Rice Field Eel Monopterus Albus Research Articles

Protein expression patterns and metal metabolites in a protogynous hermaphrodite fish, the ricefield eel (Monopterus albus)

BackgroundThe ricefield eel Monopterus albus undergoes a natural sex change from female to male during its life cycle, and previous studies have shown the potential mechanisms of this transition at the transcriptional and protein levels. However, the changes in protein levels have not been fully explored, especially in the intersexual stage.ResultsIn the present study, the protein expression patterns in the gonadal tissues from five different periods, the ovary (OV), early intersexual stage gonad (IE), middle intersexual stage gonad (IM), late intersexual stage gonad (IL), and testis (TE), were determined by untargeted proteomics sequencing. A total of 5125 proteins and 394 differentially expressed proteins (DEPs) were detected in the gonadal tissues. Of the 394 DEPs, there were 136 between the OV and IE groups, 20 between the IM and IE groups, 179 between the IL and IM groups, and 59 between the TE and IL groups. Three candidate proteins, insulin-like growth factor 2 mRNA-binding protein 3 isoform X1 (Igf2bp3), triosephosphate isomerase (Tpi), and Cu-Zn superoxide dismutase isoform X1 [(Cu-Zn) Sod1], were validated by western blotting to verify the reliability of the data. Furthermore, metal metabolite-related proteins were enriched in the IL vs. IM groups and TE vs. IL groups, which had close relationships with sex change, including Cu2+-, Ca2+-, Zn2+- and Fe2+/Fe3+-related proteins. Analysis of the combined transcriptome data revealed consistent protein/mRNA expression trends for two metal metabolite-related proteins/genes [LOC109953912 and calcium Binding Protein 39 Like (cab39l)]. Notably, we detected significantly higher levels of Cu2+ during the sex change process, suggesting that Cu2+ is a male-related metal metabolite that may have an important function in male reproductive development.ConclusionsIn summary, we analyzed the protein profiles of ricefield eel gonadal tissues in five sexual stages (OV, IE, IM, IL, and TE) and verified the plausibility of the data. After preforming the functional enrichment of metal metabolite-related DEPs, we detected the contents of the metal metabolites Zn2+, Cu2+, Ca2+, and Fe2+/Fe3+ at these five stages and screened for (Cu-Zn) Sod1 and Mmp-9 as possible key proteins in the sex reversal process.

Effects of Corbicula fluminea meal in high-fat diet on growth, lipid metabolism and intestinal microbiota of juvenile rice field eel Monopterus albus

Corbicula fluminea meal was added to a high-fat diet for eight weeks to determine its effects on growth, lipid metabolism, and intestinal microbiota in juvenile rice field eel (Monopterus albus). C. fluminea meal was made of dried and crushed soft parts of freshwater clam. The study included four diets: a normal fat diet (6.63% lipid, CON), a high fat diet (12.61% lipid, HFD), and a high fat diet supplemented with 1% or 2% C. fluminea meal (CFM1 and CFM2, respectively). The results showed that: (1) the growth performance was higher in the HFD, CFM1 and CFM2 group compared with the CON group, the hepatosomatic index (HSI) was significantly higher in fish fed the high-fat diet than in those fed other diets. (2) Compared with the CON group, The levels of serum triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C) contents, glutamate pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and the number of hepatic lipid droplets were significantly increased in the HFD group. The addition of C. fluminea meal both reduced liver damage and fat deposition. (3) The mRNA expressions of pparγ, fas, acc, me1, srebp1, g6pdh, hsl, lpl, cpt1, and cpt2 in the HFD group were significantly upregulated, while HFD supplemented with C. fluminea meal significantly downregulated the mRNA expressions of pparγ, fas, acc, me1, srebp1 and g6pdh and significantly upregulated the mRNA expressions of hsl and lpl. (4) The alpha diversity of intestinal microbial community in the HFD group was higher than those in the CON and CFM1 groups. The beta diversity revealed the microbial composition differed in the HFD group from the CON and CFM1 groups. The abundance of Firmicutes in the HFD group increased significantly compared with the CON and CFM1 groups, while the abundances of Cetobacterium decreased. In conclusion, the high-fat diet resulted in abnormal liver fat deposits and intestinal microbiota dysfunction in M. albus, but HFD supplemented with 1% or 2% C. fluminea meal reduced these negative effects.

Neuropeptide B (NPB) and NPB receptor 2b (NPBWR2b) in the ricefield eel Monopterus albus: expression and potential involvement in the regulation of gonadotropins.

The neuropeptide B/W signaling system is composed of neuropeptide B (NPB), neuropeptide W (NPW), and two cognate receptors, NPBWR1 and NPBWR2, which are involved in diverse physiological processes, including the central regulation of neuroendocrine axes in vertebrates. The components of this signaling system are not well conserved during vertebrate evolution, implicating its functional diversity. The present study characterized the ricefield eel neuropeptide B/W system, generated a specific antiserum against the neuropeptide B/W receptor, and examined the potential roles of the system in the regulation of adenohypophysial functions. The ricefield eel genome contains npba, npbb, and npbwr2b but lacks the npw, npbwr1, and npbwr2a genes. The loss of npw and npbwr1 probably occurred at the base of ray-finned fish radiation and that of npbwr2a species specifically in ray-finned fish. Npba and npbb genes are produced through whole-genome duplication (WGD) in ray-finned fish. The ricefield eel npba was expressed in the brain and some peripheral tissues, while npbb was predominantly expressed in the brain. The ricefield eel npbwr2b was also expressed in the brain and in some peripheral tissues, such as the pituitary, gonad, heart, and eye. Immunoreactive Npbwr2b was shown to be localized to Lh and Fsh cells but not to Gh or Prl cells in the pituitary of ricefield eels. Npba upregulated the expression of fshb and cga but not lhb mRNA in pituitary fragments of ricefield eels cultured in vitro. The results of the present study suggest that the NPB system of ricefield eels may be involved in the neuroendocrine regulation of reproduction.

Transcription factors Dmrt1a, Foxl2, and Nr5a1a potentially interact to regulate cyp19a1a transcription in ovarian follicles of ricefield eel (Monopterus albus)

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.

Expression Patterns and Gonadotropin Regulation of the TGF-β II Receptor (Bmpr2) during Ovarian Development in the Ricefield Eel Monopterus albus

Bmpr2 plays a central role in the regulation of reproductive development in mammals, but its role during ovarian development in fish is still unclear. To ascertain the function of bmpr2 in ovarian development in the ricefield eel, we isolated and characterized the bmpr2 cDNA sequence; the localization of Bmpr2 protein was determined by immunohistochemical staining; and the expression patterns of bmpr2 in ovarian tissue incubated with FSH and hCG in vitro were analyzed. The full-length bmpr2 cDNA was 3311 bp, with 1061 amino acids encoded. Compared to other tissues, bmpr2 was abundantly expressed in the ovary and highly expressed in the early yolk accumulation (EV) stages of the ovary. In addition, a positive signal for Bmpr2 was detected in the cytoplasm of oocytes in primary growth (PG) and EV stages. In vitro, the expression level of gdf9, the ligand of bmpr2, in the 10 ng/mL FSH treatment group was significantly higher after incubation for 4 h than after incubation for different durations. However, bmpr2 expression in the 10 ng/mL FSH treatment group at 2 h, 4 h and 10 h was significantly lower. Importantly, the expression level of bmpr2 and gdf9 in the 100 IU/mL hCG group had similar changes that were significantly decreased at 4 h and 10 h. In summary, Bmpr2 might play a pivotal role in ovarian growth in the ricefield eel, and these results provide a better understanding of the function of bmpr2 in ovarian development and the basic data for further exploration of the regulatory mechanism of gdf9 in oocyte development.

Circular RNA expression profiles and CircSnd1-miR-135b/c-foxl2 axis analysis in gonadal differentiation of protogynous hermaphroditic ricefield eel Monopterus albus

BackgroundThe expression and biological functions of circular RNAs (circRNAs) in reproductive organs have been extensively reported. However, it is still unclear whether circRNAs are involved in sex change. To this end, RNA sequencing (RNA-seq) was performed in gonads at 5 sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) of ricefield eel, and the expression profiles and potential functions of circRNAs were studied.ResultsSeven hundred twenty-one circRNAs were identified, and the expression levels of 10 circRNAs were verified by quantitative real-time PCR (qRT–PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Then, the sequence length, category, sequence composition and the relationship between the parent genes of the circRNAs were explored. A total of 147 circRNAs were differentially expressed in the sex change process, and GO and KEGG analyses revealed that some differentially expressed (such as novel_circ_0000659, novel_circ_0004005 and novel_circ_0005865) circRNAs were closely involved in sex change. Furthermore, expression pattern analysis demonstrated that both circSnd1 and foxl2 were downregulated in the process of sex change, which was contrary to mal-miR-135b. Finally, dual-luciferase reporter assay and RNA immunoprecipitation showed that circSnd1 and foxl2 can combine with mal-miR-135b and mal-miR-135c. These data revealed that circSnd1 regulates foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c.ConclusionOur results are the first to demonstrate that circRNAs have potential effects on sex change in ricefield eel; and circSnd1 could regulate foxl2 expression in the sex change of ricefield eel by acting as a sponge of mal-miR-135b/c. These data will be useful for enhancing our understanding of sequential hermaphroditism and sex change in ricefield eel or other teleosts.

Molecular Cloning and Expression Analysis of Thyrotropin-Releasing Hormone, and Its Possible Role in Gonadal Differentiation in Rice Field eel Monopterus albus.

Simple SummaryThyrotropin-releasing hormone (TRH) is an important upstream regulator in the hypothalamus-pituitary-thyroid (HPT) axis in mammals. In this study, we isolated and characterized trh gene from a protogynous hermaphrodite fish rice field eel Monopterus albus. TRH had no significant effect on serum thyroid hormone levels in rice field eel. However, we found that TRH was involved in the regulation gonadal differentiation-related gene expression and serum sex steroid hormone secretion. Our results indicated that TRH may play a novel role in gonadal differentiation in rice field eel.Rice field eel (Monopterus albus), a protogynous hermaphrodite fish, is a good model for the research of sex determination and gonadal differentiation in teleosts. In this study, we cloned the full-length cDNA sequence of trh, which encoded a predicted protein with 270 amino acids. Trh mainly expressed in the brain, followed by the ovary, testis, muscle and pituitary, and had low levels in other peripheral tissues. During natural sex reversal, trh mRNA expression levels exhibited a significant increase at the late intersexual stage in the hypothalamus. In the gonad, trh mRNA expression levels showed a trend of increase followed by decrease, and only increased significantly at the middle intersexual stage. No matter static incubation or intraperitoneal (IP) injection, TRH had no significant effect on trh and thyroid-stimulating hormone β subunit (tshβ) mRNA expression levels, and serum T3, T4 and TRH release. After static incubation of ovarian fragments by TRH, the expression of gonadal soma derived factor (gsdf) was up-regulated significantly at both the doses of 10 and 100 nM. IP injection of TRH stimulated the expression of gsdf, and inhibited the expression of ovarian aromatase gene (cyp19a1a), accompanied by the increase of serum 11-KT levels. The results indicated that TRH may play a novel role in gonadal differentiation by the regulation of gonadal differentiation-related gene expression and sex steroid hormone secretion in rice field eel.

Protogynous sex change in ricefield eel Monopterus albus (Zuiew, 1793) by hormonal induction using javanese chili extract (Piper retrofractum Vahl)

The ricefield eel Monopterus albus (Zuiew, 1793) is a protogynous hermaphrodite that undergoes sex change from female at the beginning of its life cycle to becoming male after going through the intersex phase. This study aims to evaluate the sex status of ricefield eels measuring 18–55 cm and the development of protogynous sex change by hormonal induction using Javanese chili extract (Piper retrofractum Vahl) through injection. One hundred and twenty eels averaged 24.86 ± 0.76 cm of length and 10.65 ± 0.83 g of weight was used in this research. A completely randomized experimental design consisted of four treatments (different doses of Javanese chili extract) and three replications were applied in this research. The dose of Javanese chili extract given was 187.5 g.kg-1 fish (P1) and 375 g.kg-1 fish (P2) compared to the control with injection using NaCl 3 mL.kg-1 fish (P0) and without injection (K). Injections through intramuscular were given 5 times with an interval of 7 days and observations of sex status were carried out every 14 days based on the development of the gonads. In the normal conditions, the sex status of ricefield eels measuring 20-33 cm in length indicates females, while intersex has a length ranging from 33–43 cm, and males with a length range of 43–55 cm. Hormone induction by injection of Javanese chili extract at a dose of ECJ 375 g.kg-1 fish produced a sex change percentage of eels with 75% male sex status and 25% intersex with an average length of 24.88 ± 0.35 cm and a weight of 10.22 ± 0.14 g.

Crosstalk between sex-related genes and apoptosis signaling reveals molecular insights into sex change in a protogynous hermaphroditic teleost fish, ricefield eel Monopterus albus

The ricefield eel Monopterus albus is a protogynous hermaphroditic synbranchiform species that undergoes sequential sex change from female to male, offering an interesting model for study in the mechanisms of sequential hermaphroditism in vertebrates. To explore the expression patterns and molecular mechanisms of key genes underlying sex change in the ricefield eel M. albus, a comprehensive study of the transcriptomes of gonads at five sexual stages (ovary, early intersexual stage gonad, middle intersexual stage gonad, late intersexual stage gonad, and testis) was performed. In total, 107,260,808 raw reads and 101,886,556 clean reads were obtained, and 41,216 genes were detected in the gonad tissues. The expression levels of 21 differentially expressed genes were verified by quantitative real-time PCR (qRT-PCR) and found to be in accordance with the RNA-seq data, suggesting that the RNA-seq data were reliable. Furthermore, the expression of female-related genes decreased as sex change progressed, whereas male-related genes exhibited the opposite expression patterns. Notably, apoptosis-related genes had close relationships with sex change, including genes associated with endocytosis, calcium signaling pathways, and 7 other pathways. In particular, apoptosis signaling was delivered to the p53 network, which may be associated with sex change. Importantly, tgfb3, fshr, camk4 and calmodulin may be the key genes, which helps balance germ cell apoptosis and proliferation during sex change. These results reveal specific molecular signatures in the gonad that are related to sex change and apoptosis signaling, which will be useful for enhancing our understanding of sequential hermaphroditism and sex change in the ricefield eel M. albus and other teleost.

A Study on How Methionine Restriction Decreases the Body's Hepatic and Lipid Deposition in Rice Field Eel (Monopterus albus).

Methionine restriction reduces animal lipid deposition. However, the molecular mechanism underlying how the body reacts to the condition and regulates lipid metabolism remains unknown. In this study, a feeding trial was performed on rice field eel Monopterus albus with six isonitrogenous and isoenergetic feeds that included different levels of methionine (0, 2, 4, 6, 8, and 10 g/kg). Compared with M0 (0 g/kg), the crude lipid and crude protein of M. albus increased markedly in M8 (8 g/kg) (p < 0.05), serum (total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and non-esterified free fatty acids), and hepatic contents (hepatic lipase, apolipoprotein-A, fatty acid synthetase, total cholesterol, triglyceride, and lipoprteinlipase). However, in the serum, very-low-density lipoprotein and hepatic contents (hormone-sensitive triglyceride lipase, Acetyl CoA carboxylase, carnitine palmitoyltransterase, and mirosomal triglygeride transfer protein) decreased markedly in M8 (p < 0.05). The contents of hepatic C18:2n-6, C22:6n-3, and n-3PUFA in the M8 group were significantly higher than those in M0 (p < 0.05), and the contents of lipid droplets in M8 were higher than those in M0. Compared with M0, the hepatic gcn2, eif2α, hsl, mttp, ldlrap, pparα, cpt1, and cpt2 were remarkably downregulated in M8, while srebf2, lpl, moat2, dgat2, hdlbp, srebf1, fas, fads2, me1, pfae, and icdh were markedly upregulated in M8. Moreover, hepatic SREBP1 and FAS protein expression were upregulated significantly in M8 (p < 0.01). In short, methionine restriction decreased the lipid deposition of M. albus, especially for hepatic lipid deposition, and mainly downregulated hepatic fatty acid metabolism. Besides, gcn2 could be activated under methionine restriction.

IDENTIFIKASI DAN PREVALENSI ENDOPARASIT (Gnathostoma sp.) PADA BELUT SAWAH (Monopterus albus) DENGAN UKURAN BERBEDA YANG DIKIRIM KELUAR PROVINSI KALIMANTAN SELATAN

This study aims to determine the type of endoparasitic worm Gnathostoma sp and the prevalence that infects rice field eel (Monopterus albus) with different grades. the method used is the descriptive method. Samples were taken from 3 different farm locations (Nor Aini, Banjar Regency, CV. Karya Bersama, Banjarbaru City, and Saberiannor Banjarbaru City), as many as 120 individuals with each grade (A, B, C, and D) totaling 30 individuals. Samples were examined using the microscopic method with 3 replications on each target organ, namely liver, meat, intestines, and kidneys. The results of the examination showed that Gnathostoma sp worm cysts were found, namely only in the liver, while in the intestines, flesh, and kidneys were not found. Based on the identification of Gnathostoma sp endoparasites seen from morphological characteristics, third-stage larvae (L3) in the phylum Nematoda, cylindrical body shape, transparent body color, and blackish brown, head shape resembling a light bulb, mouth, and has anus at the end of its body. Prevalence value in grade A = 40%; B= 30%; C= 40%, and D= 30%, this includes the category commonly or ordinary which describes that the parasite usually infects fish with a prevalence value range of 30-49%. It is still possible to send rice field eels outside of South Kalimantan Province, although the results of the examination of the test samples indicated the presence of the parasite infection Gnathostoma sp..

A study on methionine-mediated regulation of muscle fiber growth, development and differentiation in the rice field eel (Monopterus albus)

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.

Nr5a homologues in the ricefield eel Monopterus albus: Alternative splicing, tissue-specific expression, and differential roles on the activation of cyp19a1a promoter in vitro

Nr5a (Fushi tarazu factor 1, Ftz-F1) homologues belong to the nuclear receptor superfamily, and are involved in the regulation of reproduction in vertebrates. Four genes encoding Nr5a homologues were present in the genome of ricefield eel, which are designated as nr5a1a, nr5a1b, nr5a2, and nr5a5 in the present study. Alternatively spliced transcripts were identified for nr5a1a and nr5a1b genes. Sequence analysis indicated that nr5a5 is possibly a paralog of nr5a2, and nr5a1b is lost during evolution in some teleosts including tilapia and medaka. Ricefield eel nr5a genes exhibit tissue-specific expression patterns, with nr5a1a and nr5a1b resembling that of the SF-1/Ad4BP (NR5A1) subfamily, and nr5a2 and nr5a5 resembling that of the NR5A2/LRH/FTF subfamily. Transcriptomic analysis revealed parallel expression profiles of nr5a1a, foxl2, and cyp19a1a in ovarian follicles during vitellogenesis, with peak values at the late vitellogenic stage. Real-time PCR indicated that the expression levels of nr5a1a and foxl2 in gonads were decreased significantly during the sexual transition from female to the late intersexual stage. In vitro transient transfection assay showed that Nr5a1a up-regulated ricefield eel cyp19a1a promoter activities synergistically with Foxl2. However, Nr5a1b, Nr5a2, and Nr5a5 could neither activate ricefield eel cyp19a1a promoter alone nor enhance the stimulatory effects of Foxl2 on cyp19a1a promoter activities. Collectively, the above data suggest that Nr5a homologues may have diverse and differential roles in the tissues of ricefield eels. The up-regulation of gonadal nr5a1a and foxl2 during vitellogenesis may be important for the ovarian development whereas their down-regulation during the sexual transition period may be important for the sex change process of ricefield eels, possibly through the regulation of cyp19a1a gene expression.

Integrated Analysis of miR-430 on Steroidogenesis-Related Gene Expression of Larval Rice Field Eel Monopterus albus.

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.

Effects of dietary soy isoflavone and soy saponin on growth performance, intestinal structure, intestinal immunity and gut microbiota community on rice field eel (Monopterus albus)

An eight-week feeding trial was conducted to evaluate the effects of soy isoflavone and soy saponin on growth performance, intestinal structure, intestinal immunity and gut microbiota community of rice field eel Monopterus albus (initial weight 20.12 ± 0.15 g). Six isonitrogenous and isolipidic experimental feeds were formulated, including a positive control (55% fish meal, FM), a negative control (28.6% fish meal, 37.2% soybean meal, SBM) and basic diet group (28.6% fish meal, 22.6% soy protein concentrate, SPC), the rest was supplemented with soy isoflavone (40%, 2.5 g / kg, SI), soy saponin (≥98%, 2 g / kg, SS) and their mixed in basic diet (SI + SS). Compared with FM, growth performance, intestinal immune indices (acid phosphatase, alkaline phosphatase, complement 3, complement 4 and immunoglobulin M), intestinal villus height and intestinal muscular thickness and mRNA (occludin-like, claudin 12, tight junction protein ZO-1-like, tight junction protein ZO-2-like, interleukin-10, transforming growth factor beta 1, transforming growth factor beta 2) expression intestine of M. albus fed SBM diet were significantly decreased (P < 0.05). Growth performance of M. albus in SPC group was better than that in SBM. Compared with SPC, dietary soy isoflavone and soy saponin and their mixed (SI, SS, SI + SS) decreased growth of M. albus, serum alkaline phosphatase in SI significantly increased (P < 0.05), and intestinal immunoglobulin M in SS remarkably declined (P < 0.05), intestinal complement 3, complement 4 and immunoglobulin M, amounts of intestinal goblet cells per root, gut shannon were significantly decreased in SI + SS (P < 0.05). Based on the current results, there has the trend that dietary soy isoflavone and soy saponin damaged the intestinal structure and changed gut bacteria community of M. albus, and destroyed the intestinal barrier, eventually induced intestinal inflammatory, soy isoflavone and soy saponin mixed feed has more serious effects than they single supplemented.

Using unessential sulfur amino acids to overcome methionine deficient diets on rice field eel (Monopterus albus)

An eight-week feeding trial was conducted using sulfur amino acids to overcome methionine deficient diets on rice field eel Monopterus albus (initial weight 25.12 ± 0.51 g). Six isonitrogenous and isoenergetic experimental formulations included 55% fish meal group (FM, a reference), 22% fish meal group (D0), and the rest four diets (22% fish meal) supplemented with 2 g/kg of taurine (DTau), methionine (DMet), cysteamine hydrochloride (DCsh) and cystine (DCys), respectively. Compared with FM, the growth performance of M. albus was markedly declined in D0 (P < 0.05); while the growth performance of M. albus in DMet, DCsh and DCys were remarkably higher than D0 (P < 0.05), and these groups were indifference with FM (P > 0.05). Compared with FM, the highest amount of hepatic vacuoles was observed in D0, while the proportions of vacuoles were lower in DMet, DCsh and DCys groups than that in D0, dietary sulfur amino acids also have improved hepatic transaminase and the ability of amino acid metabolism. Compared with FM, muscular texture, density and diameters of muscle fibres were significantly decreased in D0 (P < 0.05); while muscular texture were significantly increased in DCsh and DCys (P < 0.05). Compared with D0, dietary sulfur amino acids improved diameters of the muscle fibres. Myocyte myogenic regulatory (Mrf4, MyoD2, Myog and Mrf4) and myocyte enhancer factors (MEF2b and MEF2c) mRNA expression in D0 were markedly lower than that in FM (P < 0.05), compared with D0, the expression of Mrf4 and MEF2b in DMet were significantly up regulated (P < 0.05); the expressions of Mrf4, MyoD2 and MEF2b in DCsh were markedly higher than that in D0 (P < 0.05), MyoD2, Myog, MEF2b and MEF2c in DCys also were remarkably higher than that in D0 (P < 0.05). Overall, our study indicated that dietary methionine, taurine, cysteamine hydrochloride and cystine in low fish meal diet could improve amino acids metabolism and muscle growth for M. albus, moreover, cysteamine hydrochloride and cystine overcame methionine deprivation partly.

Oxytocin-like signal regulates Lh cells directly but not Fsh cells in the ricefield eel Monopterus albus†.

The synthesis and release of LH and FSH in the pituitary of vertebrates are differentially regulated during gonadal development and maturation. However, the underlying neuroendocrine mechanisms remain to be fully elucidated. The present study examined the possible involvement of isotocin (Ist), an oxytocin-like neuropeptide, in the regulation of Lh and Fsh in a teleost, the ricefield eel Monopterus albus. The immunoreactive isotocin receptor 2 (Istr2) was shown to be localized to Lh but not Fsh cells. In contrast, immunoreactive isotocin receptor 1 (Istr1) was not observed in either Lh or Fsh cells in the pituitary. Interestingly, Lh cells in female ricefield eels expressed Istr2 and secreted Lh in response to Ist challenge stage-dependently and in correlation with ovarian vitellogenesis. Moreover, Ist decreased Lh contents in the pituitary of female fish, indicating its stimulatory roles on Lh release in vivo. The induction of Lh release by Ist in dispersed pituitary cells was blocked by a PLC or IP3R inhibitor but not by a PKA or PKC inhibitor, indicating the involvement of the IP3/Ca2+ pathway. Collectively, the above results indicate that isotocin may bind to Istr2 to stimulate Lh release via the IP3/Ca2+ pathway, and play important roles in the ovarian maturation in ricefield eels. Furthermore, the present study suggests a novel neuroendocrine mechanism underlying the differential regulation of Lh and Fsh in vertebrates.

Effect of dietary lipid on growth, body composition, serum biochemistry and hepatic metabolite alteration in Chinese rice field eel (Monopterus albus) fingerlings

Five isonitrogenous and isocaloric diets containing dietary lipid concentrations from 71.90 to 142.70 g/kg were formulated and fed to Chinese rice field eel Monopterus albus fingerlings (5.00 ± 0.50 g). The highest values of weight gain, specific growth rate (SGR), together with the lowest feed conversion ratio (FCR) were found in fish fed with 89.10 g/kg lipid diet. Fish fed with 71.90 g/kg diet (F1) had higher hepatosomatic index, viscerosomatic index and whole‐body crude lipid than fish in the other four treatments (p < .05). Plasma concentration of triacylglycerol and the activity of alanine aminotransferase were also higher in fish fed with F1 diet. Whole‐body fatty acid profile varied exclusively, but with a stable value of n−3/n−6 ratio. Gas chromatography–mass spectrometry‐based metabolomics identified eighteen differential metabolites (including idose, alanine, glutamic acid, serine and hypotaurine) in liver affected by dietary lipid content using PLS‐DA analysis. The subsequent pathway enrichment revealed ten affected pathways, with the top three pathways being glycine, serine and threonine metabolism; starch and sucrose metabolism; and D‐glutamine and D‐glutamate metabolism. The broken‐line model of SGR suggested that a dietary lipid concentration of 83.50 g/kg was appropriate for M. albus fingerlings.

Evaluation of dried porcine solubles in diets of Rice field eel (Monopterus albus)

Porcine solubles, originating from porcine mucosal tissues, is a by-product of heparin production. The nutritional value and functionality of a carrier-free, spray-dried porcine soluble (DPS) product was evaluated in 56-day experiment with juvenile rice field eel Monopterus albus (initial weight: 15.5 ± 0.19 g) fed diets with optimal (42%) and a low level (22%) of fishmeal (FM) inclusion. Six isonitrogenous and isolipidic experimental diets were formulated with two levels of FM (22% and 42%) and three levels of DPS (0%, 2.5% and 5%). Growth performance, humoral immunity, digestive enzyme activities, intestinal histology and expression of intestinal tight junction protein-related mRNA were analyzed at the end of the trial. Similar to FM, inclusion of 5% DPS significantly enhanced growth rate and reduced feed conversion ratio of rice field eel, although no significant interaction between FM and DPS on growth performance were observed. Positive influences of DPS on fish health and immunity were seen in that inclusion of DPS significantly enhanced serum complement (C3, C4), immunoglobulin M (IgM) levels and lysozyme activities, and altered total cholesterol and triglyeride contents in the serum of rice field eel. In regard to gut health, villus height, goblet cell quantity and expression of ZO-1 transcripts significantly (P < 0.05) increased with DPS inclusion in fish fed low-FM diets but was not observed in fish fed diets containing the optimal level of fishmeal. Expression of ZO-2 and CL-12 transcripts in intestine also was significantly (P < 0.05) elevated by DPS inclusion in fish fed diets containing low or optimal FM. Moreover, FM and DPS showed significant (P < 0.05) interactions in the expression of inflammatory cytokines mRNA in the intestine of rice field eel with 5% DPS inclusion significantly (P < 0.05) promoting the expression of Tgfb2 and IκBe mRNA, while repressing IL-1β, IL-8, IL-12 and IFNγ expression in fish fed diets containing low FM. This study is the first to show functionality of DPS supplementation at 5% of diet in any aquatic species. Based on these results, DPS supplementation should be considered as an effective, innovative and sustainable strategy to improve health, as well as reduce intestinal inflammation and maintain normal intestine structure of rice field eel fed low-FM diets.

Differential distribution and potential regulatory roles of estrogen receptor 2a and 2b in the pituitary of ricefield eel Monopterus albus

Estrogens play important regulatory roles in the pituitary of vertebrates. Two forms of estrogen receptor 2 (Esr2), namely Esr2a and Esr2b, are identified in teleosts, but their differential roles remain to be fully elucidated. In the present study, expression and potential functional roles of Esr2a and Esr2b were characterized in ricefield eels. esr2a and esr2b mRNA were broadly distributed in tissues, with high levels observed in the brain, pituitary, and gonads. In order to examine the cellular localization of Esr2a and Esr2b in the pituitary, specific antisera against ricefield eel Esr2a and Esr2b were generated, respectively. Interestingly, immunohistochemistry and Western blot analysis revealed that Esr2a and Esr2b were differentially distributed in the pituitary, with the former localized to the adenohypophysis while the latter to the neurohypophysis. Dual fluorescent immunostaining showed that immunoreactive Esr2a was present in Gh and Prl cells, but not in Lh and Fsh cells. Estradiol (E2) stimulated lhb and prl gene expression in dispersed pituitary cells of intersexual ricefield eels, but had no effects on gh, fshb, and gnrhr2 gene expression and Gh release. Results of the present study are helpful for further understanding the roles and mechanisms of estrogen signals in the pituitary.