温度、盐度变化及鳗弧菌刺激对菲律宾蛤仔I型溶菌酶基因表达的影响

为研究在环境因素变化后,菲律宾蛤仔Ⅰ型溶菌酶参与免疫应答反应的变化模式,实验采用cDNA末端快速扩增技术,从菲律宾蛤仔体内克隆获得了一种Ⅰ型溶菌酶基因的全长cDNA序列(命名为RpiLYZ-2),该序列开放阅读框(ORF)为471 bp,编码156个氨基酸.RpiLYZ-2基因在所检测组织中均有表达,其中在外套膜中表达量最高,在肌肉组织中表达量最低.采用荧光定量PCR法,研究了不同温度[(29±1)、(21±1)和(13±1)℃]、盐度(32、22和12)及鳗弧菌刺激对菲律宾蛤仔Ⅰ型溶菌酶基因表达的影响.结果显示,在温度21℃和盐度22胁迫处理后,RpiLYZ-1基因的表达量呈现先上升后下降的趋势,而RpiLYZ-2基因的表达量则呈现先下降后上升的趋势.在高温29℃和低盐12胁迫条件下,RpiLYZ-1基因表达量总体呈下降趋势,而RpiLYZ-2基因表达量总体呈上升趋势;经鳗弧菌刺激后,RpiLYZ-1基因的表达量得到显著诱导,而RpiLYZ-2基因表达量则表现为先降低后上升的趋势.研究表明,环境因子变化和病原菌刺激能够显著影响菲律宾蛤仔两种Ⅰ型溶菌酶基因的表达量,且RpiLYZ-1和RpiLYZ-2可能存在着功能分化.     

团头鲂促肾上腺皮质激素释放激素受体基因序列特征及表达分析

促肾上腺皮质激素释放激素受体(corticotropin-releasing hormone receptor,CRHR)是鱼类下丘脑–垂体–头肾调控轴上的重要应激调节因子。本研究通过c DNA末端快速扩增(RACE)技术成功克隆出团头鲂(Megalobrama amblycephala)CRHR1 m RNA全长序列,应用生物信息学方法对其序列特征进行解析;同时采用荧光定量PCR技术分析了团头鲂CRHR1的组织分布图谱及外源性皮质醇注射模拟应激处理下团头鲂CRHR1 m RNA的表达变化。研究结果表明,CRHR1 m RNA序列开放阅读框(open reading frame,ORF)为1290 bp,编码429个氨基酸。团头鲂CRHR1氨基酸序列与鲤科鱼类的CRHR1氨基酸序列同源性最高;该受体具有7次横跨膜结构和氨基端激素受体结构域。CRHR1在垂体中表达量最高,其次为下丘脑,在心脏、肝、脾等组织中表达丰度较低。经外源性皮质醇注射后,实验组血糖和血清皮质醇显著高于对照组,在处理2 h后达到峰值;实验组血清ACTH水平与对照组差异总体不显著,但呈现先升高后下降。应激处理后,CRHR1转录水平在4种组织中的表达变化存在差异;垂体中CRHR1在早期出现明显的表达抑制,在下丘脑中则呈现先缓慢升高后缓慢下降的趋势,而心脏和头肾中CRHR1在早期则表现出表达迅速上调而后缓慢下降的趋势。本研究进一步丰富了CRHR在鱼类研究方面的基础资料,为鱼类应激调控提供了理论基础。     

Effects of salinity and temperature during incubation on hatching and development of lingcod Ophiodon elongatus Girard, embryos

The interactive effects of salinity and temperature on development and hatching success of lingcod, Ophiodon elongatus Girard, were studied by incubating eggs at four temperatures (6, 9, 12 and 15°C) and five salinities (15, 20, 25, 30 and 35 g L^?1). Hatch did not occur in any of the 15°C treatments. Degree days (°C days) to first hatch was not influenced by temperature or salinity, however, calendar days to first hatch differed significantly for temperature (P<0.0001, 61±1, 44±1 and 35±1 days for 6, 9 and 12°C respectively). Degree days to 50% (427.1±4.2) hatch was not significantly influenced by temperature but was by salinity (P=0.0324). Viable hatch (live with no deformities, 74.1±4.0%) was greatest at 9°C and 25 g L^?1 but not significantly different in the range of 20–30 g L^?1. Larval length (9.4±0.13 mm) was greatest at 9°C and 20–30 g L^?1. Temperature and salinity significantly influenced all categories of deformities with treatments at the upper (12°C and 35 g L^?1) and lower limits (6°C and 15 g L^?1) producing the greatest deformities. The optimal temperature and salinity for incubating Puget Sound lingcod eggs was found to be 9°C and 20–30 g L^?1.     

黄条鰤胚胎发育和早期仔鱼生长的温盐适应特性

采用实验生态学、形态测度和分子生物学的方法,研究了温度、盐度对大洋性经济鱼类黄条鰤(Seriola aureovittata)胚胎孵化率、初孵仔鱼畸形率、内源性营养吸收利用、生长基因表达、存活指数(SAI)和饥饿不可逆点(PNR)的影响,并对早期仔鱼活力进行了评价。结果显示,在最适水温20~22 ℃条件下,胚胎孵化率最高,达75%~81%,且初孵仔鱼畸形率低于6.7%,胚胎发育的温度系数Q10值最接近2,且初孵仔鱼全长和卵黄囊体积最大。受精卵在盐度＞30时漂浮在水面,而在盐度为20~25时悬浮在水中,在盐度为10~15时下沉于水底部。受精卵胚胎发育的最适盐度范围为30~35,胚胎孵化率达79%~80%,初孵仔鱼畸形率低于6.0%。在4个不同温度条件下(18、20、22、24 ℃)初孵仔鱼卵黄囊吸收利用速率随着温度的升高而加快。不同盐度条件下,初孵仔鱼的SAI值表明,盐度为30~35时,仔鱼的SAI值较高且峰值出现在盐度为30组,而盐度为10组仔鱼SAI值最低。在水温为20~22 ℃时,6 d仔鱼的初次摄食率最高(78%),PNR出现在7~8 d。初孵仔鱼在水温为20~24 ℃、盐度为30~35条件下,IGF-1 mRNA表达水平显著高于其他实验组。饥饿条件下,IGF-1 mRNA在饥饿后第2天显著升高,其后在第3~4天显著下降,但仍保持较高表达水平,随着饥饿的进行继续下降至显著低于开口期表达水平。本研究明确了黄条鰤受精卵孵化的最适温度为20~22 ℃、最适盐度为30~35,并建立了初孵仔鱼活力评价的指标,研究结果可为建立规范化的黄条鰤胚胎孵化和苗种培育技术提供依据。     

Effects of rearing water quality on expression of brain corticotropin-releasing hormone and gonadal development in goldfish (Carassius auratus)

The effects of changes in water quality in a recirculating system on brain corticotropin-releasing hormone (CRH) gene expression in goldfish were examined. In the experimental group, fish were reared without water change, while in the control group, 50% of water was changed everyday. In the experimental group, total ammonia-nitrogen, nitrate and phosphate were increased over the experimental period. Brain CRH mRNA levels in the control group seemed higher than those in the experimental group, but differences were confounded by variation among individuals. There were no differences in gonadal development between both groups.     

温度和盐度对中间球海胆胚胎发育早期进程联合效应

为探讨高温和低盐对中间球海胆(Strongylocentrotus intermedius)早期胚胎发育进程的影响, 本研究利用中心复合设计(CCD)和响应曲面分析法(RSM), 开展不同温度(12~26 ℃)和盐度(22~34)对中间球海胆胚胎发育早期进程的联合效应研究, 旨在建立温度和盐度对中间球海胆胚胎发育进程的定量关系模型, 并通过统计优化方法得出温度和盐度的最佳组合。结果显示, 在实验设定的温度和盐度范围内, 随着温度的升高, 中间球海胆早期胚胎发育时间呈现出先缩短后延长的趋势; 随着盐度的降低, 中间球海胆胚胎发育早期时间延长。温度的一次效应、二次效应和盐度的一次效应均显著影响(P<0.05)中间球海胆胚胎发育早期进程; 温度一次项系数的绝对值均大于盐度的一次项系数; 温盐的联合效应对中间球海胆胚胎发育早期进程的影响不显著(P>0.05)。实验建立的 2 细胞期、8 细胞期、16 细胞期、囊胚期、上浮期和四腕幼虫期发育进程模型方程决定系数分别为 0.9576、0.9508、0.9689、0.9932、 0.9681 和 0.9763。模型优化和验证试验得出, 温度 20.47 ℃和盐度 31.46 时, 中间球海胆 2 细胞期、8 细胞期、16 细胞期、囊胚期、上浮期、四腕幼虫期的发育时间最短, 分别为 1.28 h、2.07 h、3.31 h、4.14 h、11.28 h 和 47.31 h。 研究结果表明高温和低盐会延长中间球海胆早期胚胎发育时间。     

Impact of salinity changes on growth,oxygen consumption and expression pattern of selected candidate genes in the orange mud crab (Scylla olivacea)

Change in environmental salinity level is a major limiting factor for the aquaculture productivity because it imposes severe stress on organisms that in turn retards growth. The orange mud crab (Scylla olivacea) is an important coastal aquaculture species (farming is practised in 10‰–20‰ salinity levels) in Bangladesh. The present study was conducted to investigate the changes in growth, O₂ consumption and mRNA expression levels of five selected genes in the orange mud crab (S. olivacea) exposed to three different experimental salinity levels (0‰, 10‰ and 20‰) for three months. Crabs reared at 10‰ and 20‰, showed significantly higher (p < .05) growth performance and expression of growth regulatory genes (Actin and α‐amylase). The highest levels (p < .05) of O₂ consumption and expression of ion regulatory genes (Na⁺‐K⁺‐ATPase, V‐type H⁺‐ATPase and Diuretic Hormone) were obtained at 0‰. Moderate levels of growth and expression of selected candidate genes were observed at 10‰ treatment while the highest levels of growth and gene expression were obtained at 20‰ (control salinity). Strong interactions were observed between growth performance and expression of growth genes (R² = 0.81–0.91), and rate of O₂ consumption and expression of ion regulatory genes (R² = 0.83–0.93), implying that the selected genes are important candidates for growth and ionic balance in S. olivacea. Growth performance was found to be very low at 0‰ initially, after 30 days crabs showed better growth performance at this salinity level. It is thus inferred that orange mud crab individuals might require 3–5 days for acclimation to salinity stress but it can take at least 30 days for acclimation to regular growth. Results indicate that with proper acclimation, the orange mud crab (Scylla olivacea) can be farmed at low salinity conditions and possibly in freshwater condition.     

温、盐度对鲻鱼胚胎发育和孵化的影响

本文探讨了不同温度、盐度条件下鲻鱼胚胎发育和孵化情况,结果表明:(1)鲻鱼胚胎发育的适宜水温为18—25℃;(2)适宜盐度为25—40;(3)最适孵化条件为温度25℃和盐度30—35。     

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning,tissue expression,and the function of feeding regulation

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.     

Interactive effects of salinity and complete fishmeal replacement on growth,food consumption,and gene expression of hepatic IGF‐I,IGF‐II and growth hormone receptors in Nile tilapia,Oreochromis niloticus (L.)

A 12‐week feeding trial was conducted using Nile tilapia, Oreochromis niloticus (L.) to evaluate the interactive effects of fishmeal replacement and salinity on growth, feed utilization efficiencies and relative expression of growth related genes. Two iso‐nitrogenous and iso‐energetic diets were prepared (32% protein). The control diet included 15% fishmeal (FM diet) and fishmeal component in non‐fishmeal diet (NFM) was eliminated by a mixture of poultry by‐product meal, high protein distillers dried grains and distillers dried grains with soluble. The NFM diet was supplemented with DL‐methionine and L‐lysine. Duplicated group of fish with initial mean weight of 6 g, reared in four salinity levels (0, 4, 8 and 12 g/L) were fed one of the two diets twice a day to near satiety. At the end of the experiment, growth, feed utilization efficiency and expression of growth related genes were compared. The specific growth rate (SGR), mean feed intake (MFI) and feed conversion ratio (FCR) were not affected by the diets while salinity effects were significant. The fish in the 4 g/L salinity showed the highest SGR and MFI while fish in the 0 g/L treatment showed the lowest FCR. Relative expression of hepatic IGF‐I and IGF‐II was regulated by salinity but not by the diet. Expression of growth hormone receptor gene was not affected by either diet or salinity. The present findings provide evidence for the possibility of total fishmeal replacement in saline waters (0–12 g/L) without compromising growth, feed utilization and body composition of Nile tilapia.     

温度和盐度急性胁迫对岩牡蛎存活及免疫指标的影响

为探究岩牡蛎(Crassostreanippona)对不同温度和盐度急性变化的适应性,本研究通过将岩牡蛎从暂养环境(27℃,盐度32)直接转移至5个温度梯度(15℃、19℃、23℃、31℃、35℃)和6个盐度梯度(16、20、24、28、36、40),研究了急性胁迫对岩牡蛎超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)、溶菌酶(LSZ)活性、丙二醛(MDA)含量及存活的影响。温度实验结果显示,15～31℃组岩牡蛎存活率均高于71.11%,35℃组存活率最低(34.44%),而15℃组免疫指标活性均受到明显抑制, MDA含量在144 h时达到最大值(46.93 nmol/mg prot);温度19℃、23℃、31℃组SOD、CAT、AKP和LSZ活性及MDA含量受应激显著上升后均逐渐恢复至对照水平。盐度实验结果表明, 40组盐度变化对岩牡蛎存活抑制明显,显著低于其他处理组(P0.05),适宜岩牡蛎存活的盐度变化为20～36组;盐度16组SOD、CAT、AKP和LSZ活性144 h时均显著低于对照组(P0.05), MDA含量与胁迫时间成正相关最终达到最大值(45.76 nmol/mg prot)。研究表明,温度和盐度突变均会对岩牡蛎5项免疫指标和存活率产生显著影响,岩牡蛎对温度19～31℃组和盐度20～36组突变具有较强的适应能力。     

Regulation of gonadotropin subunit genes expression by 11-ketotestosterone during early spermatogenesis in male red seabream, Pagrus major

To examine the roles of gonadal steroids in the regulation of expression of gonadotropin subunit genes, male red seabream were gonadectomized and a sub-group was treated with 11-ketotestosterone (11-KT). Castration of males during the early stage of spermatogenesis elicited a significant increase in FSHβ mRNA levels, which was prevented by 11-KT replacement. By contrast, LHβ mRNA levels were not changed by castration or 11-KT replacement. In addition, administration of 11-KT to sham-operated males suppressed the steady-state FSHβ and LHβ mRNA levels. These results indicate that 11-KT may function as a negative feedback regulator of FSHβ gene expression, and may act through the testis to down-regulate LHβ mRNA levels in male red seabream during this period.     

黄条鰤hsp70基因克隆及其在早期生长发育过程中的表达调控特性

热休克蛋白(heat shock proteins, HSPs)在鱼类的应激与免疫反应中发挥重要的生理调控作用,HSP70是该家族的重要成员。为探讨热休克蛋白在大洋性经济鱼类黄条鰤 (Seriola aureovittata)生长发育中的生理作用,本研究克隆获得了黄条鰤hsp70基因的全长cDNA序列,并采用定量PCR技术测定了其组织分布及在早期生长发育过程中的表达特征。结果显示,黄条鰤 hsp70基因的cDNA序列全长为2 332 bp,其中,5′-UTR长度为187 bp,ORF长度为1 920 bp,3′-UTR长度为225 bp,编码639个氨基酸,蛋白质分子量为70.1 kDa,等电点为5.16。黄条鰤 hsp70 mRNA的组织表达具有性别二态性差异,其中,在雌性鳃、心、脾脏和卵巢组织中显著高表达(P<0.05),且以卵巢中表达量最高;雄性垂体、鳃、头肾和精巢组织显著高表达(P<0.05),且以鳃中表达量最高。胚胎发育过程的表达检测显示,在卵裂前的受精卵中可检测到hsp70的表达,表明其具有亲本遗传的特性。同时,在胚胎发育过程的各个时期都可检测到hsp70 mRNA的表达,且在低囊胚期之前的各发育阶段一直保持较低表达水平,在原肠前期开始显著上调表达(P<0.05),其后保持相对较高表达水平,至胚胎孵化出膜期达峰值。在仔稚幼鱼中,hsp70 mRNA在初孵仔鱼和1 d仔鱼中高表达,其后在4 d仔鱼中显著降低(P<0.05),其后显著上调表达,至15 d仔鱼达峰值,其后在20 d仔鱼显著下降,并在25 d后稚鱼和幼鱼中保持相对较低表达水平。研究结果可为深入认识黄条鰤hsp70基因的结构特征、发生发育及其早期生长发育阶段的表达调控功能提供依据。     

Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1–25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.     

Effect of salinity and temperature on marine leech,Zeylanicobdella arugamensis (De Silva) under laboratory conditions

The high prevalence (80–100%) of the marine leech Zeylanicobdella arugamensis (De Silva) on cage‐cultured Asian sea bass Lates calcarifer (Bloch) led us to investigate the percentage of juvenile leeches hatched from deposited cocoons, survival of juvenile and adult marine leeches at different salinity and temperature. The results showed that the hatching percentage of juvenile leeches was highest at salinity of 30 ppt (32.5 ± 2.8%) followed by 20 ppt (18.0 ± 4.3%) and 10 ppt (12.1 ± 1.4%), respectively. It was found that the adult and juvenile leeches could live up to an average range of 4–7 days at salinity ranging from 10 to 40 ppt. The juvenile leeches were able to hatch at temperature ranging from 25 to 35 °C but unable to hatch at 40 °C. The survival period of adult and juvenile leeches ranged from 11 to 16 days at 25 °C, which was comparatively longer than 5–13 days and 10 h – 5 days at 27–30 °C and 35–40 °C, respectively. The study provided the information on the physical parameters of salinity and temperature which are most optimal for the marine leech Z. arugamensis to propagate.