人工繁育条件下的褐菖鲉(Sebastiscus marmoratus)繁殖特性研究

为进一步了解和掌握人工繁育条件下褐菖鲉(Sebastiscus marmoratus)繁殖特性,从2021年5月至2023年6月采集不同月份的褐菖鲉性腺样本,对不同阶段的性腺表型和组织变化、发育周期、生殖腺指数及绝对繁殖力进行分析。结果表明,褐菖鲉为典型的卵胎生鱼类,精巢为全囊成熟型,可分为6期;卵巢为部分同步型,交尾受精后存在妊娠期,共分为7期。各月份性腺分期及生殖腺指数统计结果显示,褐菖鲉繁殖高峰期为每年12月至翌年4月,其中雄性1龄性成熟,雌性2龄性成熟,生殖腺指数在发育阶段显著上升(P<0.05),在交尾排精和产仔后显著下降。此外,1~4龄褐菖鲉的平均绝对繁殖力分别为7087、(19726±4470)、(35924±4190)、(43203±6204)粒,各年龄组存在显著性差异,方程拟合得出绝对繁殖力与体长、体质量呈二项式相关。首次对人工繁育条件下的褐菖鲉性腺发育过程和繁殖特性进行两周年的持续追踪和研究,结果将有助于加深对其繁殖特性的了解,提升规模化繁育技术水平。

Antioxidant responses to benzo[a]pyrene,tributyltin and their mixture in the spleen of Sebasticus marmoratus

It has been reported that there is an interaction between Benzo[a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, and tributyltin （TBT）, an organometal used as an antifouling biocide. This study was therefore designed to examine the potential in vivo influence of BaP, TBT and their mixture on splenic antioxidant defense systems of Sebastiscus marmoratus. The fish were exposed to water containing environmentally relevant concentrations of BaP, TBT and their mixture. Spleens were collected for biochemical analysis after exposure for 7, 25, 50 d and after recovery for 7, 20 d. Cotreatment with BaP and TBT for 7 d potentiated the induction of glutathione peroxidase （GPx） activity by BaP or TBT alone. The cotreatment for 25 and 50 d resulted in inhibition of GPx activity, which was similar to the effect of TBT. Splenic glutathione S-transferase （GST） activities were significantly elevated in S. marmoratus exposed to BaP starting from 7 d and remained high up to 25 d. However, no further activity change was found with prolonged exposure. Cotreatment of BaP and TBT primarily inhibited the GST activity, which was similar to the effect of TBT. Cotreatment with BaP and TBT for 25 or 50 d potentiated the depletion of GSH （glutathione） by BaP or TBT alone. MDA （malondialdehyde） contents in spleen of S. marmoratus were not significantly altered compared with the control during the test period. Spleen, as an immune organ, is sensitive to exposure of BaP or TBT. It should have an effective mechanism to counteract oxidative damage. Antioxidative defense systems in spleen of S. marmoratus should be considered as potential biomarkers. Short-term exposure of BaP or TBT could result in induction of antioxidant defense system. A significant decrease of these indices, such as GSH, GST, GPx might indicate more severe contamination.

Relation of hepatic EROD activity and cytochrome P4501A level in Sebastiscus marmoratus exposed to benzo[a]pyrene

This study was designed to investigate the in vivo effects of benzo[a]pyrene （BaP） on hepatic ethoxyresorufin-O-deethylase （EROD） activity and its correlation with cytochrome P4501A （CYP1A） protein levels in Sebastiscus marmoratus, which were exposed through a water column to BaP （10, 100, 1000 ng/L, respectively） or were treated with intraperitoneal injections of BaP （0.5, 1, 5, 10 mg/kg, respectively） every 7 d. The results showed that after 25 d of waterborne exposure to 1000 ng/L BaP, fish hepatic CYP1A levels and EROD activity were significantly induced. In contrast, EROD activity was not altered 7 d after second intraperitoneal injections, whereas, CYP1A protein levels were increased. Dose-dependent increase of biliary BaP metabolites demonstrated that the catalytic activity of CYP1A was induced by treatment with BaP. The lowest observable effect concentration with regard to biliary BaP metabolites （100 ng/L） was much lower than that with reference to EROD activity （1000 ng/L）. The results suggest that biliary polycyclic aromatic hydrocarbon （PAH） metabolites were shown to better reflect the contamination gradients of PAHs than EROD activity. It appeared to be necessary to measure CYP1A protein levels to complement the EROD activity in relevant toxicological assessments.

Patterns of genetic and morphometric diversity in the marbled crab(Pachygrapsus marmoratus, Brachyura, Grapsidae)populations across the Tunisian coast

The present study reports on population structure analysis of the marbled crab Pachygrapsus marmoratus （Fabricius, 1787） from the Tunisian coast, an appropriate location to study biogeographical processes because of the presence of a well-known discontinuous biogeographic area （the Siculo-Tunisian Strait）. Patterns of morphological and genetic variation of this highly dispersive and continuously distributed decapod species were assessed among its geographically close populations which cover almost the entire Tunisian coastline. A total of 386 specimens from nine sites were collected and examined for morphometric variability at 14 morphometric traits. The results of multivariate analyses of linear morphometric traits showed the existence of sexual dimorphism in this species by PERMANOVA （Permutational multivariate analysis of variance）. In addition, both CDA （Canonical discriminant analysis） and NPMANOVA （Non parametric MANOVA test） analyses revealed statistically significant differences among the studied locations for both sexes. Overall, the outcome of CDA analysis showed that over 87% of individuals could be assigned correctly to three regional groups in both sexes （North, Center and South）. Specifically, SIMPER （Similarity Percentages） analysis showed that carapace length, carapace width and merus length were major contributors to the morphometric separation between populations. The pattern of phenotypic variation suggested by morphometric analyses was found to be highly discordant with that suggested by the analysis of a mitochondrial marker （cytochrome oxidase I, COI）. Indeed, the results inferred from restriction fragment analysis of the COI in 180 crabs, suggested high genetic homogeneity. Very low levels of haplotype diversity （h） were found in almost all the studied populations, associated with non significant genetic distances for nearly all population comparisons. Explanations to these morphometric and mtDNA patterns as well as the discrepancy between them are discussed.

Exposure to bifenthrin disrupts the development of testis in male Sebastiscus marmoratus

Bifenthrin（BF） is a pyrethroid insecticide that is widely used in agriculture, horticulture, and for residential purposes. However, few studies addressing the reproductive toxicity of BF on fishes are available. The present study was conducted to investigate the effects of BF on testicular development in Sebastiscus marmoratus and to gain insight into its mechanism. After exposure to 1, 10 and 100 ng/L BF for 50 days, there was a reduced number of mature sperm and an abundance of the late stages of spermatocysts in the testes. The levels of 17β-estradiol and testosterone were decreased significantly after BF exposure. The activity of caspase-3 was increased in a dosedependent manner after BF exposure, TUNEL assay indicated that BF exposure resulted in the occurrence of apoptosis in the testes, which might be main reason for the inhibition of spermatogenesis.

Distribution of Chitinolytic Enzyme in the Organs and Molecular Cloning of a Novel Chitinase Gene from the Kidney of Marbled Rockfish <i>Sebastiscus marmoratus</i>

Actinopterygii express two types of chitinase (acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2)) that are active at acidic pHs and involved in digestion in the stomach. We proposed the existence of a new fish chitinase that has a non-digestive function. In this study, we used Sebastiscus marmoratus, for which characteristics and cDNA cloning of chitinase isozymes (SmChi-1, SmChi-2) in the stomach have been reported. Initially, we examined the distribution of chitinase and β-N-acetylhexosaminidase (Hex) in the body and then we tried to clone novel chitinase cDNA from the kidney. Chitinase and Hex activities were measured using pNP-(GlcNAc)n, (n = 2, 3) and pNP-GlcNAc as substrates, respectively. Total RNA was extracted from the kidney. RT-PCR was performed to obtain chitinase cDNA fragments using reverse transcriptase with an oligo dT primer. The RACE method was used to obtain sequences of the upstream and downstream regions of cDNA. The full-length chitinase cDNA was determined using PrimeSTAR&reg;?Max DNA polymerase with proofreading activity. High chitinase activity was observed in the stomach, as previously reported. In addition, relatively high activity was observed in the liver, spleen, kidney, and heart. In contrast, Hex activity was detected in all organs. This result is consistent with the report that Hex is related to body-wide metabolism. Full-length cDNA (SmChi-3) of the novel chitinase was obtained from the kidney, which contained 1440 bp open reading frames. The domain structure of SmChi-3 was assumed to be similar to those of SmChi-1 and SmChi-2. SmChi-1 and SmChi-2 have a serine and glycine-rich linker region, which is characteristic of AMCase. In contrast, SmChi-3 contained no apparent sequence in the linker region. Phylogenetic analysis revealed the existence of a new chitinase group, which was named fish chitinase-3 (FCase-3) and differed from AFCase-1 and AFCase-2.

Influence of dietary n-3 LC-PUFA on growth, nutritional composition and immune function in marine fish Sebastiscus marmoratus

Abstract A 60-day feeding experiment was conducted to investigate the influence of dietary omega-3 long-chain polyunsaturated fatty acids （n-3 LC-PUFA） on growth, nutritional composition and immune function of marine fish Sebastiscus marmoratus. Five diets containing 3.6, 10.2, 18.2, 26.5, or 37.0 g/kg n-3 LC-PUFA were prepared. The results reveal significant influences of dietary n-3 LC-PUFA on the final weight, weight gain, specific growth rate, feed conversion ratio, and condition factor. As dietary n-3 LC- PUFA increased, weight gain and specific growth rate increased and were significantly higher in groups fed 18.2, 26.5 and 37.0 g/kgthan in groups fed 3.6 and 10.2 g/kg （P〈0.05）; there was no significant difference between groups fed 18.2, 26.5, or 37.0 g/kg （P〉0.05）. With increasing dietary n-3 LC-PUFA, eicosapentaenoic acid and docosahexenoic acid content in muscle and liver increased significantly, immunoglobulin class M content gradually increased from 9.1 to 14.8 Ixg/L, and lysozyme activity content increased from 1 355 to 2 268 U/mL. Broken line model analysis according to weight gain indicated that a dietary n-3 LC-PUFA level of 18.2 g/kg is essential for normal growth at a fat level of 125 g/kg. Therefore, appropriate dietary n-3 LC-PUFA not only promote growth and improve the n-3 LC-PUFA content, but also enhance immune function in S. marmoratus.

Genomic organization and transcriptional modulation in response to endocrine disrupting chemicals of three vitellogenin genes in the self-fertilizing fish Kryptolebias marmoratus

Vitellogenin（Vtg）is the precursor of egg yolk proteins,and its expression has been used as a reliable biomarker for estrogenic contamination in the aquatic environment.To examine the biomarker potential of the self-fertilizing killifish Kryptolebias marmoratus Vtgs（Km-Vtgs）,full genomic DNAs of Km-Vtgs-Aa,Km-Vtgs-Ab,and Km-Vtgs-C were cloned,sequenced,and characterized.Three Vtg genes in K.marmoratus are tandemly placed in a550 kb section of the same chromosome.In silico analysis of promoter regions revealed that both the Km-Vtgs-Aa and Km-Vtgs-Ab genes had an estrogen response element（ERE）,but the Km-Vtgs-C gene did not.However,all three Km-Vtgs genes had several ERE-half sites in their promoter regions.Phylogenetic analysis demonstrated that the three deduced amino acid residues were highly conserved with conventional Vtgs protein,forming distinctive clades within teleost Vtgs.Liver tissue showed the highest expression of Km-Vtg transcripts in all tested tissues（brain/pituitary,eye,gonad,intestine,skin,and muscle）in response to endocrine disrupting chemical（EDC）-exposed conditions.Km-Vtg transcripts were significantly increased in response to 17β-estradiol（E2）,tamoxifen（TMX）,4-n-nonylphenol（NP）,bisphenol A（BPA）,and octylphenol（OP）over 24 hr exposure.The Km-Vtg-A gene was highly expressed compared to the control in response to NP and OP.EDC-induced modulatory patterns of Km-Vtg gene expression were different depending on tissue,gender,and isoforms.

云斑尖塘鳢精子的超微结构

采用扫描电镜和透射电镜观察了云斑尖塘鳢(Oxyeleotris marmoratus)的精子结构。结果显示:扫描电镜下云斑尖塘鳢精子由头部和尾部(鞭毛)组成。头部近似圆形,直径1.98～2.12μm,尾部鞭毛长23.22～32.92μm。透射电镜下云斑尖塘鳢精子由头部、中段和尾部三部分组成。头部细胞核呈扁卵圆形,前端不具顶体,染色质致密,细胞核内偶见小空隙;核膜与质膜间有间隙,其间分布有细胞质、囊泡、溶酶体,在细胞核后端,还分布有5～7个呈环形单层排列的线粒体;植入窝较浅,中段不明显,包括中心粒复合体和袖套;精子尾部的鞭毛细长,轴丝为典型的"9+2"型结构;轴丝外侧具有较发达的侧鳍。

云斑尖塘鳢仔鱼饵料动物的非纯化培养及摄食试验

利用塘泥和水体中的浮游动物种源，对云斑尖塘鳢（Oxyeleotris mermoratus）仔鱼的饵料动物进行非纯化培养并做了仔鱼的摄食试验。结果表明：培养过程中，各类饵料动物组成1～8d以轮虫类占绝对优势，比例达到50％以上，9～10d以枝角类和桡足类无节幼体占优势，比例达到30％以上；各类饵料动物密度变化曲线呈倒“V”字型，其中饵料动物总密度、轮虫类和枝角类密度均在第7d达到最高值，分别为47．6个／ mL、29．5个／mL和16．5个／mL。从仔鱼对饵料的摄食比率看，大小范围为50～100μm的饵料动物仅适合9日龄以下的仔鱼摄食，随着仔鱼的发育，趋向于摄食较大个体的饵料动物。

褐菖鲉网箱养殖试验初报

2001年4月30日在浙江南麂海域3m×3m×3m网箱中放养0．9g自然海区捕获的褐菖鲇（Sebastiscus marmoratus）苗种，进行为期2年多的养殖试验。结果表明，该鱼生长缓慢，养殖38d体质量为8．3g，养殖240d为24．8g，养殖787d为112．3g。总养殖成活率为20．1％，平均日增体质量0．142g，平均日增体质量率0．62％。秋末至夏初低水温期是褐菖鱼由的主要生长期，在9～13℃较低水温下亦会摄食与生长。饵料系数为3．6。冬春季养殖成活率高于秋夏季，第一年夏季养殖成活率低，仅28．1％。养殖方式以混养为宜。

云斑尖塘鳢池塘育苗研究

从商业化生产的目的出发,研究池塘云斑尖塘鳢(Oxyeleotris marmoratus)仔幼鱼的培育。育苗池经清塘、培水后,当幼小轮虫生物量达10 eggs/mL以上时,按225～375尾/m2放养刚开口的仔鱼,饲养30 d,长成全长约1.5 cm的稚鱼,开始驯食人工混合饲料,再池养30～35 d,育成全长2.75～3.35 cm幼鱼。2006年共放养仔鱼434万尾,育成早期幼鱼119.7万尾,成活率27.6%。在傍晚时拉网起捕,“三网率”达95%。

云斑尖塘鳢人工育苗技术初步研究

对云斑尖塘鳢(Oxyeleotris marmoratus(Bleeker))人工育苗技术进行了初步的研究,利用燃油热水炉加温进行亲鱼越冬,采取人工辅助控温、保温的方法,配合其生态条件的调控和投喂优质饲料,培育亲鱼成熟并能自然产卵,使用人工饵料、代用饵料、生物饵料等混合饵料互相搭配组合,经过2个月左右的培育鱼苗全长达3 cm左右。亲鱼具多次产卵习性,观察到一年当中最多产卵4次。利用外源激素催产率达到95.0%,自然产卵的受精率和孵化率均高于人工催产。

Combined effect of tributyltin and benzo[a] pyrene on the levels of sex hormone and vitellogenin in female Sebastiscus marmoratu

Tributyltin（TBT）, an organometal used as an antifouling biocide, has been reported to induce masculinization of fish. Benzo [a]pyrene （BaP）, a widespread carcinogenic polycyclic aromatic hydrocarbon, has been reported that its microsomal metabolites can produce an estrogenic response when tested in vitro. This study was therefore designed to examine the potential in vivo influence of TBT, BaP and their mixture on sex hormone levels in serum of Sebastiscus marmoratus, which were given 2 separate intraperitoneally （ip） injections（a single injection every 7 d） of TBT（0.5, 1, 5 and 10 mg/kg）, BaP（0.5, 1, 5 and 10 mg/kg）, or both in combination（0.5, 1, 5 and 10 mg/kg）; control fish received olive oil vehicle only. Six days after the 2nd injection, serum samples were collected and analyzed for sex hormone levels and alkali labile protein phosphorus （ALPP）, which is related to the yolk precursor protein vitellogenin. The pollutants at all doses significantly reduced serum testosterone, estradiol and ALPP content after 2 injections compared with the corresponding controls. The reduction of the estradiol levels should be response for the decrease of the vitellogenin levels. The results in the present study suggested that aromatase seems not the major target acted by TBT and BaP in fish. This study demonstrated that TBT or BaP exposure both inhibit the reproductive potential in female Sebastiscus marmoratus. Combined effect of TBT and BaP on the serum testosterone, estradiol and ALPP was not antagonism from the anticipation.

Description of a New Species of Amolops Cope, 1865(Amphibia: Ranidae) from Nepal and Nomenclatural Validation of Amolops nepalicus Yang, 1991

A new species,Amolops mahabharatensis sp.nov.,is described from Mahabharat range(middle mountain)of Nepal.The new species is a member of the A.marmoratus group and is distinguished from its congeners using morphological and molecular data.The new species is diagnosed by having numerous warts on dorsum and flanks,dorsal body greyish olivebrown/light brown,presence of vomerine teeth series,presence of a pair of subgular vocal sacs in males and tadpole withⅢ:5+5/1+1:Ⅱ–labial tooth row.In addition,evidenced by the phylogenetic and morphological analyses in this study,Amolops nepalicus is shown to be a valid species.A molecular phylogenetic analysis based on 16 S ribosomal gene for six members of the A.marmoratus group suggested that the new species is sister to a clade containing A.nepalicus and A.panhai.The uncorrected genetic divergences between the new species and its closest congeners A.nepalicus,A.panhai and A.marmoratus based on 16 S rRNA gene were 10.5%,11.6%,and 8.6%respectively.The new species showed wide distribution in the Mahabharat range of Nepal.