虹鳟pmela基因的克隆及其在不同发育阶段和组织中的表达分析

前黑素小体蛋白a (premelanosome protein,pmela)基因是黑色素合成通路中的关键基因之一,对动物的体色有着重要影响。为探讨pmela基因在虹鳟(Oncorhynchus mykiss)体色变异中的作用,本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术对pmela基因cDNA全长进行克隆并运用生物信息学方法分析该基因的序列特征,同时采用qRT-PCR比较pmela在野生型虹鳟(虹鳟)、黄色突变型虹鳟(金鳟)和杂交F₁代受精期至孵化后3个月不同发育时期及成鱼不同组织中的相对表达量。研究获得pmela基因cDNA全长序列3 476 bp,包含2 532 bp开放阅读框,编码843个氨基酸。序列分析发现,Pmela为疏水性蛋白且存在PKD功能结构域。同源性比对发现,虹鳟Pemla氨基酸序列与红鲑(Oncorhynchus nerka)的同源性高达97.75%;进化分析结果显示,虹鳟与红鲑的亲缘关系最近,与人类(Homo sapiens)和小鼠(Mus musculus)的亲缘关系最远。...     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

牙鲆tyrp1a和tyrp1b的鉴定及tyrp1a与mmu-miR-143-5p_R+2的调控关系

为了研究牙鲆白化发生过程中的分子调控机制,本研究在获得正常和白化牙鲆转录组及micro RNA(mi RNA)深度测序数据的基础上,对tyrosinase related protein 1(tyrp1)和mmu-mi R-143-5p_R+2(mmu-143)进行了表达模式、靶基因预测及验证分析。首先通过RACE方法克隆得到白化相关基因tyrp1的2个转录本,进化树分析表明这2个转录本分别是tyrp1a和tyrp1b,利用RNAhybrid软件预测到mmu-143可能与tyrp1a基因存在互作关系,通过双荧光素酶实验初步验证了这一靶向关系。进一步的荧光定量结果显示,tyrp1a基因在正常牙鲆皮肤中的表达量显著高于白化牙鲆皮肤的表达量,正常牙鲆皮肤中mmu-143的表达量显著低于白化牙鲆皮肤中的表达量。本研究发现,牙鲆tyrp1存在2个转录本,分别是tyrp1a和tyrp1b。双荧光素酶实验和定量PCR分析初步证实,tyrp1a是mmu-143的靶基因,mmu-143是通过调控tyrp1a基因的表达来影响牙鲆白化的。此研究结果为深入揭示牙鲆白化发生的分子机制提供重要的基础资料。     

Nile tilapia broodfish fed high‐protein diets: Digestive enzymes in eggs and larvae

The aim of this study was to assess the activity of gastric, pancreatic and intestinal digestive enzymes during the embryonic and larval development of Nile tilapia (Oreochromis niloticus) GIFT strain Aqua America^® 1 obtained from a broodstock fed two levels of crude protein (CP). A total of 72 females and 24 males, 10 hapas, two CP levels (32% and 38%) and six phases of embryonic (cleavage, blastula, gastrula) and larval (hatching, 7 and 10 days post hatch, dph) stages were used. The eggs were collected in cleavage, blastula and gastrula stages, 300 mg was collected, and kept in cryogenic tubes in liquid nitrogen. For the samples at larval stage, the remaining eggs were separated according to crude protein level and kept in hatcheries and samples were collected on 7 and 10 dph the same way as before. A total of 48 samples were collected: at each protein level (32% and 38% CP), four samples were collected in each phase of embryonic and larval development. Statistical differences were not observed during embryonic development for acid proteases, trypsin, amylase and lipase activity at both levels of crude protein (32 and 38% CP). Differences for acid proteases were noticed on 7 dph; trypsin and amylase on 7 dph and 10 dph. Significant differences on blastula and 7 dph for protease; as for aminopeptidase, there was significant difference on 7 dph. The data indicated early appearance of digestive enzymes in Nile tilapia broodfish receiving 32% CP taking into account the rapid growth and development of this species.     

Thermal tolerance of a rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> strain selected by high-temperature breeding

ABSTRACT:   Thermal tolerance was studied in a rainbow trout strain successively selected through high temperature breeding at 20–27°C since 1966 in Miyazaki Prefecture, Japan. The hatching rate and fry mortality at high temperatures were examined in the selected strain along with normal strains cultured at water temperature of 9–17°C. The hatching rate of embryos fertilized at either 10 or 14°C and subsequently subjected to high temperatures in the blastula or neurula stage of the selected strain, was marginally higher than that of the normal strain counterparts. The upper 50% lethal temperatures (LT₅₀) for embryos in the early segmentation, blastula and neurula stages of the selected strain were also higher than those of the normal strain counterparts. Death temperatures and LT₅₀ of fries acclimated to 20°C of the selected strain were significantly higher than those of the normal strains. However, no difference in the critical thermal maximum was detected between the different strains. These results suggest that the selected strain of rainbow trout established by selecting successively for many generations at high temperatures acquired a degree of thermal tolerance.     

Are environmental conditions in Finnish streams limiting to early life-history survival in the nonnative rainbow trout?

The nonnative rainbow trout Oncorhynchus mykiss has been an unsuccessful invader in North European streams, although it has been widely introduced. Here we studied whether early life history stages (egg incubation and hatching, first overwintering) act as filters for the establishment of hatchery rainbow trout. Survival of hatched alevins was approximately 80%, whereas only 47% of the embryos survived. However, the latter value was impacted by the high number of unfertilized eggs. Correlation coefficients with embryo survival rate and environmental variables (pH and temperature) were statistically insignificant. In the overwintering experiments, the survival of rainbow trout was 93%. The growth was generally slowed during the winter, but in the spring the growth of rainbow trout exceeded that of the native brown trout. Our data demonstrated that the survival and growth of rainbow trout during early life-history stages were relatively high and comparable to those of the native brown trout. Based on the variables considered in our study, our results suggest that environmental conditions during early life-history stages are not detrimental for rainbow trout in the study streams.     

Aggressive behaviour and cannibalism in greater amberjack,Seriola dumerili : effects of stocking density,feeding conditions and size differences

Aggressive behaviour, assessed by 24‐h observations, and survival rates, determined in a 2‐week rearing experiment, were examined in greater amberjack, Seriola dumerili, subjected to single and multiple factors (high density, restricted feeding and diverse size). Aggressive behaviour frequency in the group subjected to both restricted feeding and size diversity (FS) was significantly higher than that in the other groups (Tukey, n=4, P<0.05). In the FS group, aggressive behaviour increased with the starvation time from 19 days post hatching (dph) to 23 dph (two‐way analysis of variance, Tukey, n=4, P<0.05), but decreased gradually thereafter to 46 dph. In the rearing experiment, the survival rates for the diverse size and FS groups were lower than those of the control or restricted feeding groups (Kaplan–Meier, log‐rank test, n=100, P<0.01). Daily mortality in all groups was the highest at 24 dph; although dead fish were usually small, disappearance due to cannibalism was not observed during the rearing period. The results indicate that aggressive behaviour in greater amberjack is induced by hunger and size diversity in the early juvenile stages (23 dph). Mass mortality during seedling production was mainly caused by injury and the death of small fish due to aggressive behaviour/cannibalism.     

三倍体雌性虹鳟性腺发育阶段细胞色素相关基因的表达

Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等细胞色素相关基因能够调节硬骨鱼类性类固醇的合成,对性腺发育和性别决定产生影响。本研究以全雌三倍体虹鳟(Oncorhynchus mykiss)为研究对象,正常雌性二倍体虹鳟为对照,选取31~68 dpf(days post fertilization)时间段的虹鳟仔鱼脑组织,采用q RT-PCR和酶联免疫的方法研究以上几种基因的表达状况和脑芳香化酶的活性变化,以期探明导致三倍体雌性虹鳟性腺发育异常的关键原因。q RTPCR结果显示,二倍体中Cyp19a1b在30~50 dpf时表达量上调并且维持在较稳定水平,但50~56 dpf时表达量逐渐下调,之后56~68 dpf表达量持续上调;三倍体中Cyp19a1b表达量在30~35 dpf开始上调,35~47 dpf逐渐下调,47~55 dpf开始第二次上调,之后维持在较稳定水平直至68 dpf,但三倍体Cyp19a1b的表达量显著(P0.05)低于同期二倍体的。二倍体Cyp11a1表达量在34 dpf出现峰值,三倍体Cyp11a1在38 dpf时出现峰值。二倍体Hsd3b1表达量在33~42 dpf时维持在较高水平,在38 dpf时出现高峰;三倍体Hsd3b1表达量在47~59 dpf时较高,在49 dpf出现高峰。二倍体中Cyp11b2在37 dpf出现峰值,之后开始下调;三倍体在40 dpf出现峰值,之后逐渐下调,但三倍体Cyp11b2表达量显著低于同期二倍体。二倍体Cyp17a1的表达量在35~46 dpf时逐渐上升,在45 dpf时达到高峰之后直至69 dpf逐渐下降,并且维持在较为平稳的水平上;但是在相同的实验条件下未检测到同一时期三倍体Cyp17a1的表达量。酶联免疫结果显示,在40 dpf时二者的脑芳香化酶活性到达高峰,但在40~60 dpf时期,二倍体虹鳟脑芳香化酶活性显著(P0.05)高于三倍体虹鳟,尤其在45~50 dpf时,该酶活性分别较三倍体的高1.15倍和1.12倍。以上结果表明三倍体虹鳟早期性腺发育迟缓的原因之一是Cyp19a1b、Cyp11a1、Cyp11b2、Cyp17a1、Hsd3b1等基因的表达晚于二倍体,且表达量低于二倍体,造成雌二醇不能正常合成,最终导致性腺发育迟缓。     

虹鳟胚胎受精率适宜发育期的研究

用鉴别液J1、J2与Bouin’s液固定剥膜比较方法,测定了虹鳟(Oncorhynchus mykiss)不同发育期胚胎的受精率。结果表明:用鉴别液J1处理后测得的二细胞期、四细胞期、八细胞期、桑葚期、高囊胚期、原肠中期胚胎的受精率分别为53.21%、77.89%、82.00%、88.2%、87.71%和81.43%;用鉴别液J2处理测得的受精率分别为51.64%、77.86%、81.43%、89.07%、87.50%和81.86%。二者与Bouin’s液固定剥膜后测定的受精率差异不显著(P>0.05),说明这两种鉴别液均可用于生产中测定受精率。在不同的发育时期,鉴别的准确性有所不同,细胞分裂期的准确性远低于桑葚期至囊胚期,这可能是由于胚胎发育不同步和早期胚胎细胞较少所致,因此,鉴别虹鳟鱼卵的受精率最好在桑葚期和囊胚期之间(12～24℃.d)的时间点。     

MITFa及TYR基因在红色锦鲤体色发生不同阶段的表达分析

本研究描述了红色锦鲤从出膜到体色形成的过程,总结和归纳不同发育阶段体色变化异同点,筛选出6个体色变化较显著时期,分别为1、2、3、4、12、48日龄。利用荧光定量PCR分析MITFa及TYR基因在红色锦鲤6个体色变化时期的表达情况。结果显示,MITFa基因在1日龄时表达量最高,显著高于48日龄时(P0.05),极显著高于其他4个时期(P0.01)。48日龄时表达量次之,亦极显著高于2、3、4、12日龄4个时期(P0.01)。2、3、4、12日龄4个时期MITFa基因表达量较低且不存在显著差异(P0.05)。TYR基因在1日龄时表达量最高(P0.01),4日龄时表达量显著高于12、48日龄(P0.05),与2、3日龄不存在显著差异(P0.05)。TYR基因表达量在2、3、12、48日龄4个时期差异不显著(P0.05)。MITFa和TYR基因在红色锦鲤体色形成过程中,表达水平整体呈现降低的趋势,其中MITFa基因表达量表现为先降后升,TYR基因则先降后升再降。以上结果显示MITFa、TYR基因与锦鲤体色形成具有一定相关性,但MITFa和TYR基因在体色发生中的相互作用还有待进一步研究证实。     

Dietary supplementation of probiotics and influence on feed efficiency,growth parameters and reproductive performance in female rainbow trout (Oncorhynchus mykiss) broodstock

The aim of this study was to determine the effects of dietary probiotics on reproductive performance, growth, haematological and biochemical parameters of female rainbow trout broodstock. A total of 60 broodstocks with an average initial weight of 2,267.4 ± 54.9 g were divided into four groups and three replicates in raceway ponds. Fish were fed with diets containing 0 (control), 1 × 10⁹ (P1), 2 × 10⁹ (P2) and 4 × 10⁹ (P3) CFU probiotic per kg diet twice a day for 8 weeks before spawning season. Results revealed that higher levels of dietary probiotics (P3) enhanced protein efficiency ratio and decreased feed conversion ratio (p < .05). There were no significant differences in haematological and other growth parameters among different treatments. According to the results, probiotic‐fed treatments had the lowest levels of cholesterol (237.7 ± 4.3 mg/dl) and the highest levels of total protein (11.3 ± 0.3 g/dl) and albumin (6.9 ± 0.3 g/dl), so that differences were found between P3 and control groups in all cases (p < .05). Based on the observations from reproductive parameters, the highest egg diameter, working and relative fecundities, fertilization and hatching rates, eyed eggs survival and alevins survival rates up to the absorption of the yolk sac were observed in P3 treatment (p < .05). Moreover, eyeing, hatching and yolk sac absorbing stages in P3 treatment were earlier than those in other treatments (p < .05). The results of present study demonstrated that probiotic supplementation improved reproductive performance and some growth and biochemical parameters in female rainbow trout broodstock and the best performance was observed in fish fed 4 × 10⁹ CFU probiotic per kg diet. Due to an increase in egg production and egg size in probiotic‐fed treatments, this feed additive can be used as a suitable ingredient to increase high‐quality egg production in rainbow trout breeders.     

Efficacy of clove oil and ethanol against Saprolegnia sp. and usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss (Walbaum) eggs

Inhibitory concentrations of clove oil and ethanol against growth of Saprolegnia sp. hyphae were screened by a modification of the hemp (Cannabis sativa L.) seed MicroPlate (HeMP) method and their usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss eggs was tested. In vitro experiment showed that in continuous static exposure, clove oil at 100 mg L^?1 significantly inhibited the growth of Saprolegnia, whereas in bath exposures, clove oil at 500 mg L^?1 had no significant effect at any exposure time tested (15, 60 and 240 min), but clove oil at 10 000 mg L^?1 significantly inhibited growth at all exposure times. Clove oil and ethanol treatments had no visible effects on the onset or spread of the fungus during incubation of rainbow trout eggs. Clove oil at 1000 mg L^?1 resulted in 95–100% mortality before the eyed stage was reached. Sublethal concentrations of clove oil and ethanol had no effects on the development rate of the embryo or growth and yolk utilization efficiency after hatching. This study suggests that clove oil and ethanol may not be options in controlling aquatic fungi infestations during incubation of rainbow trout eggs.     

Effect of different permeable and non-permeable cryoprotectants on the hatching rate of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) embryos

The purpose of the present study was to investigate the toxicity of cryoprotectants on the hatching rate of rainbow trout (Oncorhynchus mykiss) embryos. Epiboly and first eye pigmentation stage embryos were immersed in six permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), propylene glycol (PG), ethylene glycol (EG), and acetamide (Ac), in concentrations of 1–5 M for 5 or 10 min and two non-permeable cryoprotectants, sucrose (Suc) (10%, 15%, 20%) and polyvinyl pyrrolidone (PVP) (5%, 10%, 15%) for 5 min. The embryos were then washed and incubated until hatching occurred. The toxicity of the cryoprotectant was assessed by the hatching rate. The results illustrated that permeable cryoprotectant toxicity for rainbow trout embryos increased in the order of PG < DMSO < MeOH < Gly < EG < Ac. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. There were no significant decreases in hatching rate of embryos treated with sucrose and PVP with the increase of concentration; sucrose had higher hatching rates than PVP. Rainbow trout embryos at first eye pigmentation stage exhibited greater tolerance to cryoprotectants than embryos at epiboly stage.     

Production Characteristics of Rainbow Trout,Oncorhynchus mykiss and Brook Trout,Salvelinus fontinalis Under Seasonal Pond Conditions

Abstract

The production characteristics of juvenile rainbow trout, Oncorhynchus mykiss and brook trout, Salvelinus fontinalis were compared under winter pond conditions. Juvenile rainbow trout (55.1 ±1.5 g) and brook trout (28.9 ±0.4 g) were stocked at a density of 8,750 fish/ha into six 0.04-ha ponds. After 163 days, survival, growth, and feed conversion were similar (P >0.05). The results of this study suggest that brook trout may attain growth rates similar to rainbow trout under winter pond conditions in temperate regions of North America.     

Are native brook charr and introduced rainbow trout differentially adapted to upstream and downstream reaches?

Abstract— Due to species introductions, brook charr (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss) occur together in many North American streams and typically exhibit a pattern of distribution in which brook charr numerically dominate headwaters and rainbow trout dominate downstream reaches. It has been suggested that 1) the two species compete or 2) the two species do not compete because they are differentially adapted to environmental conditions found in upstream and downstream zones. We assessed whether there were differences in growth and macrohabitat (pool, run and riffle) selection of brook charr and rainbow trout in upper, middle and lower stream zones of a small Pennsylvania stream. Brook charr and rainbow trout placed in replicate paired enclosures set in upstream and downstream reaches showed no significant differences in growth and survival rates upstream, but brook charr had significantly greater growth rates than rainbow trout downstream. Enclosed fish and free-ranging fish both had negative growth rates during the summer. Enclosed fish lost significantly less weight than free-ranging fish. Instantaneous growth rates of free-ranging adult brook charr and rainbow trout from May to August were negative for both species in all stream zones. Underwater observations of adult brook charr and rainbow trout showed both species occurred significantly more often in pool macrohabitats than expected on the basis of macrohabitat availability, except for rainbow trout in the upstream zone. The proportion of pool macrohabitat was not significantly different among stream zones. Brook charr do not appear to be better adapted to upstream environments in Powdermill Run based on growth, survival and macrohabitat selection during summer. Negative biotic interactions acting along with differential environmental adaptations may explain the pattern of distribution of brook charr and rainbow trout in streams, but long-term transplant experiments with additional life stages will be necessary to examine this hypothesis.     

肌间刺缺失突变对斑马鱼胚胎发育过程中肌肉发育的影响

为阐明肌间刺缺失对斑马鱼(Danio rerio)胚胎发育时期肌肉发育的影响,采用qRT-PCR的方法,检测分析了斑马鱼不同肌间刺表型间5个肌肉特异性基因(mef2、myf5、myod、myog和sox6)在囊胚期(3hpf,hourspost fertilization)、原肠胚期(6 hpf)、体节期(12 hpf)、咽囊期(24 hpf)和孵化期(72 hpf)5个发育时期的表达情况,对比分析了肌间刺缺失对斑马鱼胚胎发育过程中肌肉发育特异性基因的时序表达差异和肌肉发育的影响,以及对受精率、孵化率和畸形率的影响。结果显示, 5个特异性基因在肌间刺缺失突变型与野生型斑马鱼各发育时期的表达量无显著性差异(P0.05)。其中,myf5、myod基因在原肠胚期表达量升高,在体节期和咽囊期开始逐步下降;mef2、myog和sox6基因在囊胚期和原肠胚期表达量很低,在体节期和咽囊期表达量迅速升高,并在孵化期开始下降。此外,受精率、孵化率和畸形率的统计结果显示,肌间刺突变型与野生型间亦无显著性差异(P0.05)。综上,推测肌间刺的缺失对斑马鱼胚胎发育及胚胎发育过程中肌肉的发育没有影响。     

Feeding increasing levels of corn gluten meal induces suboptimal muscle pigmentation of rainbow trout (Oncorhynchus mykiss)

A 24‐week growth trial was conducted to evaluate the effects of feeding levels of corn gluten meal (CGM) on growth performance and pigment deposition in the muscle of rainbow trout (Oncorhynchus mykiss). Three isonitrogenous and isoenergetic (digestible energy basis) experimental diets were formulated to contain increasing levels of CGM (0%, 9% and 18%) and 50 mg kg^?1 of astaxanthin. Each diet was fed in triplicate to groups of 75 fish (initial average body weight = 549 g fish^?1) reared at 8.5°C. The inclusion of CGM did not significantly (P > 0.05) affect final body weight, thermal growth efficiency (TGC) or feed efficiency. Carotenoid concentration determined by liquid chromatography showed a significant (P < 0.05) linear reduction in the concentration of one astaxanthin isomer, all‐trans astaxanthin and all‐trans lutein in the muscle of fish in response to increasing levels of CGM. Tristimulus colour analysis of the muscle showed a significant (P < 0.05) linear reduction in a* (redness) and C*ab (chroma). Salmofan^? score showed a significant (P < 0.05) linear and quadratic reduction in response to increasing levels of CGM. In conclusion, the inclusion of CGM up to 18% does not significantly impact growth performance of rainbow trout. However, the concentration of all‐trans astaxanthin as well as the expression of important colour attributes of the muscle can be negatively affected at levels exceeding 9% of CGM in the diet. More research on this topic is needed to discern the mechanism(s) behind the negative effects of dietary CGM and/or its intrinsic yellow pigments on muscle pigmentation of rainbow trout.     

Supplementing rainbow trout (Oncorhynchus mykiss) broodstock diets with choline and methionine improves growth in offspring

Nutritional programming represents the mechanism through which broodstock feeding strategies have lifelong effects on the offspring generation. The objective of this study was to determine whether supplementing rainbow trout, Oncorhynchus mykiss broodstock diets with choline and methionine affects offspring growth performance. Experimental diets were produced by top-dressing a commercial broodstock diet with (a) 12,000 ppm methionine, (b) 7,400 ppm choline, (c) 12,000 ppm methionine and 7,400 ppm choline, or (d) water (control). Six rainbow trout families were fed experimental diets beginning 18 months post-hatch through spawning; the offspring consumed a commercial diet. The broodstock diet did not affect maternal body weight, body condition, egg size, or egg yield (p > .05). Eggs from choline-treated broodstock contained 10% more choline than control eggs (p < .05). Offspring from choline-treated broodstock were smaller than controls at 146 days post-hatch (dph) (p < .05). At the final harvest (439 dph), offspring from the control broodstock weighed 12–18% less than offspring from broodstock consuming the supplemented diets (p < .05). However, there was a significant diet-by-family interaction on offspring growth (p < .05); supplemented diets improved performance in three of the six broodstock families. These findings indicate that supplementing broodstock diets with methionine and choline causes a nutritional programming effect that benefits growth in offspring.