Transcriptome Analysis Reveals the Immunosuppression in Tiger Pufferfish (Takifugu rubripes) under Cryptocaryon irritans Infection.

The tiger pufferfish (Takifugu rubripes), also known as fugu, has recently suffered from severe C. irritans infections under aquaculture environment, yet the underlying immune mechanisms against the parasite remain poorly understood. In this study, we conducted a comprehensive transcriptome analysis of the gill tissue from infected and uninfected fish using PacBio long-read (one pooled sample each for seriously infected and healthy individuals, respectively) and Illumina short-read (three pools for mildly infected, seriously infected, and healthy individuals, respectively) RNA sequencing technologies. After aligning sequence data to fugu's reference genome, 47,307 and 34,413 known full-length transcripts were identified and profiled in healthy and infected fish, respectively. Similarly, we identified and profiled 1126 and 803 novel genes that were obtained from healthy and infected fish, respectively. Interestingly, we found a decrease in the number of alternative splicing (AS) events and long non-coding RNAs (lncRNAs) after infection with C. irritans, suggesting that they may be involved in the regulation of the immune response in fugu. There were 687 and 1535 differentially expressed genes (DEGs) in moderately and heavily infected fish, respectively, compared to uninfected fish. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that immune-related DEGs in the two comparison groups were mainly enriched in cytokine-cytokine receptor interactions, ECM-receptor interactions, T-cell receptor signaling pathways, Th1 and Th2 cell differentiation, and Th17 cell differentiation pathways. Further analysis revealed that a large number of immune-related genes were downregulated in infected fish relative to uninfected ones, such as CCR7, IL7R, TNFRSF21, CD4, COL2A1, FOXP3B, and ITGA8. Our study suggests that C. irritans is potentially a highly efficient parasite that may disrupt the defense mechanisms of fugu against it. In addition, in combination of short-read RNA sequencing and previous genome-wide association analyses, we identified five key genes (NDUFB6, PRELID1, SMOX, SLC25A4, and DENND1B) that might be closely associated with C. irritans resistance. This study not only provides valuable resources of novel genic transcripts for further research, but also provides new insights into the immune mechanisms underlying C. irritans infection response in farmed fugu.

Surface electromyography analysis of ankle flexor and extensor activity under different standing stability levels.

Background: To observe the activation strategies of the ankle muscles using surface electromyography (sEMG) during single-leg standing (SLS) and both-leg standing (BLS) on flat ground (FG), soft mat (SM), and BOSU ball (BB) surfaces. Methods: Thirty healthy young adults participated in the study. The muscle activities of the tibialis anterior (TA) and gastrocnemius medial (GM) were measured on the three surfaces during SLS and BLS. Electromyographic evaluations of the TA and GM were recorded during maximum voluntary isometric contractions (MVIC). Muscle activation was evaluated using MVIC%, and muscle co-contraction was evaluated using the co-contraction index (CI). Results: A statistically significant increase was observed in the MVIC% of the TA, GM, and CI on the three surfaces during SLS compared to BLS, except for the comparison of CI on BB between SLS and BLS (t = -1.35, p = 0.19). The MVIC% of the TA and GM during SLS and BLS on BB was significantly increased in comparison with FG and SM. The CI during BLS on BB increased compared to FG (t = 3.19, p < 0.01) and SM (t = 4.64, p < 0.01). The CI during BLS on SM (t = -1.46, p = 0.15) decreased when compared to FG but without statistical significance. Conclusions: SLS and unstable surfaces can induce greater muscle activation, and SLS can have a greater influence on ankle muscles.

Intestinal microbiome-mediated resistance against vibriosis for Cynoglossus semilaevis.

BACKGROUND: Infectious diseases have caused huge economic loss and food security issues in fish aquaculture. Current management and breeding strategies heavily rely on the knowledge of regulative mechanisms underlying disease resistance. Though the intestinal microbial community was linked with disease infection, there is little knowledge about the roles of intestinal microbes in fish disease resistance. Cynoglossus semilaevis is an economically important and widely cultivated flatfish species in China. However, it suffers from outbreaks of vibriosis, which results in huge mortalities and economic loss. RESULTS: Here, we used C. semilaevis as a research model to investigate the host-microbiome interactions in regulating vibriosis resistance. The resistance to vibriosis was reflected in intestinal microbiome on both taxonomic and functional levels. Such differences also influenced the host gene expressions in the resistant family. Moreover, the intestinal microbiome might control the host immunological homeostasis and inflammation to enhance vibriosis resistance through the microbe-intestine-immunity axis. For example, Phaeobacter regulated its hdhA gene and host cyp27a1 gene up-expressed in bile acid biosynthesis pathways, but regulated its trxA gene and host akt gene down-expressed in proinflammatory cytokines biosynthesis pathways, to reduce inflammation and resist disease infection in the resistant family. Furthermore, the combination of intestinal microbes and host genes as biomarkers could accurately differentiate resistant family from susceptible family. CONCLUSION: Our study uncovered the regulatory patterns of the microbe-intestine-immunity axis that may contribute to vibriosis resistance in C. semilaevis. These findings could facilitate the disease control and selective breeding of superior germplasm with high disease resistance in fish aquaculture. Video Abstract.

Transcriptomic analysis reveals the gene expression profiles in the spleen of spotted knifejaw (Oplegnathus punctatus) infected by Vibrio harveyi.

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.

Dietary bile acid supplementation reveals beneficial effects on intestinal healthy status of tongue sole (Cynoglossus semiliaevis).

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 effects of core stability training in nurses with nonspecific low back pain.

ABSTRACT: To investigate the effect of core stability training on nonspecific low back pain (NSLBP) in nurses.The data were collected retrospectively by reviewing the patient's medical records and pain questionnaires in our rehabilitation center. A total of 40 nurses with NSLBP were included and divided into observation group and control group. Each group were given routine health education for NSLBP. Core stability training was performed in observation group for 4 weeks. Surface electromyography (sEMG) evaluation of erector spine and multifidus muscle, pain Numeric Rating Scale (NRS) and Japanese Orthopaedic Association (JOA) scores were evaluated and analyzed before and 4 weeks after intervention.There was no significant difference of NRS score and JOA score between two groups before intervention (P > .05, respectively). The NRS and JOA scores were significantly improved in both two groups after 4 weeks of intervention (P < .05, respectively). Moreover, the improvement of NRS and JOA scores in the observation group were better than those of the control group (P < .05, respectively). No significant difference of average electromyography (AEMG) or median frequency (MF) were noted between the healthy side and the affected side in both groups before or after intervention (P > .05, respectively). After 4 weeks of intervention, the AEMG of the healthy and the affected side of the two groups showed an improved trend without significant difference (P > .05, respectively). The MF of affected side was significantly higher 4 weeks after intervention than those before treatment in the observation and control group (P < .05, respectively).Core stability training can alleviate pain, improve the fatigue resistance of the core muscles and the balance of the functions of bilateral multifidus muscles in nurses with NSLBP.

Identification and functional analysis of the perforin-1 like gene in disease resistance in half smooth tongue sole (Cynoglossus semilaevis).

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.

Development of a 38 K single nucleotide polymorphism array and application in genomic selection for resistance against Vibrio harveyi in Chinese tongue sole, Cynoglossus semilaevis.

Based on 1572 re-sequenced Chinese tongue sole (Cynoglossus semilaevis), we investigated the accuracy of four genomic methods at predicting genomic estimated breeding values (GEBVs) of Vibrio harveyi resistance in C. semilaevis when SNPs varying from 500 to 500 k. All methods outperformed the pedigree-based best linear unbiased prediction when SNPs reached 50 k or more. Then, we developed an SNP array "Solechip No.1" for C. semilaevis breeding using the Affymetrix Axiom technology. This array contains 38,295 SNPs with an average of 10.5 kb inter-spacing between two adjacent SNPs. We selected 44 candidates as the parents of 23 families and genotyped them by the array. The challenge survival rates of offspring families had a correlation of 0.706 with the mid-parental GEBVs. This SNP array is a convenient and reliable tool in genotyping, which could be used for improving V. harveyi resistance in C. semilaevis coupled with the genomic selection methods.

Identification of Potential Blind-Side Hypermelanosis-Related lncRNA-miRNA-mRNA Regulatory Network in a Flatfish Species, Chinese Tongue Sole (Cynoglossus semilaevis).

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 (Cynoglossus semilaevis). Differentially expressed long non-coding RNAs (DElncRNAs), miRNAs (DEmiRNAs), and differentially expressed genes as well as their competing endogenous RNA (ceRNA) networks were identified. A total of 34 DElncRNAs, 226 DEmiRNAs, and 610 DEGs were identified. Finally, lncRNA-miRNA-mRNA regulatory networks (involving 29 DElncRNAs, 106 DEmiRNAs, and 162 DEGs) associated with blind-side hypermelanosis were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of 162 DEGs in ceRNA networks identified DEGs (e.g., oca2, mc1r, and ihhb) in pigmentation-related biological processes and DEGs (e.g., ca4, glul, and fut9) in nitrogen metabolism, glycosphingolipid biosynthesis, and folate biosynthesis pathways, as well as their corresponding DElncRNAs and DEmiRNAs to potentially play key regulatory roles in blind-side hypermelanosis. In conclusion, this is the first study on the ceRNA regulatory network associated with blind-side hypermelanosis in flatfish. These new findings expand the spectrum of non-coding regulatory mechanisms underpinning blind-side hypermelanosis, which facilitates the further exploration of molecular regulatory mechanisms of malpigmentation in flatfish.

Prediction of genomic breeding values based on pre-selected SNPs using ssGBLUP, WssGBLUP and BayesB for Edwardsiellosis resistance in Japanese flounder.

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.

Genome-Wide Association Mapping and Gene Expression Analyses Reveal Genetic Mechanisms of Disease Resistance Variations in Cynoglossus semilaevis.

The sustainable development of aquaculture has been impeded by infectious diseases worldwide. However, the genomic architecture and the genetic basis underlying the disease resistance remain poorly understood, which severely hampers both the understanding of the evolution of fish disease resistance traits and the prevention of these diseases in the aquaculture community. Cynoglossus semilaevis is a representative and commercially-important flatfish species. Here we combined genome-wide association study and Fst and nucleotide diversity filtration to identify loci important for the disease resistance. Based on 1,016,774 single-nucleotide polymorphisms (SNPs) identified from 650 Gb genome resequencing data of 505 individuals, we detected 33 SNPs significantly associated with disease resistance and 79 candidate regions after filtration steps. Both the allele frequencies and genotype frequencies of the associated loci were significantly different between the resistant and susceptible fish, suggesting a role in the genetic basis of disease resistance. The SNP with strongest association with disease resistance was located in Chr 17, at 145 bp upstream of fblx19 gene, and overlapped with the major quantitative trait locus previously identified. Several genes, such as plekha7, nucb2, and fgfr2, were also identified to potentially play roles in the disease resistance. Furthermore, the expression of some associating genes were likely under epigenetic regulations between the bacterial resistant and susceptible families. These results provide insights into the mechanism that enable variation of disease resistance to bacterial pathogen infection. The identified polymorphisms and genes are valuable targets and molecular resources for disease resistance and other traits, and for advanced breeding practice for superior germplasm in fish aquaculture.

Differences in DNA Methylation Between Disease-Resistant and Disease-Susceptible Chinese Tongue Sole (Cynoglossus semilaevis) Families.

DNA methylation, the most widely studied and most well-understood epigenetic modification, has been reported to play crucial roles in diverse processes. Although it has been found that DNA methylation can modulate the expression of immune-related genes in teleosts, a systemic analysis of epigenetic regulation on teleost immunity has rarely been performed. In this research, we employed whole-genome bisulfite sequencing to investigate the genome-wide DNA methylation profiles in select disease-resistant Cynoglossus semilaevis (DR-CS, family 14L006) and disease-susceptible C. semilaevis (DS-CS, family 14L104) against Vibrio harveyi infection. The results showed that following selective breeding, DR-CS had higher DNA methylation levels and different DNA methylation patterns, with 3,311 differentially methylated regions and 6,456 differentially methylated genes. Combining these data with the corresponding transcriptome data, we identified several immune-related genes that exhibited differential expression levels that were modulated by DNA methylation. Specifically, DNA methylation of tumor necrosis factor-like and lipopolysaccharide-binding protein-like was significantly correlated with their expression and significantly contributed to the disease resistance of the selected C. semilaevis family. In conclusion, we suggest that artificial selection for disease resistance in Chinese tongue sole causes changes in DNA methylation levels in important immune-related genes and that these epigenetic changes are potentially involved in multiple immune responses in Chinese tongue sole.

Expression analysis and characterization of dmrt2 in Chinese tongue sole (Cynoglossus semilaevis).

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.

Genomic Selection Using BayesCπ and GBLUP for Resistance Against Edwardsiella tarda in Japanese Flounder (Paralichthys olivaceus).

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.

Establishment and characterization of a kidney cell line from kelp grouper Epinephelus moara.

A novel cell line, Epinephelus moara kidney cell line (EMK), was established from kidneys of kelp grouper E. moara. Cells were cultured at 24 °C in Leibovitz's L-15 medium (L15) supplemented with antibiotics, basic fibroblast growth factor (bFGF), foetal bovine serum (FBS) and 2-mercaptoethanol (2-ME). EMK cells, fibroblastic in morphology, proliferated to 100% confluency in 3-4 days and were subcultured for over 50 passages. The cells could grow from 18 to 30 °C, with optimal growth at 24 °C. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 42. Green fluorescent signals could be observed in EMK cells when the cells were transfected with pEGFP-N3 plasmid. Moreover, a significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or nervous necrosis virus (NNV), and viral replication was confirmed by quantitative real-time PCR (qPCR). These results suggested the potential of the EMK cell line for studies of transgene and pathogenesis of SGIV and NNV.

Molecular characterization and expression analysis of a novel r-spondin member (rspo2l) in Chinese tongue sole (Cynoglossus semilaevis).

Numerous studies suggest R-spondins (Rspos) plays a role in mammalian sex development and differentiation by activating WNT signaling pathways. However, Rspos are frequently less reported in teleosts. In this study, a molecular characterization and expression analysis was conducted with a new rspondin member in the Chinese tongue sole, rspondin2-like (rspo2l). The length of rspo2l cDNA is 1251 bp with 732 bp of coding sequence. A qRT-PCR analysis revealed that the transcription of rspo2l was distributed in various tissues, with high transcription levels in the liver, skin, and gills which might indicate a possible role in immunity. We next examined a time-course of transcription levels in four immune tissues (gill, liver, spleen, and kidney) after Vibrio harveyi challenge. It was found that rspo2l was up-regulated in the gills, spleen, and kidney and down-regulated in the liver, and the greatest responses occurred at 24 and 48 h after bacterial challenge. An assessment of ß-catenin, the key regulator of the canonical WNT signaling pathway, at different time points in four immune organs revealed that its transcription profile was similar to that of rspo2l after bacterial challenge. The results suggest that tongue sole rspo2l might play a role in immune responses after bacterial challenge, while the potential link with the WNT signaling pathway still requires further investigation. This is the first report about the involvement of rspondins in fish immune responses.

Characterization and expression pattern of r-spondin1 in Cynoglossus semilaevis.

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, ß-catenin, foxl2, and StAR were highly affected; wnt4a and ß-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.

Locus Mapping, Molecular Cloning, and Expression Analysis of rps6kb2, a Novel Metamorphosis-Related Gene in Chinese Tongue Sole (Cynoglossus semilaevis).

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.

Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis).

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.

The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry.

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. 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.