Low salinity increases survival,body weight and development in tadpoles of the Chinese edible frog Hoplobatrachus rugulosus

Water salinity affects survival, growth and metamorphosis of anuran tadpoles. Hoplobatrachus rugulosus is considered not only as a freshwater amphibian but is also found in brackish wetlands. However, whether salinity change interferes with hatching, survival, body mass and development of H. rugulosus tadpoles is unknown. We found that salinity levels of <4‰ did not affect of survival or hatching of H. rugulosus eggs. At an early larval stage, tadpoles could tolerate up to 9‰ salinity for 96 h; however, body water content decreased when salinity was >5‰. After a 3‐week experiment, body weights of tadpoles exposed to 2‰ and 4‰ salinities were higher but that of the 6‰ group was lower compared with the 0‰ group. More than 90% of tadpoles exposed to 2‰ and 4‰ salinity showed complete metamorphosis. Salinity levels <4‰ promoted survival of tadpoles better than 0‰, whereas none of tadpoles in the 6‰ group became juvenile frogs in 50 days. Time taken to reach metamorphosis was shorter for 2‰ and 4‰ (47.22 ± 0.28 and 47.26 ± 0.33 days, respectively) than for 0‰ (49.31 ± 0.35 days). Juvenile frogs in the 2‰ group had greater body weight than the control. It could be concluded that salinity of <4‰ increased survival and body weight of H. rugulosus tadpoles, and shortened the time taken to reach metamorphosis.     

Arachidonic acid metabolism in gill homogenate and isolated gill cells from rainbow trout,Oncorhynchus mykiss: the effect of osmolality,electrolytes and prolactin

An assay method based on thin layer chromatography to study the arachidonic acid (AA) metabolism in gill tissues was optimized and the effect of osmotically different incubation mediums on AA metabolism was evaluated. Rainbow trout gill tissues metabolize AA into PGE₂ in highest concentration followed by PGD₂, PGF₂ and 6-keto-PGF₁ (the stable metabolite of PGI₂) among the prostanoids tested. Approximately 40% of PGE₂ is synthesized within the first minute of incubation and is directly dependent on the substrate concentration (AA). As in mammalian tissues, PGE₂ synthesis in fish gills is inhibited by the cyclooxygenase inhibitor indomethacin. PGE₂ synthesis in gill homogenate and isolated gill cells incubated in trout Ringer was 0.45 and 1.9 ng/mg protein, respectively, and increased to 8.9 and 4.3 ng/mg protein, respectively, when incubated in KPO₄ buffer, due to a ten-fold increase in the free AA. The hydroxy acid synthesis of the gill homogenate was higher (13%), and that of the isolated gill cells incubated in KPO₄ buffer was lower (44%) compared to gill homogenate and cells incubated in trout Ringer. Gill homogenate incubated in 50 mM phosphate buffer with increasing sodium or potassium concentrations (up to 250 mM) exhibited a concentration-dependent increase in PGE₂ synthesis (220% and 72%, respectively). Prolactin stimulated the PGE₂ synthesis up to 30% while PGD₂, PGF₂ and 6-keto-PGF₁ synthesis was not affected. This effect of prolactin was maximal when PGE₂ synthesis was estimated 30 minutes after prolactin addition and diminished after two hours. These results suggest that rainbow trout gills possess the ability to metabolize AA through the cyclooxygenase and lipoxygenase pathways. PGE₂ synthesis may be under the influence of ion balance and prolactin availability, indicating the probable involvement of AA metabolites in the regulation of ion balances across the gill membrane.     

牙鲆let-7基因的克隆与表达及其靶基因预测

let-7是时序发育过程中的重要调控因子,在促进幼体向成体转变过程中起着极其重要的作用.为探讨let-7对牙鲆(Paralichthys olivaceus)从幼体到成体转变的变态发育过程的影响,本实验对let-7进行了克隆与表达研究,并预测了其可能的靶基因.利用普通PCR技术从牙鲆中成功鉴定出let-7及其前体序列,...     

Differences in the total lipid and lipid class composition of larvae and metamorphosing sea lampreys, Petromyzon marinus

This study compared the alterations in total lipid and lipid class composition of kidney, liver, and intestine from sea lamprey, Petromyzon marinus, during their nontrophic metamorphosis with these parameters in unmetamorphosed larvae. Total lipid in kidney and liver initially was higher by 104 and 66%, respectively, in the earliest metamorphic stage (3) examined compared to larvae and then decreased by 73 and 37%, respectively, from stage 3 to stage 7. Total lipid in intestine, on the other hand, was 53% lower at stage 3 compared to larvae and then significantly increased by 260% from stage 3 to stage 7. Large amounts of triacylglycerol (TG) in kidney and liver implicate these organs as lipid depots; much of the change in total lipid content of kidney and liver could be explained by alterations in TG, although significant variations in other lipid classes (e.g., phospholipid, cholesterol) also were noted. These results suggest that lamprey metamorphosis may proceed in two metabolic phases in a tissue-specific manner and that lipid depletion results from specific catabolism of stored TG reserves.     

Growth and reproduction of the red frog crab, <Emphasis Type="Italic">Ranina ranina</Emphasis> (Linnaeus, 1758), in the Andaman Sea off Thailand

ABSTRACT:   Studies were conducted on growth and reproduction of the red frog crab, Ranina ranina (Linnaeus, 1758), in the Andaman Sea off Thailand from 1998 to 1999. Samples were collected from Ko Similan, Ko Surin and Thai-Myanmar boundary waters by gill net. Results showed that the relationship between carapace length (CL) and body weight (BW) for males (CL, 5.84–14.10 cm) was BW = 0.2598 CL^3.0931, and for females (CL, 5.35–10.97 cm) was BW = 0.4280 CL^2.8656. Growth in each gender showed allometric growth. The average CL of males was significantly larger than that of females. Average male and female crab sizes in 1999 were smaller than in 1998. The spawning season was found to be from November to February; maturing male crabs were abundant from September to December. Average sizes of CL at first maturity for males and females were 7.44 and 7.22 cm, respectively. Fecundity ranges of ovigerous female crabs were 74 600–167 900 eggs with an average egg diameter of 0.62 mm. The monthly sex ratios (male : female) varied between 1:0.56 and 1:2.77.     

Effects of salmon prolactin and growth hormone on plasma osmolality, Na+ concentration and protein content in the saffron cod

In saffron cod, Eleginus gracilis, the antifreeze glycoprotein (AFGP) is present during the winter. The effects of salmon prolactin (sPRL) and growth hormone (sGH) on plasma osmolality, sodium concentration and protein content of the saffron cod were investigated. In the winter, plasma osmolality and protein content decreased following the intraperitoneal injection of sPRL (20 µg kg^-1 day^-1 × 3) but injection of the same dose of sGH had no effect on either plasma osmolality or protein content. In similar experiments performed during the summer, sPRL and sGH had no effect on these parameters. The results suggest that PRL may act on the kidney, possibly clearing antifreeze glycoprotein (AFGP) by increasing glomerular filtration. The possible role of PRL and GH in the disappearance of AFGP during the summer are discussed.     

Insulin-like growth factor I (IGF-I) mRNA expression in coho salmon,Oncorhynchus kisutch: Tissue distribution and effects of growth hormone/prolactin family proteins

To examine the hormonal and nutritional regulation of insulin-like growth factor I (IGF-I) mRNA expression, a sequence-specific solution hybridization/RNase protection assay for coho salmon IGF-I mRNA was developed. This assay is both rapid and sensitive and has low inter- (less than 15%) and intra-assay variations (less than 5%). Using this assay, the tissue distribution of IGF-I mRNA and effects of growth hormone (GH), prolactin (PRL) and somatolactin (SL) on hepatic IGF-I mRNA expression in coho salmon were examined in vivo. Liver had the highest IGF-I mRNA level of 16 pg/μg DNA. Significant amounts of IGF-I mRNA were also found in all other tissues examined (intestine 4.1, kidney 3.8, gill arch 2.4, brain 2.4, ovary 2.3, muscle 2.1, spleen 1.7 and fat 1.1 pg/μg DNA). Injection of coho salmon GH at doses of 0.1 and 1 μg/g body weight significantly increased the hepatic IGF-I mRNA levels in a dose-dependent manner. Injection of coho salmon SL, a recently discovered member of the GH/PRL family, stimulated the IGF-I mRNA expression at the higher dose (1 μg/g), whereas coho salmon PRL had no effect at either dose. Concentration-dependent stimulation by coho salmon GH was also obtained in vitro in primary culture of salmon hepatocytes in concentrations ranging from 0.01 to 1 μg/ml. These results indicate that IGF-I mRNA expression occurs in a variety of tissues in coho salmon, and that at least the hepatic expression is under the regulation of GH and possibly other hormones. The sequence-specific assay established in the present study can be used for accurate quantitation of IGF-I mRNA in salmonid species, and can contribute to a better understanding of the physiology of IGF-I in salmonids.

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Résumé Afin d'étudier les régulations homronales et nutritionnelles de l'expression des ARNm de l'IGF-I (insulin-like growth factor I), un dosage spécifique par hybridation en solution des ARNm d'IGF-I de saumon coho et protégé des RNases, a été développé. Ce dosage, à la fois rapide et sensible, présente un faible coefficient de variation inter- (< 15%) et intra- (< 5%) dosage. L'étude de la distribution tissulaire des ARNm de l'IGF-I et des effets de l'hormone de croissance (GH), de la prolactine (Prl) et de la somatolactine (SI) sur l'expression hépatique des ARNm de l'IGF-I, a été entreprise in vivo chez le saumon coho en utilisant ce dosage. Le foie présente les plus grandes quantités d'ARNm d'IGF-I (16 pg/μg d'ADN). Des quantités significatives d'ARNm d'IGF-I ont été également détectées dans tous les autres tissus étudiés (intestin 4,1; rein 3,8; branchie 2,4; ovaire 2,3; muscle 2,1; rate 1,7 et graisse 1,1 pg/μg d'ADN). L'injection à des saumons coho, de GH à des doses de 0,1 et 1 μg/g de poids vif, augmente significativement et de manière dose dépendante les niveaux hépatiques d'ARNm d'IGF-I. L'injection de SI de saumon coho, un membre récemment découvert de la famille GH/Prl, stimule avec la plus haute dose utilisée, l'expression des ARNm d'IGF-I alors que la Prl n'a aucun effet. La GH augmente de manière dose dépendante (0,01–1 μg/ml) l'expression in vitro des ARNm d'IGF-I par des ARNm d'IGF-I par des hépatocytes de saumon coho en culture. Ces résultats indiquent que, chez le saumon coho, l'expression des ARNm d'IGF-I est présente dans le nombreaux tissus et que, l'expression hépatique est, au moins en partie, régulée par la GH et peut-être par d'autres hormones. Le dosage par séquence spécifique mise au point dans le présent travail, peut-être utilisé pour la quantification précise des ARNm, d'IGF-I de salmonidés et devrait permettre une meilleure connaissance de la physiologie de L'IGF-I chez les salmonidés.

     

中国大鲵生长激素受体的克隆与表达分析

利用中国大鲵(Andrias davidianus)性腺转录组测序获得的生长激素受体(GHR)基因部分序列,克隆获得基因全长2992 bp,开放阅读框1812 bp,编码604个氨基酸,该蛋白具有高度保守的FGEFS基序与BOX框。系统进化分析结果显示,中国大鲵GHR氨基酸序列与两栖类非洲爪蟾(Xenopus laevis)同源性最高,蜥形纲锦龟(Chrysemys picta bellii)次之,哺乳类水牛(Bubalus bubalis)和鱼类半滑舌鳎(Cynoglossus semilaevis))最低。实时定量PCR结果表明,GHR基因在肝中表达最高,肌肉、垂体、肾、性腺中的表达量次之,其他各组织表达量较低。在精巢发育早期GHR表达较高,随后表达量逐渐降低,在卵巢中表达量随时间增长而增加;17α-甲基睾丸酮(MT)与芳香化酶抑制剂来曲唑(LE)短暂处理后GHR基因在脑与卵巢中表达量发生变化,MT处理后,脑与卵巢中GHR表达量增加,LE处理后脑与卵巢中表达量降低。研究表明,GHR基因在大鲵性腺发育中可能发挥作用,且MT与LE可能通过不同的途径调节GHR基因的表达。     

三疣梭子蟹B型清道夫受体蛋白的原核表达及细胞、组织分布研究

B型清道夫受体是清道夫受体超家族成员,可识别多种配体,在机体脂类转运及免疫防御过程中发挥重要作用。为深入研究三疣梭子蟹B型清道夫受体(Pt-SRB)的功能,本实验在前期工作基础上构建了Pt-SRB胞外结构域原核表达载体,并成功获得了重组蛋白rPt-SRB。利用亲和层析方法获得纯化的rPt-SRB,并将纯化rPt-SRB蛋白免疫大鼠获得抗rPt-SRB重组蛋白免疫抗血清以用于后续研究。SDS-PAGE检测发现,体外诱导表达重组rPt-SRB以包涵体的形式出现在大肠杆菌BL21裂解液的沉淀中,分子量大小约为49.18 ku。Western-Blot分析表明,大鼠抗rPt-SRB血清能与rPt-SRB特异性结合。本实验还利用免疫荧光技术,对Pt-SRB在三疣梭子蟹血淋巴细胞的定位及不同组织中的分布进行研究。结果显示,Pt-SRB在三疣梭子蟹消化道、肝胰腺、鳃、心脏、肌肉组织中均有分布,但不同组织中Pt-SRB的分布不同。免疫荧光结果显示,绿色荧光信号在消化道及腺体结缔组织中较强,另外在肝小管上皮、鳃丝上皮细胞中亦有较强的阳性信号,表明PtSRB蛋白在上述组织结构中分布较广;在血淋巴细胞中,绿色荧光信号主要分布于细胞质和细胞膜,在细胞核中没有明显荧光信号,提示Pt-SRB在三疣梭子蟹血淋巴细胞的细胞质和细胞膜上表达。本结果将为三疣梭子蟹B型清道夫受体蛋白的生理及免疫学功能的研究奠定基础。     

Effects of acute and chronic zinc exposure on steroid hormone receptor expression in the lined seahorse,Hippocampus erectus

The element zinc (Zn) can elicit a variety of physiological responses in animals, particularly through its effects on steroid hormone receptor expression. Here, we evaluated the effects of Zn on the expression profiles of steroid hormone receptors in the lined seahorse, Hippocampus erectus, following experimental acute and chronic Zn exposure. Complex response patterns were observed; however, steroid hormone receptor expression in liver and kidney following 12‐day Zn injection (acute exposure) differed significantly from the control treatments. Apparent effects on hepatic lipid metabolism and osmotic regulation in the kidney need detailed investigation. Furthermore, 49‐day (chronic) waterborne Zn exposure produced a significant decrease in both estrogen receptor (ER) expression levels and estradiol concentrations. In additional, chronic exposure led to a substantial reduction of hepatic adipose cells, implying that Zn may regulate hepatic lipid metabolism via ER and estradiol. Our study demonstrated the effects of both acute and chronic Zn exposure on expression patterns of steroid hormone receptors, which will contribute to understanding the mechanism of Zn‐induced hepatic lipid metabolism and other physiological responses in seahorses.     

Effects of dietary protein levels on the growth,body composition,serum biochemistry and digestive enzyme activity in Chinese rice field eel (Monopterus albus) fingerlings

A 12‐week feeding trial was conducted to determine the influence of dietary protein levels on growth performance, body composition, haematological parameters and digestive enzyme activity of Chinese rice field eel (Monopterus albus) fingerlings. Six isolipidic experimental diets (27.89%, 32.23%, 35.95%, 40.06%, 43.31% and 46.53%) were formulated to randomly assign to triplicate groups of 50 rice field eel fingerlings (5.0 ± 0.5 g) cultured in plastic cages. Results indicated that the best growth performance of fish with the highest WG (158.68 ± 31.59%) and SGR (2.28 ± 0.21%) was obtained at 40.06% group. Significant alterations were demonstrated on whole‐body crude protein, crude lipid and amino acid profile by different dietary protein levels. Besides, haematological parameters including triacylglycerol, aspartate aminotransferase and alkaline phosphatase decreased with various levels by the increasing of dietary protein levels. The lowest pepsin activity was observed in 27.89% protein group, which suggested lower availability of protein as substrate for protease activity. Meanwhile, the highest amylase activity in fish of 27.89% group indicated higher carbohydrate level in this group. Based on the polynomial regression analysis of WG and SGR, the dietary protein requirement for fingerlings of Monopterus albus was estimated to be 386.4–390.8 g/kg, respectively, with the lipid level of 10.08%.     

中华绒螯蟹和罗氏沼虾cyclin B蛋白的原核表达、抗体制备及其鉴定

从中华绒螯蟹(Eriocheir sinensis)和罗氏沼虾(Macrobrachium rosenbergii)卵巢cDNA中克隆了cyclin B基因的开放阅读框,分别连接到pGEX-2T和pET-32a表达载体,转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白GST-EsCB和Trx-MrCB.通过优化培养温度、IPTG浓度以及诱导时间,得出重组蛋白的最佳表达条件分别为:0.1mmol/L IPTG、37℃、4h和0.1 mmol/L IPTG、30℃、6h.重组蛋白主要以包涵体形式存在.亲和层析纯化重组蛋白,作为抗原免疫家兔,制备GST-EsCB和Trx-MrCB多克隆抗体.ELISA检测抗体效价均高达1:80 000,Western blot鉴定该抗体能特异性识别重组蛋白,而且均能够从卵巢总蛋白中检测到cyclin B蛋白.在中华绒螯蟹未成熟卵巢中cyclin B蛋白出现2种亚型,在罗氏沼虾未成熟卵巢中只有1种亚型,推测虾和蟹cyclin B蛋白调控卵母细胞成熟的分子机制不同.本研究制备了cyclin B抗体,旨为在蛋白水平研究cyclin B在卵母细胞成熟过程中的作用机制提供基础条件.     

Molecular characterization and increased expression of the Nile tilapia, Oreochromis niloticus (L.), T-cell receptor beta chain in response to Streptococcus agalactiae infection

The complete cDNA sequence of the Nile tilapia T-cell receptor (TCR) β chain was cloned using 5' RACE. The full-length, 1263-bp cDNA contained a 942-bp open reading frame (ORF) encoding a 314-amino-acid protein. Sequence analyses revealed that the Nile tilapia TCR β chain contains four conserved cysteine residues involved in the formation of disulphide bridges and a conserved amino acid motif believed to be important for assembly and signalling of the TCR αβ/CD3 complex, both of which are normally found in the TCR β chain of other vertebrates. As detected using semi-quantitative and quantitative RT-PCR, the highest expression level of TCR β was detected in the thymus. Interestingly, Streptococcus agalactiae significantly induced the up-regulation of the TCR β chain, and the strongest up-regulation was detected in the brain and peripheral blood leucocytes (PBLs). In in vitro experiments, concanavalin A and Aeromonas hydrophila were found to significantly increase the expression of the TCR β chain in PBLs after 48 h (P < 0.01) and 72 h (P < 0.05), respectively. Furthermore, real-time PCR analysis showed that intraperitoneal injection (IP) of 10(7) cfu mL(-1) of S. agalactiae could induce TCR β expression that was greater than the expression observed following administration of 10(9) cfu mL(-1). The presence of the TCR β chain in fish detected in this study suggests the presence of T-cell populations that have been found in higher vertebrates, which may play a crucial functional role in the response to fish pathogens.     

Effects of total fish oil replacement to vegetable oils at two dietary lipid levels on the growth,body composition,haemato‐immunological and serum biochemical parameters in caspian brown trout (Salmo trutta caspius Kessler, 1877)

This research aimed to evaluate the effects of two dietary fat levels [low fat (LF) (10%), high fat (HF) (20%)] and sources [fish oil (FO), vegetable oil (VO)] on the growth and some physiological parameters of Caspian brown trout fingerlings for 60 days. Tuna oil or blends of canola and soybean oils (85:15) were added to diets to design four feeds namely LFFO, HFFO, LFVO and HFVO according to the fat levels and sources. The fish fed the LFFO diet had lower weight gain than the other fish (P<0.05). The total n‐6 fatty acids increased in fish fed diets with the blends of VO, while the total n‐3 fatty acids decreased in these fish (P<0.05). Serum lysozyme activity was higher in fish fed the HFVO diet than the other fish (P<0.05). Serum glucose, total cholesterol, triglyceride and very low‐density lipoprotein were lower in fish fed LFFO than the other fish (P<0.05). The present study demonstrates that in terms of fish growth, VOs can be used as an alternate source of dietary fat, whereas fish health and nutritional value are improved with the LFFO diet. According to these results, a partial substitution of FO by VO in high‐level fat diets is suggested for long‐term feeding of Caspian brown trout.     

Influence of different dietary lipid sources on the growth,tissue fatty acid composition,histological changes and peroxisome proliferator‐activated receptor γ gene expression in large yellow croaker (Pseudosciaena crocea R.)

A 9‐week feeding trial was conducted to investigate the impact of dietary lipid sources on the lipid mechanisms of large yellow croaker by feeding three isonitrogenous and isoenergetic diets containing fish oil (FO), soybean oil (SO) and beef tallow (BT) respectively. The effects of the diets on the growth performance, somatic indices, tissue fatty acid composition, histological changes and peroxisome proliferator‐activated receptor (PPAR)γ expression were evaluated. Experimental diets were all well accepted by fish and no significant (P>0.05) differences were found in the weight gain, growth rate and feed conversion rate. The fatty acid profile of the fish fillet and liver reflected the fatty acid composition of the diets. Specific‐fatty acids were selectively retained, however, in the flesh of the fish; in particular, docosahexaenoic acid and arachidonic acid concentrations were higher than the dietary concentrations. When FO was replaced by SO or BT diets, the reduction in eicosapontaenoic acid in fish tissue was more pronounced, suggesting a preferred utilization of this fatty acid. The consumption of SO displayed intense lipid accumulation in the liver of the fish. The expression of PPARγ increased significantly in the SO group compared with the FO and BT groups (P<0.05).