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To investigate the impact of the effect of high temperature stimulation on Monopterus albus larvae after a certain period of time, five experimental groups were established at different temperatures. Then, the M. albus under high temperature stress was fed at 30°C for 70 days. After that, the growth index of the M. albus was counted and analyzed. In terms of growth index, high temperature stress had significant effects on FCR, FBW, WGR, and SGR of M. albus (p < 0.05). The SR increased after being stimulated by temperature (p < 0.1). The study revealed that liver cells of M. albus were harmed by elevated temperatures of 36°C and 38°C. In the experimental group, the activities of digestive enzymes changed in the same trend, reaching the highest point in the 32°C group and then decreasing, and the AMS activity in the 38°C group was significantly different from that in the 30°C group (p < 0.05). The activities of antioxidase in liver reached the highest at 34°C, which was significantly different from those at 30°C (p < 0.05). In addition, the expression levels of TLR1, C3, TNF-α, and other genes increased in the experimental group, reaching the highest point at 34°C, and the expression level of the IL-1β gene reached the highest point at 32°C, which was significantly different from that at 30°C (p < 0.05). However, the expression level of the IRAK3 gene decreased in the experimental group and reached its lowest point at 34°C (p < 0.05). The expression level of the HSP90α gene increased with the highest temperature stimulus and reached its highest point at 38°C (p < 0.05). In the α diversity index of intestinal microorganisms in the experimental group, the observed species, Shannon, and Chao1 indexes in the 34°C group were the highest (p < 0.05), and β diversity analysis revealed that the intestinal microbial community in the experimental group was separated after high temperature stimulation. At the phylum level, the three dominant flora are Proteus, Firmicutes, and Bacteroides. Bacteroides and Macrococcus abundance increased at the genus level, but Vibrio and Aeromonas abundance decreased. To sum up, appropriate high-temperature stress can enhance the immunity and adaptability of M. albus. These results show that the high temperature stimulation of 32°C–34°C is beneficial to the industrial culture of M. albus. [ABSTRACT FROM AUTHOR]

Abstract Background Monopterus albus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown. Methods We produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively. Results This effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmβ8 and Ptk2β were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation. Conclusions This study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M. albus, which will also enrich the sex differentiation mechanism of teleosts.

Metamifop (MET) is a widely used herbicide. It is likely for it to enter water environment when utilized, thus potential impacts may be produced on aquatic animals. Little information is available about its effects on the endocrine system of fish to date. In the current study, female rice field eels (Monopterus albus) were exposed to different MET concentrations (0, 0.2, 0.4, 0.6, 0.8 mg L −1) for 96 h to examine the effect of MET on the hypothalamic-pituitary-gonadal (HPG) axis and sexual reversal. The results showed that high concentrations of MET exposure increased vitellogenin (VTG) levels in liver and plasma, but plasma sex hormone levels were not affected by MET exposure. MET exposure increased the expression of CYP19A1b and CYP17 that regulate sex hormone production in the brain, but the expression of genes (CYP19A1a, CYP17, FSHR, LHCGR, hsd11b2, 3β-HSD) associated with sex hormone secretion in the ovary and the estrogen receptor genes (esr1, esr2a, esr2b) in the liver were all suppressed. In addition, the expression of sexrelated gene (Dmrt1) was suppressed. This study revealed for the first time that MET has estrogen-like effects and has a strong interference with the expression of HPG axis genes. MET did not show the ability to promote the sexual reversal in M. albus, on the contrary, the genes expression showed that the occurrence of male pathway was inhibited. [ABSTRACT FROM AUTHOR]

In immature lymphocytes, recombination activating genes 1 and 2 are necessary for antigen receptor V (D) J recombination, representing immature lymphocyte biomarkers. Herein, we cloned and sequenced rice-field eel rag1 and rag2 genes. Their expressions in the thymus, liver, and kidney were significant from 0 days post hatching (dph) to 45 dph, peaking at 45 dph in these three tissues. In situ hybridization detected high rag1 and rag2 expressions in the liver, kidney, and thymus of rice-field eel from 2 to 45 dph, suggesting that multiple tissues of rice-field eel contain lymphocyte lineage cells and undergo lymphopoiesis. Tissue morphology was used to observe lymphopoiesis development in these three tissues. The thymus primordium began to develop at 2 dph, while the kidney and liver have generated. Our findings verified that the thymus is the primary lymphopoietic tissue and suggested that, in rice-field eel, lymphocyte differentiation also occurs in the liver and kidney. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named Ma TLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that Ma TLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that Ma TLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that Ma TLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of Ma TLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the Ma TLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant Ma TLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of Ma TLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that Ma TLR14 recruited MyD88 as adaptor. When overexpressed, Ma TLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, Ma TLR14 plays an important role in the microorganism recognition and signaling transduction. • Ma TLR14 is expressed in the intestinal epithelium of hindgut and mainly localized to internal cellular compartments. • Ma TLR14 is upregulated at mRNA expression level after bacterial and PAMPs stimulation. • Ma TLR14 can bind gram-negative bacteria as well as gram-positive bacteria through the LRR domain. • Ma TLR14 significantly induces the expression of tnf-α and il-6 via NF-κB and MAPK pathways. [ABSTRACT FROM AUTHOR]

An 8‐week trial investigated how andrographolide effected lipopolysaccharide (LPS)‐induced serum biochemical indices, immune responses and the intestinal inflammatory response of Monopterus albus. The study included control and andrographolide groups (150 mg/kg andrographolide) with three replicates per group. Samples were taken 0, 3, 6, 12, 24 and 48 h after LPS injection. The results showed that the levels of GLU, COR, AST, ALT, IgM, LZM, AKP, C3 and C4 in serum showed an initial increasing that then decreased. Compared with that of the control group, the levels of serum LZM, AKP, IgM, C3 and C4 in the andrographolide group increased quicker after LPS injection, while the levels of GLU, COR, AST and ALT increased more slowly; further, these indices recovered to normal levels quicker. The expression of several genes in the intestine first increased and then decreased after LPS injection. Compared with that of the control group, il‐10, tgf‐β1, tgf‐β2 and tgf‐β3 gene expression in the andrographolide group increased quicker after LPS injection, while that of il‐1β, il‐8, il‐12β, nf‐κb, tlr‐3, tlr‐7 and tlr‐8 increased more slowly; these indices recovered to normal levels more quickly. In conclusion, LPS injection induced immune stress and inflammatory response in M. albus. Dietary supplementation with 150 mg/kg andrographolide effectively alleviated these responses. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila causes sepsis and is responsible for significant economic losses in Asian finless eel (Monopterus albus) aquaculture. Previous studies have found that macrophage migration inhibitory factor (MIF) and Cathepsin B (CTSB) may be expressed in many vertebrate tissues and organs. However, there have been no reports discussing the importance of MIF and CTSB in Asian swamp eel. The complete ORF sequences of MAMIF and MACTSB were found to be 348 and 993 bp, respectively. The adaptive analysis showed that the sequences of MAMIF and MACTSB were highly conserved, and only two positive selection sites were identified in MAMIF. Quantitative real time polymerase chain reaction (qRT‐PCR) demonstrated that MAMIF and MACTSB genes were expressed in all tissues evaluated with the highest expression occurring in the head kidney, spleen and liver. Challenge with Aeromonas hydrophila resulted in a significant increase in the expression of MAMIF and MACTSB in skin, liver, head kidney and spleen that peaked at 12‐h post‐infection. The results show that MAMIF and MACTSB play important roles in the immune function of Monopterus albus. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated protein that plays critical roles in innate immunity and pathogenesis of various diseases. To date, teleost STING against viral stimulation has been identified, whereas STING signaling events in fish against bacteria are not well understood. In the present study, the open reading frame (ORF) of STING from Asian swamp eel (Monopterus albus) was cloned (named Ma STING) and its roles in bacterial infection were investigated. Amino acid sequence alignment and phylogenetic analysis revealed that Ma STING had conserved structures with mammalian STING and shared the closest relationship with mandarin fish STING. Subcellular localization analysis showed that Ma STING distributed in the whole cytoplasm and mainly co-localized with ER. Expression pattern analysis found that Ma STING was constitutively expressed in all the examined tissues with the highest expression in the liver and spleen. Post stimulation with bacteria and various PAMPs, the expression of Ma STING was induced at indicated time points in the immune-related organs and isolated peripheral blood leucocytes. Furthermore, the mechanism underlying Ma STING against bacterial infection was further studied. The qPCR analysis showed that Ma STING overexpression promoted 2′3′-cGAMP induced the expression of IFN-1, ISG15, Viperin, Mx, IL-1β and TNF-α. Western blotting assay suggested that Ma STING significantly enhanced the phosphorylation of TANK-binding kinase 1 (TBK1) and p65. Ma STING also significantly increased the luciferase activity of IFN-1 and NF-κB promoters. Taken together, Ma STING is involved in host defense against bacterial infection by inducing the inflammatory response. • The STING gene is cloned from Asian swamp eel (Monopterus albus) STING (named Ma STING). • Ma STING is mainly distributed in immune-associated tissues and significantly upregulated upon bacterial infection. • Ma STING can activate the expression of IFN-1 and proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a , foxl2 , and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10–50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100–200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well. • cyp19a1a , nr5a1a , foxl2 , and dmrt1a were expressed in follicular cells. • Foxl2 up-regulated cyp19a1a transcription via the conserved Foxo site. • Nr5a1a and Foxl2 bound to cyp19a1a promoter to exert their full synergism. • The sequence between Region III and I-box of Nr5a1a was key to the synergism. • Dmrt1a abolished the synergism of Nr5a1a and Foxl2 on c yp19a1a transcription. [ABSTRACT FROM AUTHOR]

Background: The natural sex reversal severely affects the sex ratio and thus decreases the productivity of the rice field eel (Monopterus albus). How to understand and manipulate this process is one of the major issues for the rice field eel stocking. So far the genomics and transcriptomics data available for this species are still scarce. Here we provide a comprehensive study of transcriptomes of brain and gonad tissue in three sex stages (female, intersex and male) from the rice field eel to investigate changes in transcriptional level during the sex reversal process. Results: Approximately 195 thousand unigenes were generated and over 44.4 thousand were functionally annotated. Comparative study between stages provided multiple differentially expressed genes in brain and gonad tissue. Overall 4668 genes were found to be of unequal abundance between gonad tissues, far more than that of the brain tissues (59 genes). These genes were enriched in several different signaling pathways. A number of 231 genes were found with different levels in gonad in each stage, with several reproduction-related genes included. A total of 19 candidate genes that could be most related to sex reversal were screened out, part of these genes’ expression patterns were validated by RT-qPCR. The expression of spef2, maats1, spag6 and dmc1 were abundant in testis, but was barely detected in females, while the 17β-hsd12, zpsbp3, gal3 and foxn5 were only expressed in ovary. Conclusion: This study investigated the complexity of brain and gonad transcriptomes in three sex stages of the rice field eel. Integrated analysis of different gene expression and changes in signaling pathways, such as PI3K-Akt pathway, provided crucial data for further study of sex transformation mechanisms. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous small RNAs that can regulate target mRNAs by binding to their sequences in the 3′ untranslated region. The expression of miRNAs and their biogenetic pathway are involved in sexual differentiation and in the regulation of the development of germ cells and gonadal somatic cells. The rice field eel (Monopterus albus) undergoes a natural sexual transformation from female to male via an intersex stage during its life cycle. To investigate the molecular mechanisms of this sexual transformation, miRNAs present in the different sexual stages of the rice field eel were identified by high-throughput sequencing technology. A significantly differential expression among the 3 genders (p < 0.001) was observed for 48 unique miRNAs and 3 miRNAs*. Only 9 unique miRNAs showed a more than 8-fold change in their expression among the 3 genders, including mal-miR-430a and mal-miR-430c which were higher in females than in males. However, mal-miR-430b was only detected in males. Several potential miRNA target genes (cyp19a, cyp19b, nr5a1b, foxl2 amh, and vasa) were also investigated. Real-time RT-PCR demonstrated highly specific expression patterns of these genes in the 3 genders of the rice field eel. Many of these genes are targets of mal-miR-430b according to the TargetScan and miRTarBase. These results suggest that the miR-430 family may be involved in the sexual transformation of the rice field eel. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We isolated the complete Foxl2 (Foxl2a) cDNA from the Monopterus albus ovary. An alignment of known Foxl2 amino-acid sequences confirmed the conservation of the Foxl2 open reading frame, especially the forkhead domain and C-terminal region. The expression of Foxl2 was detected in the brain, eyes, and gonads. A high level of Foxl2 expression in the ovary before sex reversal, but its transcripts decreased sharply when the gonad developed into the ovotestis and testis. The correlation between the Foxl2 expression and the process of sex development revealed the important function of Foxl2 during the sex reversal of M. albus. Immunohistochemical analysis showed that Foxl2 was expressed abundantly in granulosa cells and in the interstitial cells of the ovotestis and testis. These results suggest that Foxl2 plays a pivotal role in the development and maintenance of ovarian function. Foxl2 may be also involved in the early development of testis and the development of ocular structures of M. albus. [ABSTRACT FROM AUTHOR]

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l−1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g−1). Na+/K+-ATPase (Nka) is an essential transporter in brain cells, and since NH4+ can substitute for K+ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K+ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na+/K+-ATPase and Na+/NH4+-ATPase activities over a range of K+/NH4+ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l−1 NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na+/NH4+-ATPase activities were significantly lower than the Na+/K+-ATPase activities assayed at various NH4+/K+ concentrations. Furthermore, the effectiveness of NH4+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K+ specificity of K+ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus. [ABSTRACT FROM AUTHOR]

Abstract: CYP19A1 in the brain and pituitary of vertebrates is important for reproductive and non-reproductive processes. In teleosts, it is broadly accepted that estradiol (E2) up-regulates cyp19a1b gene via a positive autoregulatory loop. Our present study, however, showed that E2 did not up-regulate ricefield eel cyp19a1b in the hypothalamus and pituitary, whereas dihydrotestosterone (DHT) or testosterone (T) stimulated cyp19a1b expression only in the pituitary. Two tissue-specific promoters, namely promoter I and II directing the expression in the brain and pituitary respectively, were identified. Promoter I contained a non-consensus estrogen response element (ERE), and consequently did not respond to E2. Promoter II contained an androgen response element (ARE) and consequently responded to DHT. Taken together, these results demonstrated a novel steroidal regulation of cyp19a1b gene expression and an alternative usage of tissue-specific cyp19a1b promoters in the brain and pituitary of a teleost species, the ricefield eel. [Copyright &y& Elsevier]

An eight-week feeding trial was performed to determine how methionine regulates muscle growth in the rice field eel Monopterus albus (initial weight 25.08 ± 0.31 g). Six isonitrogenous and isoenergetic experimental feeds were formulated, and different levels of methionine (0, 2 g/kg, 4 g/kg, 6 g/kg, 8 g/kg, or 10 g/kg) were added to the basic diet (110 g/kg fish meal, 400 g/kg soy concentrate meal). The weight gain rate (WGR) of M. albus markedly increased as methionine concentrations increased to 6 g/kg (P < 0.05) and gradually decreased as methionine concentrations increased to 10 g/kg. The feed conversion rate of M. albus showed a trend opposite to the WGR. Muscle glycogen markedly increased as methionine concentrations increased to 2 g/kg (P < 0.05), while the creatine kinase, lactic acid and lactate dehydrogenase contents of muscle markedly decreased as dietary methionine concentrations increased to 2 g/kg (P < 0.05). The content of amino acids, total essential amino acids, total nonessential amino acids and total amino acids in muscle was increased by supplementation with 8 g/kg and 10 g/kg methionine. The springiness of muscle markedly increased as methionine supplementation increased to concentrations of 6 g/kg (P < 0.05). The hardness, gumminess and chewiness of muscle markedly increased as methionine concentrations increased to 8 g/kg (P < 0.05). When supplemented with 8 g/kg and 10 g/kg methionine, the density of muscle fibers and the percentage of muscle fibers under 60 μm were remarkably decreased, while the percentage of muscle fibers greater than or equal to 100 μm was remarkably increased (P < 0.05). When 8 g/kg and 10 g/kg methionine were added, gcn2 and mstn mRNA expression was significantly downregulated (P < 0.05), while gh , ghr , TnT , mhc , mlc , myf5 , myod2 , myog , pax7 and mef2a mRNA expression was remarkably upregulated (P < 0.05). Correlative analysis of muscle-related gene expression showed that myf5 , mlc , and myog expression was negatively correlated with gcn2 expression (P < 0.05), and ghr , mef2b , gh , and mhc expression was negatively correlated with gcn2 expression (P < 0.01), while mstn expression was positively correlated with gcn2 expression (P < 0.01). In conclusion, dietary methionine increased the growth performance of M. albus , especially for muscle growth and development, improved muscle texture and main nutritional factors (amino acids, glycogen), and reduced the content of lactic acid in muscle. • Dietary methionine enhanced muscle growth by increasing diameters of muscle fibers. • Dietary methionine improved muscle texture, amino acids and glycogen of muscle. • gcn2 gene could be activated when M. albus feed intake methionine deficiency diet. [ABSTRACT FROM AUTHOR]

• Krill meal (KM) is a promising fish meal replacement and the optimal replacement ratio is 22.49 %. • Replacing fishmeal with KM improved the growth performance and improve immune function of Monopterus albus. • Replacing fishmeal with KM increased the muscle textural quality of Monopterus albus. • Replacing fishmeal with KM upregulated the mRNA expression levels of myogenic regulation factors of Monopterus albus. An 8-week feeding experiment was conducted to evaluate the effects of replacing fish meal (FM) with krill meal (KM) on the growth, immunity, muscle textural quality, and expression of myogenic regulation factors of Monopterus albus (initial body weight: 23.00 ± 0.10 g). Five experimental diets consisted of different proportions of FM (0, 25, 50, 75, and 100 g/kg) were substituted with KM, corresponding to dietary inclusion levels of 0 (KM0), 28.8 g/kg (KM29), 57.6 g/kg (KM58), 86.4 g/kg (KM86), and 115.2 g/kg (KM115). The results showed that: (1) the weight gain rate (WGR) of the fish in the KM58, KM86, and KM115 groups were significantly higher than those of the KM0 group, whereas the opposite result was observed for the feed conversion ratio. (2) Serum complement 3 content and superoxide dismutase activity in the KM58 group and alkaline phosphatase activity in the KM29, KM58, and KM115 groups were significantly higher than those in the KM0 group. However, there were no significant differences in serum immunoglobulin M, complement 4, malondialdehyde content, and catalase activity among all groups. (3) The highest levels of hardness, cohesiveness, springiness, chewiness, gumminess, and adhesiveness of muscle tissues of M. albus fed with the KM58 diet were significantly higher than those of the other treatments. (4) No remarkable difference was detected in MEF 2A, MEF 2D, MyoG, and Myf 5 mRNA expression levels in the muscle tissues among the dietary treatments. However, MyoD 2 , Myh 6, MyoD 1, MRF 4, MEF 2B, and MEF 2C mRNA expression levels were significantly upregulated by KM replacement. In conclusion, these results suggested that dietary inclusion levels of 57.6–115.2 g/kg KM improved the growth performance, immune function, muscle textural quality, and expression of myogenic regulation factors of M. albus. Based on the WGR, the optimal level of KM was 103.08 g/kg. [ABSTRACT FROM AUTHOR]

The present study aims to reveal the mechanism by which miR-430s regulate steroidogenesis in larval rice field eel Monopterus albus. To this end, M. albus embryos were respectively microinjected with miRNA-overexpressing mimics (agomir430a, agomir430b, and agomir430c) or miRNA-knockdown inhibitors (antagomir430a, antagomir430b, and antagomir430c). Transcriptome profiling of the larvae indicated that a total of more than 149 differentially expressed genes (DEGs) were identified among the eight treatments. Specifically, DEGs related to steroidogenesis, the GnRH signaling pathway, the erbB signaling pathway, the Wnt signaling pathway, and other pathways were characterized in the transcriptome. We found that steroidogenesis-related genes (hydroxysteroid 17-beta dehydrogenase 3 (17β-hsdb3), hydroxysteroid 17-beta dehydrogenase 7 (17β-hsdb7), hydroxysteroid 17-beta dehydrogenase 12 (17β-hsdb12), and cytochrome P450 family 19 subfamily a (cyp19a1b)) were significantly downregulated in miR-430 knockdown groups. The differential expressions of miR-430 in three gonads indicated different roles of three miR-430 (a, b, and c) isoforms in regulating steroidogenesis and sex differentiation. Mutation of the miR-430 sites reversed the downregulation of cytochrome P450 family 17 (cyp17), cyp19a1b, and forkhead box L2 (foxl2) reporter activities by miR-430, indicating that miR-430 directly interacted with cyp17, cyp19a1b, and foxl2 genes to inhibit their expressions. Combining these findings, we concluded that miR-430 regulated the steroidogenesis and the biosynthesis of steroid hormones by targeting cyp19a1b in larval M. albus. Our results provide a novel insight into steroidogenesis at the early stage of fish at the molecular level. [ABSTRACT FROM AUTHOR]