of CS-dmrt2 is 2834 bp in length, with a 251 bp 5′-untranslated region (UTR), a 1086 bp 3′-UTR and a 1503 bp open reading frame (ORF) that encodes a 501-amino-acid peptide. qPCR revealed that CS-dmrt2 was mainly expressed in C. semilaevis testes. In situ hybridization (ISH) showed CS-dmrt2 expression throughout early gonadal development (36 days after hatching (dah) and 86 dah), but the expression was higher in male gonads than in female gonads. CS-dmrt2 mRNA was highly expressed in male germ cells. Comparison of methylation levels between females and males demonstrated hypo-methylated levels of the CS-dmrt2 promoter in the male gonads, which is consistent with the high mRNA expression. These results suggest that the CS-dmrt2 gene may play a functional role in gonadal differentiation/development and germ cell maturation in the testis.

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine flatfish which is now severely threatened by various bacterial pathogens (especially at juvenile stage) in China. As we all known, it is of great importance to increase the natural disease resistance of farmed fish. So, the aim of this study was to verify the exitance of genetic variance of natural disease resistance and to detect the selection response by using juvenile natural survival data (involving four year-classes and three generations with 221 full-sib families, 195,589 individuals). Survival was defined as binary trait (dead/alive) fitted in two cross-sectional models (i.e. cross-sectional linear sire-dam model (CLM) and cross-sectional threshold (logit) sire-dam model (CTM)). Heritabilities of survival were estimated with each generation dataset and with complete dataset. Heritability estimates varied among generations regardless of model used, i.e., 0.01–0.17 and 0.03–0.25 for CLM and CTM respectively. On the observed (CLM) and underlying (CTM) scale with complete dataset, the heritabilities were 0.09 ± 0.04 and 0.13 ± 0.06 respectively. Both models performed nearly identical and very high selection accuracy (> 0.99), the accuracy of selection obtained from CLM (0.993) was slightly higher than CTM (0.991). By cross-validation, the prediction accuracy of CLM is 21% higher than CTM, which was 0.885 and 0.730 for CLM and CTM respectively. The average of predicted genetic gain for each generation was 14.89%, and the average of realized genetic gain was 8.10% per generation for juvenile survival. These results confirmed the existence of genetic variation for juvenile natural survival and highlighted the enormous potential for improving natural survival by selective breeding in tongue sole.

Half-smooth tongue sole (Cynoglossus semilaevis) is a commercially important fish species. There were no reports about accurate genetic parameters of growth traits in C. semilaevis, despite efforts aimed at the culture of this species. Therefore, the main goal of this study was to estimate the parameters for the early growth traits (total length, body depth and body weight) of C. semilaevis based on the data obtained from 80 families. The trait of fish with pure white bottom or not (named as the trait of bottom color) was also analyzed. All the heritabilities estimated for the traits were medium, which varied from 0.210 to 0.362. Genetic correlations among growth traits were highly positive, varying from 0.913 to 0.959, indicating that selection of one of the three traits would result in correlated increase in the others. Also, genetic correlations between bottom color and growth traits were positive and varying from 0.241 to 0.353, suggesting that selection for the bottom color can be utilized to enhance the selection of growth traits. In addition, fish from the top performing 16 full-sib families were selected as broodstock, based on the above analysis and the comparison of family breeding value. This research provides an important basic material to implement selective breeding in C. semilaevis.

To elucidate the effect of thyroid hormone receptor α A (thraa) on metamorphosis, the full length cDNA of half-smooth tongue sole Cynoglossus semilaevis was cloned. The relative gene transcript level of thraa at different development stages was quantified using real-time PCR. Transcription of thraa increased and declined rapidly during metamorphosis. Hyperthyroidism was induced in juveniles and larvae with exposure to T3 and T4, and hypothyroidism with thiourea (TU), 2-mercapto-1-methylimidazole (MMI). thraa mRNA was higher in fish treated for 6 days with MMI than in untreated controls, although inhibited larvae did not complete metamorphosis. The addition of exogenous T4 reversed this effect in the MMI-treated group, but not in the TU-treated group. In situ hybridization revealed progressive tail end of body growth and change during developmental stages, with corresponding changes in thraa expression. This process may be induced by thyroid hormones with thraa as a major mediator. The morphological changes of tip of the tail may be associated with the development of lateral swimming.

Edwardsiella tarda is one of the most prevalent and economically important diseases worldwide in aquaculture, including olive flounder (Paralichthys olivaceus). Genetic improvement for resistance to E. tarda is an alternative strategy for the prevention of disease outbreaks. The objective of this work was to evaluate the genetic parameters of disease resistance by challenge test data. Mortality was recorded daily for 16 days (day1 to 16). At day 4 and 16, the cumulative mortality was 52%, and 87%, respectively. Resistance was defined as binary test survival (BTS), day of death (DD), and binary test day survival (BTDS). For DD, only day 16 was set as the cut-off day, while for the other two traits, each test day was set as a cut-off day. These traits were fitted in four statistical models for estimating variance components and calculating genetic parameters. Namely, 1) cross-sectional linear model for BTS (CLM) and DD (LM), 2) threshold (logit) model (CTM) for BTS, and 3) linear (logit) repeatability model (LRM) for BTDS. In LM, the heritability of resistance was 0.23 ± 0.06. In other three models, heritabilities were estimated at each cut-off point (test day) respectively, and heritabilites fluctuated daily. At day 4 (with approximately 50% overall mortality), the heritability was 0.17 ± 0.11 for CLM, 0.20 ± 0.14 for CTM and 0.09 ± 0.08 for LRM. Heritabilities obtained based on LM (at day16) and other three model estimated at day 4 are considered to be accurate. The selection accuracies of LM and LRM are nearly identical (0.735 and 0.755 respectively) and are 9.5% to13.9% higher than values of CLM and CTM (0.663 and 0.671 respectively). The rank correlations between EBVs obtained from CLM, CTM and LRM were very high (ranging from 0.97–1.00), indicating a near identical ranking of families. However, the correlations between EBVs from LM and from other three models were substantially lower (ranging from 0.79–0.82), which imply a substantial reranking of families. Low to moderate heritabilities indicated that resistance against E. tarda can be genetically improved, and selection by using models (i.e. LM and LRM) which take time to death into account is more accurate than cross-sectional models.

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

r-spondin1 (rspo1) encodes a secreted protein that is involved in the determination and differentiation of the mammalian ovary. However, little information is yet available for teleosts. Here, we identified a homologue of rspo1 in Cynoglossus semilaevis. The full-length cDNA of rspo1 had a length of 2,703 bp with an open reading frame of 834 bp, encoding a protein with a length of 277 amino-acids. rspo1 expression was detected via qRT-PCR in various tissues, and significant sexually dimorphic expression was observed in the gonads. Furthermore, ISH located rspo1 in germ cells such as spermatogonia, spermatocytes, spermatids, spermatozoa, and oocytes, as well as in somatic cells of the gonads. Following knockdown of rspo1 in an ovarian cell line, the expressions of wnt4a, β-catenin, foxl2, and StAR were highly affected; wnt4a and β-catenin were significantly downregulated, whereas foxl2 and StAR were significantly upregulated. In summary, these data suggest that rspo1 may be involved in the regulation of ovarian development and differentiation through a conserved pathway, while the function of the gene in the testis remains elusive.

Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.

E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis.

Logistics network assignment problem is an important research field on logistics optimization. According to user equilibrium (UE) theory, The equilibrium assignment framework of logistics network is a basic problem for it. The framework structure of logistics network consisted of carriers, logistics service providers, the shippers, and the modified wardrop user equilibrium of logistics network is proposed. Based on the modified UE condition, the optimum condition/equilibrium status of carriers, logistics service providers and the shippers in the equilibrium situation are analyzed.