Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass.

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Transcriptome analysis revealed gene expression feminization of testis after exogenous tetrodotoxin administration in pufferfish Takifugu flavidus.

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.

The role of autophagy on eye migration during the metamorphosis of Paralichthys olivaceus.

The metamorphosis of flatfish is unique, especially its eye migration. Autophagy has been found to be involved in a variety of organisms' metamorphosis. In order to explore the relationship between autophagy and flatfish metamorphosis, we investigated the expression of autophagy marker gene lc3b during the metamorphosis using real-time quantitative polymerase chain reaction and in situ RNA hybridization. Besides, we inhibited cell division, which was reported as the force source of eye migration, and autophagy around the mobile eye by microinjecting the inhibitors to explore the effects on autophagy expression and eye migration. We found that autophagy taking place during the metamorphosis, particularly in the areas around the eyes. In addition, the eye migration could be blocked by inhibiting the autophagy in the supraorbital area of the blind side, and after we blocked the eye migration by inhibiting cell proliferation in the infraorbital area of the blind side, the autophagy around the eye was partially inhibited. These findings indicate that the autophagy around the eyes caused by eye migration. Moreover, the cell death caused by autophagy loosen the orbital tissue to create space for the eye migration.

Isolation of prolactin gene and its differential expression during metamorphosis involving eye migration of Japanese flounder Paralichthys olivaceus.

Eye migration during flatfish metamorphosis is driven by asymmetrical cell proliferation. To figure out Prolactin (PRL) function in this process, the full-length cDNA of prl was cloned from Japanese flounder (Paralichthys olivaceus) in our study. The deduced PRL protein shares highly conserved sequence with other teleosts, but has several amino acids loss compared with higher vertebrates, including amphibians, reptiles, avian and mammals. Spatio-temporal expression of prl gene displayed its extensive expression in the early development stages, while the limited expression of prl was observed in the pituitary, brain, and intestine of adult fish. In situ hybridization showed the asymmetrical distribution patterns of prl gene around the eyes during metamorphosis, which was coincident with the cell proliferation signals. Colchicine inhibited cell proliferation and reduced the prl gene expression, which indicates that PRL was involved in cell proliferation in the suborbital area of the migrating eye. The treatment of methimazole and 9-cis-retinoic acid respectively led to a reduction in the number of proliferating cells and the downregulation of prl expression, suggesting PRL was regulated by thyroid hormone signaling pathway and retinoic acid related signaling pathways. The results gave us a basic understanding of PRL function during flatfish metamorphosis.

Extracellular vesicles piwi-interacting RNAs from skin mucus for identification of infected Cynoglossus semilaevis with Vibrio harveyi.

Extracellular vesicles play a regulatory role in intracellular and intercellular transmission through a variety of biological information molecules, including mRNA, small RNAs and proteins. piRNAs are one kind of regulatory small RNAs in the vesicles at the post transcriptional level. Hereby, we isolated the extracellular vesicles from skin mucus and screened the piRNA profiles of these vesicles, aiming at developing biomarkers related to bacterial infections in Cynoglossus semilaevis. The different profilings of piRNAs in mucous extracellular vesicles of C. semilaevis were compared through small RNA sequencing, between fish infected with Vibrio harveyi and healthy ones. The number of clean reads on the alignment of exosome sick (ES) group was 105, 345 and that of exosome control (EC) group was 455, 144. GO and KEGG pathway enrichment analysis showed that most of the target genes were involved in cellular process, response to stimulus, biological regulation, immune system process and signal transduction, signal molecular and interaction, transport and catabolism. The 45 final candidate piRNAs related to immunity or infectious diseases included 20 piRNAs with high expression in the ES group and 25 piRNAs with a low expression in the ES group. After verification by qRT-PCR, there was significant difference of five piRNAs expression level between infected fish and healthy fish, in line with the sequencing. The expression level of piR-mmu-16401212, piR-mmu-26829319 and piR-gga-244092 in infected fish were significantly lower than that of control group, while piR-gga-71717 and piR-gga-99034 were higher, which implying that these piRNAs in mucous extracellular vesicles can be used to identify diseased fish from normal ones. This work supplied a novel class of biomarker for infection diagnosis in fish, and it will be benefit for screening disease resistant breeding of C. semilaevis.

Single-nucleotide polymorphisms responsible for pseudo-albinism and hypermelanosis in Japanese flounder (Paralichthys olivaceus) and reveal two genes related to malpigmentation.

Paralichthys olivaceus is the kind of cold-water benthic marine fish. In the early stages of development, the symmetrical juveniles transform into an asymmetrical body shape through metamorphosis for adapting benthic life. After that, one side of the fish body is attached to the ground, and the eyes turn to the opposite side which is called ocular side. The body color also appears asymmetry. The skin on the ocular side is dark brown, and the skin on the blind side is white without pigmentation. Pseudo-albinism and hypermelanosis have been considered distinct body color disorders in flatfish. Pseudo-albinism and hypermelanosis in Paralichthys olivaceus are due to abnormal or uneven pigment distribution, due to the interaction of hereditary and environmental factors, rather than a single-nucleotide mutation of a specific gene. Here, we report three single-nucleotide polymorphisms (SNPs) responsible for both pseudo-albinism and hypermelanosis, which are located on two body color-related genes involved in melanogenesis-related pathways. c.2440C>A (P. V605I) and c.2271-96T>C are located on the Inositol 1,4,5-trisphosphate receptor type 2-like (ITPR2) (Gene ID: 109624047), they are located in exon 16 and the non-coding region, respectively, and c.2406C>A (P.H798N) is located in exon 13 of the adenylate cyclase type 6-like (AC6) gene(Gene ID: 109630770). ITPR2 and AC6 expression, which both participate in the thyroid hormone synthesis pathway associated with pseudo-albinism and hypermelanosis in P. olivaceus, were also investigated using qRT-PCR. In hypermelanotic fish, there were relatively higher levels of expression in ITPR2 and AC6 mRNA of hyper-pigmented skin of blind side than that of non-pigmented skin on the blind side and pigmented skin on the ocular side, while in pseudo-albino fish, expression level of ITPR2 and AC6 mRNA in pigmented skin of ocular side was significantly higher than that in non-pigmented skin both ocular and blind side. The results indicated that the expression of the two genes in abnormal parts of body color is positively correlated with pigmentation, suggesting that the influence of abnormal expression of two genes on the pigmentation in abnormal parts of body color deserves further study.

CRISPR/Cas9-induced nos2b mutant zebrafish display behavioral abnormalities.

The immunomodulatory function of nitric oxide synthase (NOS2) has been extensively studied. However, some behavioral abnormalities caused by its mutations have been found in a few rodent studies, of which the molecular mechanism remains elusive. In this research, we generated nos2b gene knockout zebrafish (nos2bsou2/sou2 ) using CRISPR/Cas9 approach and investigated their behavioral and molecular changes by doing a series of behavioral detections, morphological measurements, and molecular analyses. We found that, compared with nos2b+/+ zebrafish, nos2bsou2/sou2 zebrafish exhibited enhanced motor activity; additionally, nos2bsou2/sou2 zebrafish were characterized by smaller brain size, abnormal structure of optic tectum, reduced mRNA level of presynaptic synaptophysin and postsynaptic homer1, and altered response to sodium nitroprusside/methylphenidate hydrochloride treatment. These findings will likely contribute to future studies of behavioral regulation.

Detecting Cynoglossus semilaevis infected with Vibrio harveyi using micro RNAs from mucous exosomes.

Exosomes are important mediators of vesicle transportation and contain microRNAs (miRNAs) that mediate transcriptional gene knockout and silencing in biological processes. Moreover, exosomic miRNAs are promising biomarkers for disease diagnosis and physiological status indication in many species, including fish. The impact of the Vibrio harveyi pathogen on Cynoglossus semilaevis aquaculture is becoming more and more serious as the industry expands. To overcome this challenge, miRNAs in mucous exosomes were screened by small RNA sequencing and verified by quantitative real-time PCR to develop biomarkers. This is the first capture of exosomes from flatfish mucus coupled with miRNA profiling. The results revealed significant differences in expression levels of some miRNAs between infected and healthy fish. Three unique miRNAs were identified for V. harveyi infection diagnosis; expression levels of dre-miR-205-5p and dre-miR-205-5p in infected fish were significantly lower than controls, while dre-miR-100-5p expression was higher. These miRNAs in mucous exosomes could be used to differentiate diseased and healthy fish in an early screening method with practical value for breeding disease-resistant C. semilaevis.

A combination of genome-wide association study screening and SNaPshot for detecting sex-related SNPs and genes in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) males and females exhibit great differences in growth rate and appearance. The species is heterogametic (ZW/ZZ) and has sex-reversed "pseudomales" that are genetically female and physiologically male. In this study, we identified eight sex-specific single nucleotide polymorphism (SNP) markers for the sex identification of C. semilaevis by using a combination of genome-wide association study (GWAS) screening and SnaPshot validation. Candidate SNPs were screened using genotyping by sequencing to perform GWAS of the differential SNPs between the sexes of C. semilaevis. The SNP loci were amplified using a multiplex PCR system and detected via SNaPshot, which enables multiplexing of up to 30-40 SNPs in a single assay and ensures high accuracy of the results. The molecular markers detected in our study were used to successfully identify normal males and pseudomales from 45 caught and 40 cultured C. semilaevis specimens. Linkage disequilibrium analysis showed that the eight SNP loci were related to each other, with a strong linkage. Moreover, we investigated the expression of prdm6 mRNA containing a missense SNP and confirmed that the gene is differentially expressed in the gonads of the different sexes of C. semilaevis; the expression of prdm6 mRNA was significantly higher in the males than in the females and pseudomales. This means prdm6 may be related to sex differentiation in C. semilaevis.

Large scale SNP unearthing and genetic architecture analysis in sea-captured and cultured populations of Cynoglossus semilaevis.

Knowledge on population structure and genetic diversity is a focal point for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Here we used the GBS approach for the genome-wide identification of SNPs in a collection of Cynoglossus semilaevis and for the assessment of the level of genetic diversity in C. semilaevis genotypes. GBS analysis generated a total of 55.12 Gb high-quality sequence data, with an average of 0.63 Gb per sample. The total number of SNP markers was 563, 109. In order to explore the genetic diversity of C. semilaevis and to select a minimal core set representing most of the total genetic variation with minimum redundancy, C. semilaevis sequences were analyzed using high quality SNPs. Based on hierarchical clustering, it was possible to divide the collection into 2 clusters. The marine fishing populations were clustered and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. These analyses showed that genotypes were clustered based on species-related features. Differential significant SNPs were also captured and validated by GBS and SNaPshot, with linkage disequilibrium and haplotype analysis, seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. And SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. These differential SNPs could be also employed as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations in future.

Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis.

BACKGROUND: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed "pseudomales" who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring. RESULTS: To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation-related and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish. CONCLUSIONS: These signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed "pseudomales" in C. semilaevis.

Identification and characterization of the melanocortin 1 receptor gene (MC1R) in hypermelanistic Chinese tongue sole (Cynoglossus semilaevis).

The Chinese tongue sole (Cynoglossus semilaevis) is a flatfish with distinctive asymmetry in its body coloration. The melanism (hyperpigmentation) in both the blind side and ocular side of C. semilaevis gives it an extremely low commercial value. However, the fundamental molecular mechanism of this melanism remains unclear. Melanocortin 1 receptor (MC1R), a GTP-binding protein-coupled receptor, is considered to play a vital role in the physiology of the vertebrate pigment system. In order to confirm the contribution of MC1R to the body coloration of C. semilaevis, the expression levels of Mc1r mRNA were measured in seven tissue types at different developmental stages of normal and melanistic C. semilaevis. The expression levels of Mc1r mRNA in the heart, brain, liver, kidney, ocular-side skin, and blind-side skin of melanistic C. semilaevis were significantly higher than that of normal C. semilaevis in all developmental stages. Moreover, the knocking down of Mc1r in the C. semilaevis liver cell line (HTLC) increased the expression of the downstream genes microphthalmia transcription factor (Mitf) and tyrosinase-related protein 1 (Tyrp1) in the pigmentation pathway. Thus, the present data suggest that MC1R might play important roles in Tyrp1- and Mitf-mediated pigment synthesis in C. semilaevis.

Seminal Plasma Exosomes: Promising Biomarkers for Identification of Male and Pseudo-Males in Cynoglossus semilaevis.

In mammals, small RNAs enclosed in exosomes have been identified as appropriate signatures for disease diagnosis. However, there is limited information on exosomes derived from seminal plasma, and few studies have reported analyzed the composition of exosomes and enclosed small RNAs in fish. The half-smooth tongue sole (Cynoglossus semilaevis) is an economically important fish for aquaculture, and it exhibits sexual dimorphism: the female gender show higher growth rates and larger body sizes than males. Standard karyotype analysis and artificial gynogenesis tests have revealed that this species uses heterogametic sex determination (ZW/ZZ), and so-called sex-reversed pseudo-males exist. In this study, we successfully identified exosomes in the seminal plasma of C. semilaevis; to the best of our knowledge, this is the first report of exosomes in fish seminal plasma. Analysis of the nucleotide composition showed that miRNAs were dominant in the exosomes, and the miRNAs were sequenced and compared to identify signature miRNAs as sexual biomarkers. Moreover, target genes of the signature miRNAs were predicted by sequence matching and annotation. Finally, four miRNAs (dre-miR-141-3P, dre-miR-10d-5p, ssa-miR-27b-3p, and ssa-miR-23a-3p) with significant differential expression in the males and pseudo-males were selected from the signature candidate miRNAs as markers for sex identification, and their expression profiles were verified using real-time quantitative PCR. Our findings could provide an effective detection method for sex differentiation in fish.

New insights on geographical/ecological populations within Coilia nasus (Clupeiformes: Engraulidae) based on mitochondrial DNA and microsatellites.

The Coilia nasus is an important fish species, which is of commercial values in China. In order to manage the fisheries resources and the development of sustainable fishing strategies to protect this species, 11 microsatellite loci polymorphisms and mtDNA COI sequences were used to examine the genetic diversity of C. nasus in Japan and China. In total, the 40 COI haplotypes and 93 microsatellite alleles were detected. The mtDNA phylogeny did not support population grouping, but the distribution patterns of mtDNA haplotypes and the results of STRUCTURE analysis based on microsatellite indicated a degree of genetic isolation in this species. Our study suggested that the lack of a population genetic structure might result in its amphidromous life cycle, and the geographical distance and habitat fragments might cause isolated populations. Thus, the sampling populations of C. nasus in Japan and China could be divided as four geographical/ecological populations.

Dorsal fin development in flounder, Paralichthys olivaceus: Bud formation and its cellular origin.

The development of the median fin has not been investigated extensively in teleosts, although in other fishes it has been proposed that it involves the same genetic programs operating in the paired appendages. Adult median fins develop from the larval bud; therefore an investigation of fin bud formation and its cellular origin is essential to understanding the maturation mechanisms. In Paralichthys olivaceus, skeletogenesis proceeds from an anterior to posterior direction providing a good opportunity to study the formation of dorsal fin bud. An apical ectodermal ridge appeared at the basal stratum of the presumptive dorsal fin was first observed at 3 days post hatching. Then the apical ectodermal fold formed as the bud outgrew in 6 days post-hatch larvae. The bud continued to grow, breaking through the dorsal fin fold in 9 days post-hatch larvae. At 13 days post-hatch, the bud grew beyond the edge of the fin fold and formed into the four future rays. Molecular markers of cell type showed the existence of neural crest cells, scleroblasts and sclerotomes in the dorsal fin bud. The earliest gene expression in the dorsal fin bud was Hoxd10 at 3 days post-hatch larvae, then Hoxd9, Hoxd11 and Hoxd12. This indicates Hoxd10 might be a candidate molecular marker of the bud formation site. Some key molecular markers for paired appendage development, such as FGF8, Wnt7, and Shh were expressed at the apical ectodermal ridge and later the apical ectodermal fold. Moreover, the form of the dorsal fin bud could be inhibited by Hh pathway inhibitor, further indicating that common basic molecular mechanisms might be utilized by median fins.

The complete mitochondrial genome sequence of Yongeichthys criniger and phylogenetic studies of Gobiidae.

Yongeichthys criniger belongs to Gobiidae, Gobioidei, and Perciformes in taxonomy. Its mitochondrial genome was first determined, which consists of 13 typical vertebrate protein-coding genes, 22 tRNA, 2 rRNA genes, and 1 control region. The mitogenome base's composition is 27.28% for A, 26.12% for T, 28.98% for C, and 17.62% for G. The phylogenic analysis involves 32 Gobiidae species in GenBank database. The results will provide more molecular information for the further studies on species identification and phylogenic evolution of Gobiidae.

Comparative analysis of the neurula transcriptomes of two species of flatfishes: Platichthys stellatus and Paralichthys olivaceus.

Flatfish with left-right eye asymmetry are the most significant among vertebrates. However, the genetic basis for the control of this characteristic is still unclear. We propose that the gene(s) for eye asymmetry initially control minor differences in cell number in the tissues around the eyes during eye development. This minor difference is then amplified, causing eye migration during metamorphosis. Therefore, comparing the neurula transcriptomes between flatfish species with different eye-reversal mutants may provide very useful information to screen for genes involved in eye asymmetry. In this study, two cDNA libraries constructed from neurulas of P. stellatus (high ratio of eye reversal) and P. olivaceus (very low ratio of eye reversal) were sequenced and compared. There were 8121 and 8108 unigenes annotated to 32 categories in P. stellatus and P. olivaceus, respectively, and the highest KEGG pathways in both species were 'signal transduction', 'immune system', and 'endocrine system'. In total, 62,692 and 18,938 putative single nucleotide polymorphisms (SNPs) were predicted in the P. stellatus and P. olivaceus transcriptomes, respectively. Furthermore, 8026 SNPs found in P. stellatus did not exist in P. olivaceus. Fifty-one SNPs were identified in nine genes (Fgf7, Wnt9, Sfrp2, Bmpr1B, Bmpr2, Pax3, Pax6, Six1 and Tgfßr2) related to eye development. In particular, Tgfßr2 with Asp77Glu found in P. stellatus but not in P. olivaceus will provide important information for screening genes associated with eye asymmetry.

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.

Change of body height is regulated by thyroid hormone during metamorphosis in flatfishes and zebrafish.

Flatfishes with more body height after metamorphosis should be better adapted to a benthic lifestyle. In this study, we quantified the changes in body height during metamorphosis in two flatfish species, Paralichthys olivaceus and Platichthys stellatus. The specific pattern of cell proliferation along the dorsal and ventral edge of the body to allow fast growth along the dorsal/ventral axis might be related to the change of body height. Thyroid hormone (T4 and T3) and its receptors showed distribution or gene expression patterns similar to those seen for the cell proliferation. 2-Mercapto-1-methylimidazole, an inhibitor of endogenous thyroid hormone synthesis, inhibited cell proliferation and decreased body height, suggesting that the change in body shape was dependent on the local concentration of thyroid hormone to induce cell proliferation. In addition, after treatment with 2-mercapto-1-methylimidazole, zebrafish larvae were also shown to develop a slimmer body shape. These findings enrich our knowledge of the role of thyroid hormone during flatfish metamorphosis, and the role of thyroid hormone during the change of body height during post-hatching development should help us to understand better the biology of metamorphosis in fishes.

The population history of Garra orientalis (Teleostei: Cyprinidae) using mitochondrial DNA and microsatellite data with approximate Bayesian computation.

BACKGROUND: The South China landmass has been characterized by a complex geological history, including mountain lifting, climate changes, and river capture/reversal events. To determine how this complexity has influenced the landmass's phylogeography, our study examined the phylogeography of Garra orientalis, a cyprinid widely distributed in South China, using sequences from the mitochondrial DNA control region and cytochrome b gene (1887 bp) and polymorphisms of thirteen microsatellite loci. RESULTS: In total, 157 specimens were collected from eight populations. All 88 mtDNA haplotypes were identified as belonging to three major lineages, and these lineages were almost allopatric in their distributions. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of G. orientalis were distributed south of the Yunkai Mountains, including on Hainan Island. The mtDNA data revealed a strong relationship between phylogeny and geography. In the microsatellite analysis, a total of 339 alleles with an average of 26 alleles per locus were observed across thirteen microsatellite loci. A clustering algorithm for microsatellite data revealed an admixture-like genetic structure. Although the mtDNA and microsatellite data sets displayed a discordant population structure, the results of an approximate Bayesian computation approach showed that these two markers revealed congruent historical signals. The population history of G. orientalis reflects vicariance events and dispersal related to the complex geological history of South China. CONCLUSION: Our results (i) found that the discordances between mtDNA and microsatellite markers were accounted for by admixtures; (ii) showed that the Wuzhishan and Yinggeling mountain ranges and Qiongzhou Strait were important barriers limiting gene exchange between populations on both sides; (iii) indicated that during glaciation and inter-glacial periods, the strait and continental shelves were exposed and sank, which contributed with the dispersion and differentiation of populations; and (iv) displayed that the admixtures between lineages took place in coastal populations and then colonized the tributaries of the Pearl River.