Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut.

Atlantic Halibut (Hippoglossus hippoglossus) has a X/Y genetic sex determination system, but the sex determining factor is not known. We produced a high-quality genome assembly from a male and identified parts of chromosome 13 as the Y chromosome due to sequence divergence between sexes and segregation of sex genotypes in pedigrees. Linkage analysis revealed that all chromosomes exhibit heterochiasmy, i.e. male-only and female-only meiotic recombination regions (MRR/FRR). We show that FRR/MRR intervals differ in nucleotide diversity and repeat class content and that this is true also for other Pleuronectidae species. We further show that remnants of a Gypsy-like transposable element insertion on chr13 promotes early male specific expression of gonadal somatic cell derived factor (gsdf). Less than 4.5 MYA, this male-determining element evolved on an autosomal FRR segment featuring pre-existing male meiotic recombination barriers, thereby creating a Y chromosome. Our findings indicate that heterochiasmy may facilitate the evolution of genetic sex determination systems relying on linkage of sexually antagonistic loci to a sex-determining factor.

Vitellogenin-derived fragment in embryos of Japanese flounder Paralichthys olivaceus with binding and bactericidal activities against an infectious bacterium via an interaction with saccharides.

Thirty- and 90-kDa proteins with binding ability to Edwardsiella tarda, a causative bacterium of Edwardsiellosis in fish, were purified from the embryo of Japanese flounder Paralichthys olivaceus. The proteins were isolated with affinity chromatography, in which the bacterium was used as a ligand and galactose, mannose, and ethylenediaminetetraacetic acid (EDTA) were used as elution agents, followed by gel filtration chromatography. N-terminal amino acid sequencing and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis revealed that the 90-kDa protein was lipovitellin heavy-chain (LvH), which is one of the proteolytically cleaved products of maternal vitellogenin (Vg) and represents the main precursor of the egg yolk in teleosts, and the 30-kDa protein was an N-terminal bit of LvH. On the other hand, Vg in the serum of the mother fish did not bind to E. tarda. While the 90-kDa protein did not show anti-bacterial activity, the 30-kDa protein strongly exhibited activity toward E. tarda, with a minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) below 0.06 µM, suggesting that the latter protein plays an important role during embryogenesis in the flounder. This is the first report showing that Vg-derived products have monosaccharides-binding activity and a fragment derived from LvH exhibits bactericidal activity.

Temporal Transcriptome Analysis Reveals Dynamic Expression Profiles of Gametes and Embryonic Development in Japanese Flounder (Paralichthys olivaceus).

The maternal-to-zygotic transition (MZT) is a crucial event in embryo development. While the features of the MZT across species are shared, the stage of this transition is different among species. We characterized MZT in a flatfish species, Japanese flounder (Paralichthys olivaceus). In this study, we analyzed the 551.57 GB transcriptome data of two types of gametes (sperms and eggs) and 10 embryo developmental stages in Japanese flounder. We identified 2512 maternal factor-related genes and found that most of those maternal factor-related genes expression decreased at the low blastula (LB) stage and remained silent in the subsequent embryonic development period. Meanwhile, we verified that the zygotic genome transcription might occur at the 128-cell stage and large-scale transcription began at the LB stage, which indicates the LB stage is the major wave zygotic genome activation (ZGA) occurs. In addition, we indicated that the Wnt signaling pathway, playing a diverse role in embryonic development, was involved in the ZGA and the axis formation. The results reported the list of the maternal genes in Japanese flounder and defined the stage of MZT, contributing to the understanding of the details of MZT during Japanese flounder embryonic development.

Expression dynamics of visual opsin genes in marbled sole Pseudopleuronectes yokohamae during metamorphosis from the larval to the juvenile stage.

We clarified the properties of visual opsin genes in the marbled sole (Pseudopleuronectes yokohamae) by cDNA sequencing, quantification of the opsin gene expression from the larval to the juvenile stage, and measurement of the maximum absorption spectra (λmax) using photopigment reconstitution. In the marbled sole eye, at least eight visual opsin genes, lws, rh2-a, rh2-b, rh2-c, sws2a, sws2b, sws1, and rh1, were expressed. Quantitative RT-PCR analysis revealed that the expression of opsin genes increased (lws, rh2-c, sws2a, and rh1) or decreased (rh2-a, rh2-b, sws2b, and sws1) from the larval to the juvenile stage. Notably, rh2-a expression was observed only in pre- to mid-metamorphic stage larvae and disappeared after metamorphosis. Thus, pre-metamorphism-specific expression of rh2-a in the marbled sole suggests that its function is restricted to the developmental stage. The reconstituted RH2-A opsin λmax was 470 nm, which is typical of acanthopterygian species. These results strongly suggest that mid-wavelength-sensitive rh2-a expression was diminished drastically in the marbled sole, probably resulting in a shift of spectral sensitivity during its metamorphosis from the larval to the juvenile stage.

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.

Olive flounder (Paralichthys olivaceus) myogenic regulatory factor 4 and its muscle-specific promoter activity.

Myogenic regulatory factor 4 (MRF4) is a basic helix-loop-helix (bHLH) transcription factor that plays crucial roles in myoblast differentiation and maturation. Here, we report the isolation of the olive flounder (Paralichthys olivaceus) mrf4 gene and the spatiotemporal analysis of its expression patterns. Sequence analysis indicated that flounder mrf4 shared a similar structure with other vertebrate MRF4, including the conserved bHLH domain. Flounder mrf4 contains 3 exons and 2 introns. Sequence alignment and phylogenetic analysis showed that it was highly homologous with Salmo salar, Danio rerio, Takifugu rubripes, and Tetraodon nigroviridis mrf4. Flounder mrf4 was first expressed in the medial region of somites that give rise to slow muscles, and later spread to the lateral region of somites that give rise to fast muscles. Mrf4 transcript levels decreased significantly in mature somites in the trunk region, and expression could only be detected in the caudal somites, consistent with the timing of somite maturation. Transient expression analysis showed that the 506 bp flounder mrf4 promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos.

Toxicity Test Assay of Waterborne Methylmercury on the Japanese Flounder (Paralichthys olivaceus) at Embryonic-Larval Stages.

Methylmercury (MeHg) is a widespread pollutant in aquatic ecosystems, but its toxicity to the early life stages of marine fish has not been adequately investigated. This study used acute toxicity tests on embryonic-larval stages of a marine flounder Paralichthys olivaceus to determine the LC50 values for embryos and larvae and evaluate the responses of several biological endpoints to subacute MeHg exposure. Under exposures (0-15 µg L-1) below LC50 values ( < 15.3 µg L-1 for embryos and 16.3 µg L-1 for larvae), embryos were more sensitive to MeHg than were the larvae. MeHg exposures at ≥ 13 µg L-1 increased morphological deformities and mortality, reduced growth and yolk absorption rate of the embryonic-larval flounder. These endpoints were sensitive to MeHg and their responses were dose-dependent. They could be used as bioindicators for assessing MeHg toxicity to the ELS of the flounder. Embryonic-larval flounder could be a useful fish for ecotoxicological assessment of MeHg in marine ecosystems.

Muscle fibre type composition in the lateral muscle of olive flounder Paralichthys olivaceus.

In this paper, a combined-method study has been made on the lateral muscle of the teleost olive flounder Paralichthys olivaceus in just-hatched and adult stages. In just-hatched stage, both slow and fast muscle fibres were detected: (1) in situ hybridization analysis indicated that slow and fast myosin heavy chain genes were specifically expressed in the superficial and deep part of the myotomal muscle, respectively; (2) immunohistochemistry analysis showed that fibres in the deep part reacted with anti-fast myosin antibody F310; (3) western blot analysis detected a weak expression of slow myosin and a strong expression of fast myosin. In adult stage, the slow and fast muscle fibres had their own distribution characteristics: (1) hematoxylin/eosin staining showed the histological characteristics of the muscle fibre composition; (2) histochemical observations showed that the deep muscle fibres, and some fibres near the epidermis, contain alkali-stable myofibrillar ATPase activity; (3) immunohistochemistry analysis indicated that all the deep muscle fibres reacted with F310 antibody and some fibres in the superficial layer of muscle also reacted with F310; (4) western blot analysis showed that fast myosin was expressed both in the blended muscles (the mix of superficial and deep muscles) and deep muscles, while slow myosin was mainly expressed in the blended muscles. These findings suggested that both slow and fast muscle fibres existed in the musculature of the olive flounder in just-hatched and adult stages. Notably, the adult fast fibres also exist in the superficial layer of the muscle.

Transient photoreception in the hindbrain is permissive to the life history transition of hatching in Atlantic halibut.

In nonmammalian vertebrates, photoreception takes place in the deep brain already early in development, but knowledge is lacking about the functions of these nonvisual photoreceptive systems. Prior to hatching, Atlantic halibut has a transient bilateral cluster of photoreceptive cells in the hindbrain. The cluster is imbedded in a neuronal network projecting to the narrow belt of hatching glands in the yolk sac. In halibut, hatching is inhibited in light and activated by transfer to darkness and c-fos analysis during hatching shows that the hindbrain cluster and hatching glands have neural activation. Unexpectedly, the hindbrain cluster expresses dual photopigments, vertebrate ancient opsin and melanopsin. Evolutionarily, these opsins are believed to belong to different classes of photopigments found in rhabdomeric and ciliary photoreceptors. The concept that an organism develops transient light sensitivity to target critical aspects of life history transitions as hatching provides a fascinating landscape to investigate the timing of other biological events.

Toxicity of the biocide polycarbamate, used for aquaculture nets, to some marine fish species.

We investigated toxic effects of the antifouling biocide polycarbamate (PC) on marine fish by conducting acute, early-life stage toxicity (ELS), and embryo toxicity tests. Mummichog (Fundulus heteroclitus) 96-h LC50 values for hatched larvae (body weight about 2.0 mg) and juveniles (660 ±â€¯36 mg) were about 12 and 630 µg/L, respectively. The ELS test using mummichog embryos yielded a lowest-observed-effect concentration of 3.9 µg/L and a no-observed-effect concentration of 2.1 µg/L with growth as the most sensitive endpoint. The embryo toxicity test for spotted halibut (Verasper variegatus) revealed a 10-d EC50 of 8.1 µg/L with abnormality as an endpoint. During the ELS and embryo toxicity tests, morphological abnormalities (notochord undulation) were induced in the embryos. Biochemical and gene-expression analysis suggest that PC-induced morphological abnormalities involve disruption of lysyl oxidase-mediated collagen fiber organization, essential for notochord formation, and inhibition of gene expression related to notochord formation.

The DNA methylation status of MyoD and IGF-I genes are correlated with muscle growth during different developmental stages of Japanese flounder (Paralichthys olivaceus).

Many genes related to muscle growth modulate myoblast proliferation and differentiation and promote muscle hypertrophy. MyoD is a myogenic determinant that contributes to myoblast determination, and insulin-like growth factor 1 (IGF-I) interacts with MyoD to regulate muscle hypertrophy and muscle mass. In this study, we aimed to assess DNA methylation and mRNA expression patterns of MyoD and IGF-I during different developmental stages of Japanese flounder, and to examine the relationship between MyoD and IGF-I gene. DNA and RNA were extracted from muscles, and DNA methylation of MyoD and IGF-I promoter and exons was detected by bisulfite sequencing. The relative expression of MyoD and IGF-I was measured by quantitative polymerase chain reaction. IGF-I was measured by radioimmunoassay. Interestingly, the lowest expression of MyoD and IGF-I emerged at larva stage, and the mRNA expression was negatively associated with methylation. We hypothesized that many skeletal muscle were required to complete metamorphosis; thus, the expression levels of MyoD and IGF-I genes increased from larva stage and then decreased. The relative expression levels of MyoD and IGF-I exhibited similar patterns, suggesting that MyoD and IGF-I regulated muscle growth through combined effects. Changes in the concentrations of IGF-I hormone were similar to those of IGF-I gene expression. Our results the mechanism through which MyoD and IGF-I regulate muscle development and demonstrated that MyoD interacted with IGF-I to regulate muscle growth during different developmental stages.

GATA4 is a transcriptional regulator of R-spondin1 in Japanese flounder (Paralichthys olivaceus).

GATA4 is a well-known transcription factor of the GATA family implicated in regulation of sex determination and gonadal development in mammals. In this study, we cloned the full-length cDNA of Paralichthys olivaceus gata4 (Po-gata4). Phylogenetic, gene structure, and synteny analysis showed that Po-GATA4 is homologous to GATA4 of teleost and tetrapod. Po-gata4 transcripts were detected in Sertoli cells, spermatogonia, oogonia and oocytes, with higher transcript levels overall in the testis than the ovary. The promoter region of P. olivaceus R-spondin1was found to contain a GATA4-binding motif. Results of CBA (cleaved amplified polymorphic sequence-based binding assay) indicated that GATA4 could indeed bind to the promoter sequence of R-spondin1. Moreover, human GATA4 recombinant protein could upregulate R-spondin1 in P. olivaceus ovary cells and FBCs (flounder brain cell line). In FBCs, overexpression of Po-gata4 resulted in elevated transcript levels of R-spondin1. Taken together, our results indicate that Po-GATA4 is involved in gonadal development by regulating R-spondin1 expression.

Germ Cell Migration, Proliferation and Differentiation during Gonadal Morphogenesis in All-Female Japanese Flounder (Paralichthys Olivaceus).

In this study, all genetic female (XX) broods of Japanese flounder were produced artificially by mating the females with sex-reversed males. The proliferation and migration of primordial germ cells (PGCs), formation of ovary and oogenesis were described in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region. At 15 days posthatching (dph), a part of PGCs were covered by a single layer somatic cells and formed the genital ridge. By 22 dph, the elongated gonadal primordia appeared under the ventral kidney, where the PGCs were totally enclosed by somatic cells. During the process of migration, PGCs were presumed to be mitotically inactive. From 63 to 73 dph, somatic cells rearrangement resulted in the formation of a narrow crevice, which became deeper and formed ovarian lumen. However, at 52 dph, dramatic mitotic proliferation of germ cell occurred and germline nest formed before the appearance of ovarian lumen. The onset of intensive germ cell proliferation and appearance of cell nests could be accepted as a criterion of initial ovarian differentiation. Then germ cells and somatic epithelial cells were gradually delimited by basement membrane and formed the germinal epithelium. In this period, results from in situ hybridization revealed that the early forkhead box L2 (pofoxl2) was expressed in somatic cells and oocytes in primary growth, which indicated the prefollicle cells formed. Then oogonia or oocytes, follicle cells, basement membrane, and theca cells composed a follicle complex. Finally, oocytes underwent meiosis and developed into to mature eggs. Anat Rec, 301:727-741, 2018. © 2017 Wiley Periodicals, Inc.

Roles of GATA6 during Gonadal Development in Japanese Flounder: Gonadogenesis, Regulation of Gender-Related Genes, Estrogen Formation and Gonadal Function Maintenance.

GATA-binding protein 6 (GATA6), a highly-conserved transcription factor of the GATA family plays an important role in gonadal cell proliferation, differentiation and endoderm development. In this study, the full-length cDNA of GATA6 of Paralichthys olivaceus (Japanese flounder) was obtained. Phylogenetic, gene structure and synteny analyses demonstrated that GATA6 of P. olivaceus is homologous to that of teleosts and tetrapods. The P. olivaceus GATA6 transcript showed higher expression in testis than in ovary, demonstrating a sexually dimorphic gene expression. During embryonic development, the expression of P. olivaceus GATA6 increased at the blastula stage, demonstrating that GATA6 is involved in morphogenesis. Results of in situ hybridization showed that GATA6 signals were detected in Sertoli cells, oogonia and oocytes. Moreover, 17α methyl testosterone, a male hormone, could moderately upregulate P. olivaceus GATA6 and downregulate P. olivaceus aromatase CYP19A1 in testis cells. These results suggest that GATA6 may play an important role in gonadal development in P. olivaceus. This study provides valuable information on the function of P. olivaceus GATA6, laying the foundation for further development of breeding techniques in this species.

Developmental toxicity in flounder embryos exposed to crude oils derived from different geographical regions.

Crude oils from distinct geographical regions have distinct chemical compositions, and, as a result, their toxicity may be different. However, developmental toxicity of crude oils derived from different geographical regions has not been extensively characterized. In this study, flounder embryos were separately exposed to effluents contaminated by three crude oils including: Basrah Light (BLO), Pyrenees (PCO), and Sakhalin Vityaz (SVO), in addition to a processed fuel oil (MFO-380), to measure developmental toxicity and for gene expressions. Each oil possessed a distinct chemical composition. Edema defect was highest in embryos exposed to PCO and MFO-380 that both have a greater fraction of three-ring PAHs (33% and 22%, respectively) compared to BLO and SVO. Observed caudal fin defects were higher in embryos exposed to SVO and MFO-380, which are both dominated by naphthalenes (81% and 52%, respectively). CYP1A gene expressions were also highest in embryos exposed to SVO and MFO-380. Higher incidence of cardiotoxicity and lower nkx 2.5 expression were detected in embryos exposed to PCO. Unique gene expression profiles were observed in embryos exposed to crude oils with distinct compositions. This study demonstrates that crude oils of different geographical origins with different compositional characteristics induce developmental toxicity to different degrees.

RNA seq- and DEG-based comparison of developmental toxicity in fish embryos of two species exposed to Iranian heavy crude oil.

To determine and compare the toxic effects of Iranian heavy crude oil (IHCO) on the embryonic development of two fish species, we examined transcriptome profiles using RNA-seq. The assembled contigs were 66,070 unigenes in olive flounder embryos and 76,498 unigenes in spotted seabass embryos. In the differential gene expression (DEG) profiles, olive flounder embryos showed different up- and down-regulated patterns than spotted seabass embryos in response to fresh IHCO (FIHCO) and weathered IHCO (WIHCO). In this work, we categorized DEG profiles into six pathways: ribosome, oxidative phosphorylation, Parkinson's disease, Alzheimer's disease, Huntington's disease, and cardiac muscle contraction, validating the expression patterns of 13 DEGs using real-time quantitative RT-PCR. The expression of the CYP1A, CYP1B1, and CYP1C1 genes in spotted seabass embryos was higher than in olive flounder embryos, whereas genes related to cell processing, development, and the immune system showed the opposite trend. Orthologous gene cluster analysis showed that olive flounder embryos were sensitive (fold change of genes with cutoff P<0.05) to both FIHCO and WIHCO, but spotted seabass embryos exhibited higher sensitivity to WIHCO than FIHCO, indicating that species-specific differences are likely to be reflected in population levels after oil spills. Overall, our study provides new insight on the different embryonic susceptibilities of two marine fish species to FIHCO and WIHCO and a better understanding of the underlying molecular mechanisms via RNA-seq and DEGs.

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.

Gene expression patterns regulating embryogenesis based on the integrated de novo transcriptome assembly of the Japanese flounder.

The Japanese flounder (Paralichthys olivaceus) is one of the most important commercial and biological marine fishes. However, the molecular biology involved during embryogenesis and early development of the Japanese flounder remains largely unknown due to a lack of genomic resources. A comprehensive and integrated transcriptome is necessary to study the molecular mechanisms of early development and to allow for the detailed characterization of gene expression patterns during embryogenesis; this approach is critical to understanding the processes that occur prior to mesectoderm formation during early embryonic development. In this study, more than 117.8 million 100bp PE reads were generated from pooled RNA extracted from unfertilized eggs to 41dph (days post-hatching) embryos and were sequenced using Illumina pair-end sequencing technology. In total, 121,513 transcripts (≥200bp) were obtained using de novo assembly. A sequence similarity search indicated that 52,338 transcripts show significant similarity to 22,462 known proteins from the NCBI non-redundant database and the Swiss-Prot protein database and were annotated using Blast2GO. GO terms were assigned to 44,627 transcripts with 12,006 functional terms, and 10,024 transcripts were assigned to 133 KEGG pathways. Furthermore, gene expression differences between the unfertilized egg and the gastrula embryo were analysed using Illumina RNA-Seq with single-read sequencing technology, and 24,837 differentially and specifically expressed transcripts were identified and included 5,286 annotated transcripts and 19,569 non-annotated transcripts. All of the expressed transcripts in the unfertilized egg and gastrula embryo were further classified as maternal, zygotic, or maternal-zygotic transcripts, which may help us to understand the roles of these transcripts during the embryonic development of the Japanese flounder. Thus, the results will contribute to an improved understanding of the gene expression patterns and signalling pathways that control the molecular mechanisms of early embryonic development.

Evolutionary Conservation of pou5f3 Genomic Organization and Its Dynamic Distribution during Embryogenesis and in Adult Gonads in Japanese Flounder Paralichthys olivaceus.

Octamer-binding transcription factor 4 (Oct4) is a member of POU (Pit-Oct-Unc) transcription factor family Class V that plays a crucial role in maintaining the pluripotency and self-renewal of stem cells. Though it has been deeply investigated in mammals, its lower vertebrate homologue, especially in the marine fish, is poorly studied. In this study, we isolated the full-length sequence of Paralichthys olivaceus pou5f3 (Popou5f3), and we found that it is homologous to mammalian Oct4. We identified two transcript variants with different lengths of 3'-untranslated regions (UTRs) generated by alternative polyadenylation (APA). Quantitative real-time RT-PCR (qRT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) were implemented to characterize the spatial and temporal expression pattern of Popou5f3 during early development and in adult tissues. Our results show that Popou5f3 is maternally inherited, abundantly expressed at the blastula and early gastrula stages, then greatly diminishes at the end of gastrulation. It is hardly detectable from the heart-beating stage onward. We found that Popou5f3 expression is restricted to the adult gonads, and continuously expresses during oogenesis while its dynamics are downregulated during spermatogenesis. Additionally, numerous cis-regulatory elements (CRE) on both sides of the flanking regions show potential roles in regulating the expression of Popou5f3. Taken together, these findings could further our understanding of the functions and evolution of pou5f3 in lower vertebrates, and also provides fundamental information for stem cell tracing and genetic manipulation in Paralichthys olivaceus.

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.