Yangzhen Li

Epigenetic modification and inheritance in sexual reversal of fish

Environmental sex determination (ESD) occurs in divergent, phylogenetically unrelated taxa, and in some species, co-occurs with genetic sex determination (GSD) mechanisms. Although epigenetic regulation in response to environmental effects has long been proposed to be associated with ESD, a systemic analysis on epigenetic regulation of ESD is still lacking. Using half-smooth tongue sole (Cynoglossus semilaevis) as a model-a marine fish that has both ZW chromosomal GSD and temperature-dependent ESD-we investigated the role of DNA methylation in transition from GSD to ESD. Comparative analysis of the gonadal DNA methylomes of pseudomale, female, and normal male fish revealed that genes in the sex determination pathways are the major targets of substantial methylation modification during sexual reversal. Methylation modification in pseudomales is globally inherited in their ZW offspring, which can naturally develop into pseudomales without temperature incubation. Transcriptome analysis revealed that dosage compensation occurs in a restricted, methylated cytosine enriched Z chromosomal region in pseudomale testes, achieving equal expression level in normal male testes. In contrast, female-specific W chromosomal genes are suppressed in pseudomales by methylation regulation. We conclude that epigenetic regulation plays multiple crucial roles in sexual reversal of tongue sole fish. We also offer the first clues on the mechanisms behind gene dosage balancing in an organism that undergoes sexual reversal. Finally, we suggest a causal link between the bias sex chromosome assortment in the offspring of a pseudomale family and the transgenerational epigenetic inheritance of sexual reversal in tongue sole fish.

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.

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.

Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination

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.

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

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.

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.

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

ABSTRACT 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 &bgr;‐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. HighlightsA new r‐spondin member, r‐spondin2‐like (rspo2l), was cloned and characterized in Chinese tongue sole.rspo2l transcription was detected in various tissues and the high levels were observed in skin and gill.rspo2l transcription showed obvious up‐regulation in gill, spleen, and kidney after Vibrio harveyi challenge.rspo2l might play a role in immune responses by cooperating with WNT signaling pathway.

Phenotypic and genetic parameter estimation of juvenile growth and bottom color traits in half-smooth tongue sole, Cynoglossus semilaevis

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.