Comparative Analysis of mRNA, microRNA of Transcriptome, and Proteomics on CIK Cells Responses to GCRV and Aeromonas hydrophila.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.

Characterization and function of Japanese flounder (Paralichthys olivaceus) slc2a6 in response to lymphocystis disease virus infection.

Slc2a6 is a member of the slc2 family (solute carrier 2 family) and previous reports have indicated its involvement in the inflammatory response. Slc2a6 is regulated by the NF-ĸB signaling pathway. This study investigated the differential expression of slc2a6 in the early embryonic development of Japanese flounder, revealing that the early gastrula stage had the highest level of slc2a6 expression. Moreover, slc2a6 expression was increased in vitro after stimulation by lymphocystis disease virus (LCDV), and in vivo experiments also showed significantly elevated levels in the spleen and muscle tissues following LCDV stimulation. Subcellular localization revealed that Slc2a6 was expressed in both the nucleus and cytoplasm of cells. The pcDNA3.1-slc2a6 overexpression plasmid was successfully constructed; the si-slc2a6 interfering strand was screened and samples were collected. The expression of NF-ĸB signaling pathway-related genes il-1ß, il-6, nf-ĸb, and tnf-α was evaluated in overexpressed, silenced, and LCDV-stimulated samples. The results showed that slc2a6 is involved in viral regulation in Japanese flounder by regulating innate immune responses.

Role of Thyroid Hormone in Dynamic Variation of gdf6a Gene during Metamorphosis of Paralichthys olivaceus.

The Japanese flounder (Paralichthys olivaceus) is a marine fish that undergoes a dramatic postembryonic metamorphosis, with the right eye shifting to the left and its lifestyle transitioning from planktonic to benthic. As the light environment of the habitat changes from bright to dim, its photoreceptor system also undergoes adaptive change. Growth differentiation factor 6a (Gdf6a) is a member of the BMP family, which plays a key role in regulating the dorsal-ventral pattern of the retina and photoreceptor fate, and the differentiation of different photoreceptors is also modulated by a thyroid hormone (TH) binding its receptor (TR). However, the relationship between gdf6a and TH and its role in the regulation of photoreceptors during flounder metamorphosis is still poorly understood. In this study, bioinformatics analysis showed that Gdf6a had a conserved TGFB structural domain and clusters with fishes. The expression analysis showed that the expression of gdf6a was highest in the eye tissue of adult flounder and tended to increase and then decrease during metamorphosis, reaching its highest levels at the peak of metamorphosis. Moreover, the expression of gdf6a increased in the early stages of metamorphosis after exogenous TH treatment, while it was inhibited after exogenous thiourea (a TH inhibitor, TU) treatment. To further investigate the targeting role of TH and gdf6a in the metamorphosis of flounder, the results of the Dual-Luciferase revealed that triiodothyronine (T3) may regulate the expression of gdf6a through TRß. In conclusion, we speculate that TH influences the development of cone photoreceptors during the metamorphosis of the flounder by regulating the expression of gdf6a.

A proteomic approach reveals biomineralization and immune response for mantle to pearl sac in the freshwater pearl mussel (Hyriopsis cumingii).

In the process of production of freshwater pearl, implanted mantle pieces undergo a series of complex physiological and biochemical processes to form pearl sac, which produce pearl. This is a very important site of occurrence due to immune-induced biomineralization, while its molecular regulatory mechanism is still unclear. Here, we use proteomics to identify differentially expressed proteins (DEPs) of the mantle and pearl sac and examine the biomineralization and immune response of the pearl sac formation process in Hyriopsis cumingii. Using iTRAQ technology and bioinformatics analysis, we obtained DEP profiles between the mantle and pearl sac. A total of 1871 proteins were identified. Of these, 74 DEPs were found between the pearl sac and outer mantle, 112 DEPs between the pearl sac and inner mantle, and 124 DEPs between the outer and inner mantles. Bioinformatics analysis revealed that the screened biomineralization-related DEPs were mainly enriched in signaling pathways associated with calcium signaling, regulation of the actin cytoskeleton and protein processing in the endoplasmic reticulum, while the immune-related DEPs were mainly enriched in the Notch, Hippo, nuclear factor kappa-B (NF-κB), and transforming growth factor-ß (TGF-ß) signaling pathways. In addition, the expression of six biomineralization-related and four immune-related proteins were verified at the transcriptional level using quantitative real-time PCR. Our findings contribute to furthering the understanding of the mechanisms of pearl formation and immune response, and have long-term implications for future studies on the production of high-quality freshwater pearls and development of the freshwater pearl industry.

Molecular identification and function characterization of four alternative splice variants of trim25 in Japanese flounder (Paralichthys olivaceus).

Trim25 is a member of Tripartite Motif (TRIM) family. Previous studies report that trim25 modulates antiviral activity by activating RIG-I. In this study we explored the four alternative splicing (AS) variants X1-X4 of Japanese flounder trim25. The sequences of the AS variants were highly conserved. Expression levels of trim25 X1-X4 were increased after 12 h of poly I:C treatment in vitro. In vivo expression of X2-X4 in liver, kidney (except X2) and blood was significantly up-regulated in early stages of poly I:C treatment. Subcellular localization analysis showed that Trim25 X1-X4 were distributed in different cellular organelles. The recombinant vector pcDNA3.1-Trim25 X1-X4 were successfully overexpressed in Flounder cells and the samples were collected. Expression patterns of RIG-I pathway genes dhx58, traf6, traf2, nfkbia and il-8 were explored in vitro and in vivo after poly I:C treatment, as well as overexpressed samples. The findings of this study imply that AS variants of trim25 confer antiviral activity in Japanese flounder by modulating innate immune response.

Molecular characterization and functional analysis of Japanese flounder (Paralichthys olivaceus) thbs2 in response to lymphocystis disease virus.

In mammals, a matricellular protein, thrombospondin 2 (Thbs2) has been reported to play important roles in modulating cell-matrix interactions, vascular integrity and thrombosis formation. However, the role of gene, thbs2 has not yet been studied in teleost. In the present study, this novel fish gene from Japanese flounder was cloned and its function in resistant to lymphocystis disease virus was elucidated. The Japanese flounder thbs2 encoded a 1176-amino acid protein with 91% identity to medaka. Amino acid sequence indicated that Japanese flounder Thbs2 contained 10 typical conserved domains. The thbs2 was expressed in all stages of embryo development, and in hatched larva stage, its expression was significantly higher than that in other stages (P < 0.05). The relative expression level of thbs2 was significantly higher in the head kidney, liver, blood, gill, and heart of the lymphocystis disease virus resistant fish than in sensitive fish (P < 0.05); and in muscle, this difference was at highly significant (P < 0.01). Additionally, the distribution of Thbs2 in tissue was evaluated by immunohistochemical staining. Subcellular localization analysis showed that Thbs2 was distributed throughout the cytoplasm of the cells. Taken together, our results provide new basic data for thbs2 function, especially its role in anti-lymphocystis disease virus immune response.

Proteomic variation in metamorphosing Paralichthys olivaceus induced by exogenous thyroid hormone.

Thyroid hormone (TH) is essential for Paralichthys olivaceus metamorphosis. Exogenous TH treatment induces premature metamorphosis in P. olivaceus larvae and a series of studies have been conducted to identify thyroid hormone-regulated functional genes and microRNAs involved in the metamorphosis of P. olivaceus; however, the proteins involved in this process remain to be fully clarified. In this study, the differential proteomic responses of P. olivaceus larvae to exogenous TH treatment were examined using tandem mass tags (TMT) for quantitation labeling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The expression levels of 629 cellular proteins were identified to be significantly affected by TH treatment. The reliability of our TMT-labeled LC-MS/MS analysis was verified by examining the mRNA and protein levels of four selected proteins using quantitative real-time reverse-transcription PCR and western blot analyses. The possible biological significance of these proteins was further investigated by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analyses. Notably, we identified and described five groups of proteins involved in different important life events that were significantly regulated by exogenous TH treatment. Our study provides an improved understanding of the molecular mechanisms by which TH regulates the metamorphosis of P. olivaceus.

Characterization and expression of lin-28a involved in lin28/let-7signal pathway during early development of P. olivaceus.

Heterochronic lin-28 is a conserved RNA-binding protein that plays a key role in the timing of developmental events in organisms. As a crucial heterochronic gene, the protein controls developmental events of the second of four larval stages in Caenorhabditi elegans. Heterochronic let-7 miRNAs are often present in various species and highly conserved in sequence and biological function and are required for various biological processes. Previous studies showed that ten let-7 miRNAs were identified in the Japanese flounder (Paralichthys olivaceus) and that they were primarily expressed during metamorphosis. In this study, we clone and characterize the lin-28a gene from P. olivaceus and exhibit its dynamic expression pattern at different developmental stages and various adult tissues. The results show that the P. olivaceus lin-28a gene has high sequence similarity with other species and is highly expressed in the embryonic stage but weakly expressed in the larval stage. In addition, lin-28a overexpression causes cell proliferation and significantly promotes the levels of pre-let-7a and pre-let-7d while markedly depressing let-7a and let-7d expression in FEC (Flounder Embryonic Cell), which indicate that lin-28 possibly blocks the maturation of let-7 miRNAs. Additionally, lin-28a is identified as a target gene of let-7 miRNAs, and let-7 miRNAs directly regulate lin-28a expression by targeting its 3' UTR. Taken together, lin-28a along with let-7 miRNA participates in a lin-28/let-7 axis pathway that regulates cell division and timing of embryonic and metamorphic events in P. olivaceus.

Coordinated expression and regulation of deiodinases and thyroid hormone receptors during metamorphosis in the Japanese flounder (Paralichthys olivaceus).

In vertebrates, thyroid hormone receptors (TRs) and deiodinases are essential for developmental events driven by the thyroid hormones (THs). However, the significance of deiodinases during the metamorphosis of the Japanese flounder (Paralichthys olivaceus) remains unclear. Moreover, regulation and response of the TRs and deiodinases to THs in this fish are poorly understood. Therefore, we detected the expression patterns of THs, deiodinases, and TRs in drug-treated larvae and untreated larvae of P. olivaceus by using enzyme-linked immunosorbent assay and quantitative real-time PCR during P. olivaceus metamorphosis. To further understand the roles of these elements, a rescue assay was performed. Our results show the importance of THs, TRs, and deiodinases in flatfish metamorphosis. Our results also confirm that D1 and D2 activate THs and D3 plays the opposite and complementary role. Moreover, we demonstrated that both TRα and TRß have important but different roles during P. olivaceus metamorphosis.

Identification and expression of SRF targeted by miR-133a during early development of Paralichthys olivaceus.

Serum response factor (SRF) is a MADS-box transcription factor that regulates the expression of genes involved in development, metabolism, cell proliferation, and differentiation. In the present study, we cloned the full-length SRF cDNA which includes the coding region of 1503 bp, a 573-bp 5'untranslated region (UTR) and a 400-bp 3'-UTR. The deduced 501 amino acid sequence of the SRF protein contained a MADS domain and NLS at the N terminus, similar to other organisms, and it also is highly phylogenetically conserved. SRF mRNA is ubiquitously expressed in various tissues, with the highest level in the kidneys, and it is also highly expressed during the embryonic and metamorphic stages. During metamorphosis, the SRF mRNA levels are down-regulated by exogenous thyroid hormone (TH) at 17 dph and by thiourea (TU) at 29, 36, and 41 dph, whereas SRF mRNA levels were significantly up-regulated by the added exogenous TH to the TU-treated larvae at 41 dph, which indicates that thyroid hormone is essential for expression of SRF mRNA, so, higher levels of TH did not result in changes of SRF mRNA levels, while TH deficiency or inhibited by the non-specific TU toxicity cause down-regulation of SRF mRNA, which indicated that TH can indirectly affect the SRF mRNA levels. Meanwhile, using a luciferase reporter assay, we verified that SRF is a common target gene of miR-133a which is a muscle-specific microRNA (miRNA), which indicated that SRF may be involved in the signaling pathway of miRNA that regulates muscle development.

A key gene of the small RNA pathway in the flounder, Paralichthys olivaceus: identification and functional characterization of dicer.

Dicer is critical for producing mature microRNAs (miRNAs) from precursor molecules and small interfering RNAs and plays an important role in controlling development and metabolism. In the present study, we cloned the flounder dicer gene, which is 6585 nucleotides (nt), including a 5'-untranslated region (UTR) of 231 nt, a 3'-UTR of 663 nt and an open reading frame of 5691 nt encoding a polypeptide of 1897 amino acids, and analyzed the conservation and expression pattern of dicer. The tissue distribution analysis indicated that dicer is abundantly expressed in the brain, heart, liver, spleen, stomach, kidney, gill, muscle, intestine and gonad of adult fish. Temporal expression analysis indicated that dicer mRNA is highly expressed during the embryonic and early larval stages, and exhibits low expression during the metamorphic stages. Treatment with thyroid hormone (TH) or thiourea indirectly or directly up-regulated dicer mRNA levels at 17 and 23 dph, whereas treatment with TH down-regulated dicer mRNA levels at 36 dph. The dicer-specific siRNA significantly down-regulated dicer mRNA and pol-let-7d levels, while pol-let-7d precursor levels were not differentially changed compared with the control (NC). These results demonstrated that dicer plays a key role in development and metabolism through the production of mature miRNAs, providing basic information for further studies concerning the role of dicer in Paralichthys olivaceus development.

Identification and characterization of TOR in Macrobrachium rosenbergii and its role in muscle protein and lipid production.

The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation.

Thyroid Hormone Signaling Is Required for Dynamic Variation in Opsins in the Retina during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus).

In the present study, we investigated the function of thyroid hormones (TH) in visual remodeling during Japanese flounder (Paralichthys olivaceus) metamorphosis through cellular molecular biology experiments. Our results showed that the expression of the five opsin genes of the flounder were highest in eye tissue and varied with the metamorphosis process. The expression of rh1, sws2aß and lws was positively regulated by exogenous TH, but inhibited by thiourea (TU) compared to the control group. In addition, there was a significant increase in sws2aß and lws in the rescue experiments performed with TU-treated larvae (p < 0.05). Meanwhile, T3 levels in flounder larvae were increased by TH and decreased by TU. Based on the differences in the expression of the three isoforms of the thyroid hormone receptor (TR) (Trαa, Trαb and Trß), we further hypothesized that T3 may directly or indirectly regulate the expression of sws2aß through Trαa. This study demonstrates the regulatory role of TH in opsins during flounder metamorphosis and provides a basis for further investigation on the molecular mechanisms underlying the development of the retinal photoreceptor system in flounders.

Presence of immune factors in freshwater mussel (Hyriopsis cumingii) entails autologous serum an essential component in the culture of mantle cells.

Mussel cell culture is a challenging problem and serum serves a crucial biological role in cell culture as an autologous supply and an immunizing agent. In this study, the biology (calcium ions, total protein, pH, and osmotic pressure) of fetal bovine serum (FBS) and Hyriopsis cumingii serum (HCS) was investigated, and the development of Hyriopsis cumingii (H. cumingii) mantle cells in HCS and FBS systems was examined. The results showed that total protein, calcium ions, and osmotic pressure varied significantly (p<0.05). The activity of mantle cells was superior in the HCS culture system to that in the FBS culture system. The label-free technique was used to distinguish the two serum proteins to investigate the supportive effect of autologous serum on cell culture. These were examined for 109 unique proteins and 35 particular HCS proteins. Most differentially expressed proteins (DEPs) were involved in immune response, cell differentiation, and calcium ion binding. Furthermore, immune factors such as HSP, CALR, APOB, C3 were identified with significant differences. HSP was significantly more present in HCS than in FBS as an endogenous protective protein that regulates immune system function, cell differentiation, transport, and activity regulation. Parallel reaction monitoring (PRM) analysis was carried out to validate the expression levels of 19 DEPs, indicating high reliability of the proteomic results. This study reveals the important role of immune factors in mussel cell culture, providing a theoretical basis for explaining the applicability of autologous serum in cell culture. It is also helpful in improving the cell culture conditions of mussels.

Enzymatic Stress Responses of Coreius guichenoti to Microplastics with Different Particle Sizes.

The wild population resources of Coreius guichenoti have sharply declined in recent decades, and any negative factors may have a significant impact on their survival. In this study, the enzymatic stress responses of C. guichenoti to 25 and 48 µm polyethylene fragments were explored for the first time. This was achieved by evaluating the changes in physiological and biochemical indicators of the species in response to the environmental stimuli of microplastics. In this study, we observed an early stress response in the external tissues of C. guichenoti following exposure to microplastics. The TP content in skin and muscle and the MDA content in skin, gill and muscle initially showed a significant increase. The skin, gill, and muscle exhibited greater stress responses to M5 particles, whereas M3 particles caused a greater response in the intestine and especially the liver. After the removal of microplastic exposure, the stress state of the C. guichenoti would be alleviated in a short period, but it could not fully recover to the pre-exposure level. In summary, microplastics pose a significant threat to C. guichenoti. While their negative effects can be alleviated by the removal of microplastics exposure, full recovery does not occur in a short period. Continuous monitoring of microplastics in natural waters and targeted aquatic ecological restoration are essential to ensure the normal growth and reproduction of the wild population of C. guichenoti.

Transcriptome analysis of growth and shell color between two genetic variants of Corbicula fluminea with different shell colors.

To clarify the molecular mechanism of the black and yellow shell coloration, we performed a transcriptome analysis of whole tissue of Corbicula fluminea in Hongze Lake (Jiangsu Province, China). After assembly, 335,247 unigenes were obtained, and 136,804 unigenes were functionally identified using public databases (NR, GO, KEGG, eggnog, and Swissprot). 1567 differentially expressed genes (DEGs) were detected through pairwise comparisons, of which 941 DEGs were up-regulated and 626 were down-regulated in the black-shelled clam. We compared the DEGs between two clams and identified some coloration-related genes. Notably, the black-shelled clam was larger than the yellow-shelled. We speculated that higher digestion and anabolic ability of black-shelled clam might lead to this phenomenon. In contrast, the yellow-shelled clam appeared to be more sensitive to environmental stress. The metabolic energy of the yellow-shelled clam was depleted to maintain or recover from stress, and provide less energy for growth. In summary, our finding provides a theoretical basis for the molecular mechanism of pigmentation and the difference of somatotype in bivalve, as well as promotes the future breeding of more elite varieties.

Correction: Li et al. Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii). Biology 2022, 11, 1369.

In the original publication [...].

A New Cell Line Derived from the Spleen of the Japanese Flounder (Paralichthys olivaceus) and Its Application in Viral Study.

A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1ß, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.

Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (Hyriopsis cumingii).

Insulin-like growth factor 1 (IGF1) plays an important regulatory role in the regulation of growth, differentiation, and anabolism in a variety of cells. In this study, the full-length cDNA of the IGF1 gene was cloned from Hyriopsis cumingii, named HcIGF1. The expression level of HcIGF1 in six tissues (adductor muscle, foot, hepatopancreas, gill, mantle, and gonad) was determined. In addition, the localization of HcIGF1 in the mantle was analyzed by in situ hybridization, and finally the function of HcIGF1 was explored by RNA interference and prokaryotic expression. The results showed that the amino acid sequence contained a typical IIGF structural domain. The phylogenetic tree showed that HcIGF1 clustered with other marine bivalve sequences. Quantitative real-time PCR and in situ hybridization analysis showed that HcIGF1 was expressed in all tissues. The highest expression was in the foot and the lowest was in the mantle. In the mantle tissue, the hybridization signal was mainly concentrated in the outer mantle. After RNA interference, the expression of IGF1 was found to be significantly decreased (p < 0.05), and its related genes IGF1R, AKT1, and cyclin D2 were downregulated, while MAPK1 were upregulated. The recombinant HcIGF1 protein was purified and its growth-promoting effect was investigated. The results showed that the recombinant HcIGF1 protein could significantly promote the proliferative activity of the mantle cells of mussels, with the best proliferative effect at 12.5 µg/mL. The results of this study provide a new method to solve the problem of weak proliferation of shellfish cells in vitro and lay the foundation for further understanding of the growth regulation mechanism of H. cumingii, as well as a better understanding of the physiological function of IGF1 in mollusks.

Transcriptome analysis provides insights into long noncoding RNAs in medaka gonads.

Long non-coding RNAs (lncRNAs) are gradually regarded as regulators in sex determination and gonad development of various animals. Medaka (Oryzias latipes) is an excellent reproductive research model with sex-determining genes. However, the regulation of gonadal lncRNAs on medaka reproductive development remains unknown. Here, 5317 lncRNAs were obtained from medaka ovary and testis by Illumina HiSeq4000, among which 177 lncRNAs were up-regulated and 120 lncRNAs were down-regulated in the testis compared to the ovary. In addition, 6904 cis-regulated target genes were predicted from 3099 lncRNAs. GO and KEGG enrichment analysis showed that these target genes were mainly involved in phosphorylation, metabolic, metabolism of xenobiotics by cytochrome P450, insulin secretion, and GnRH signaling pathways. Furthermore, six highly expressed lncRNAs were randomly selected to verify the sequencing data by quantitative real time PCR (qRT-PCR). Next, in situ hybridization revealed that one of the sex-biased lncRNA MSTRG.14827.1 was highly expressed in immature germ cells, indicating MSTRG.14827.1 may play a key role in gametogenesis. Taken together, this study provides emerging lncRNA libraries and opens new avenues for future investigation of lncRNAs in medaka.