Optimal dietary lipid level for large yellow croaker (Pseudosciaena crocea) larvae

A 30‐day feeding experiment was conducted in blue tanks (70 × 50 × 60 cm, water volume 180 L) to determine the effects of dietary lipid levels on the survival, growth and body composition of large yellow croaker (Pseudosciaena crocea) larvae (12 days after hatchery, with initial average weight 1.93 ± 0.11 mg). Five practical microdiets, containing 83 g kg^?1 (Diet 1), 126 g kg^?1 (Diet 2), 164 g kg^?1 (Diet 3), 204 g kg^?1 (Diet 4) and 248 g kg^?1 lipid (Diet 5), were formulated. Live feeds (Artemia sinicia nauplii and live copepods) were used as the control diet (Diet 6). Each diet was randomly assigned to triplicate groups of tanks, and each tank was stocked with 3500 larvae. During the experiment, water temperature was maintained at 23(±1) °C, pH 8.0 (±0.2) and salinity 25 (±2) g L^?1. The results showed that dietary lipid significantly influenced the survival and growth of large yellow croaker larvae. Survival increased with the increase of dietary lipid from 83 to 164 g kg^?1, and then decreased. The survival of larvae fed the diet with 83 g kg^?1 lipid (16.1%) was significantly lower than that of larvae fed other diets. However, the survival in larvae fed the diet with 16.4 g kg^?1 lipid was the highest compared with other artificial microdiets. Specific growth rate (SGR) significantly increased with increasing dietary lipid level from 83 to 164 g kg^?1 (P < 0.05), and then decreased. The SGR in larvae fed the diet with 164 g kg^?1 lipid (10.0% per day) was comparable with 204 g kg^?1 lipid (9.6% per day), but were significantly higher than other microdiets (P < 0.05). On the basis of survival and SGR, the optimum dietary lipid level was estimated to be 172 and 177 g kg^?1 of diet using second‐order polynomial regression analysis respectively.     

大黄鱼ATG5基因的分子特征及促进病毒增殖的作用研究

自噬是维持真核细胞稳态的重要过程，在细胞分化、发育、免疫等生理过程中发挥作用。目前，人们对于自噬相关基因（Autophagy related gene，ATG）在鱼类免疫应答中的功能仍知之甚少。本研究从大黄鱼（Larimichthys crocea）中克隆得到了自噬相关基因ATG5（LcATG5），其开放阅读框（ORF）全长828个核苷酸，编码275个氨基酸的蛋白质，预测的分子量为32.3 kD，等电点为5.7。氨基酸序列比对和系统进化分析结果显示，LcATG5与其他物种ATG5之间的序列一致性较高，含有1个高度保守的APG5结构域，并且与棘头梅童鱼（Collichthys lucidus）ATG5的亲缘关系最近。荧光定量PCR结果表明，LcATG5在所检测的11个组织或器官中均有表达，在血液中表达量最高，在脾脏中表达量最低；LcATG5在来源于大黄鱼头肾组织的原代粒细胞、淋巴细胞和巨噬细胞以及细胞系LYCK细胞中也均有表达，在原代粒细胞中表达量相对较高，而在巨噬细胞中相对较低；病毒类似物poly(I:C)刺激后，这4种免疫细胞中LcATG5的表达水平都显著上调，其在LYCK细胞中变化最为显著，刺激后12 h上调了3.93倍。过表达LcATG5的鲤上皮瘤（Epithelioma papulosum cyprinid, EPC）细胞受鲤春病毒血症病毒（Spring Viremia of Carp Virus, SVCV）感染48 h后，细胞病变效应（Cytopathic effects, CPE）明显高于对照组，细胞培养上清中SVCV的滴度为1013.82 TCID50/Ml，高于对照组109.27 TCID50/mL，同时细胞内SVCV标志基因SVCV-G、SVCV-M和SVCV-P的表达量分别上调了13.77倍、15.72倍和11.39倍，表明LcATG5过表达促进了EPC细胞中SVCV病毒增殖，这些结果为深入研究自噬和自噬相关基因在鱼类病毒感染过程中的作用及机制奠定了基础。     

Nocardiosis in large yellow croaker, Larimichthys crocea (Richardson)

An epizootic in seawater-cage reared large yellow croaker, Larimichthys crocea, in China was caused by a Nocardia sp. from August to October 2003. The cumulative mortality rate was 15% and the diseased fish were 16 months old with individual length varying from 25 to 30 cm. Multiple, white nodules, 0.1-0.2 cm in diameter, were scattered on the heart, spleen and kidney. The morphology of isolated bacteria from Lowenstein-Jensen medium and tryptic soy agar was bead-like or long, slender, filamentous rods. Experimental infection indicated that the isolated bacterium was the pathogen responsible for the mortalities. A partial sequence of the 16S rRNA gene of the organism and the type strain of Nocardia seriolae JCM 3360T (Z36925) formed a monophyletic clade with a high sequence similarity of 99.9%. Based on the morphological, physiological, biological properties and the phylogenetic analysis, the pathogenic organism was identified as N. seriolae. This is the first report on N. seriolae-infected large yellow croaker in aquaculture.     

Physiological and molecular changes in large yellow croaker (Pseudosciaena crocea R.) with high‐fat diet‐induced fatty liver disease

Fatty liver disease is regularly observed in cultured large yellow croaker, and the disease leads to lower growth rates and reduced harvest yields. The goal of this study was to achieve a more detailed understanding of the physiological and molecular changes in response to high‐fat diet‐induced fatty liver in large yellow croaker. Large yellow croaker fed a high‐fat diet (HFD) for 9 weeks developed hepatic steatosis characterized by histological observation and significantly increased plasma triglyceride levels and serum alanine aminotransferase (ALT) activities. However, no significant differences in serum total protein, glucose, cholesterol, non‐esterified free fatty acids (NEFA), aspartate aminotransferase, high‐density lipoprotein and low‐density lipoprotein were observed between the normal diet and the high‐fat diet (HFD) group. The fatty acid composition of tissue lipids was not affected significantly by dietary lipid levels. Gene expression analysis demonstrated that the HFD decreased fatty acid synthase expression and increased PPARγ expression, but had no effect on lipoprotein lipase and PPARα expression. These results suggest that the HFD‐induced physiological changes and fatty liver may be due to the alteration of related gene expression. As such, further investigations of the metabolic pathways and differentially expressed genes are of particular significance in the mechanistic study and understanding of HFD‐induced fatty liver disease.     

大黄鱼网箱养殖水体的细菌群落结构

以16S rDNA为分子标记, 通过构建克隆文库、限制性片段长度多态性(Restriction Fragment Length Polymorphism, RFLP)分析等技术手段, 研究大黄鱼(Larimichthys crocea)网箱养殖水体中细菌的群落结构。样品采自福建省宁德市三都湾富发养殖基地。随机选取5个不同养殖网箱水样混合, 取3 L混合水样过滤富集细菌后提取总DNA, 用细菌通用引物27F和1 492R扩增其16S rRNA基因, 构建克隆文库。从文库中随机挑选154个克隆子进行分析, 得到137个阳性克隆, 92种RFLP带型。对部分代表性克隆子进行测序的结果表明, 大黄鱼养殖水体中细菌多样性非常丰富。序列分析结果显示, γ-变形菌纲的假交替单胞菌属(Pseudoalteromonas)细菌为最优势菌(占γ-变形菌纲克隆子数的31.2%), 海源菌属(Idiomarina)次之(占γ-变形菌纲克隆子数的15.1%)。此外还存在6.6%的厚壁菌门(Firmicutes)、5.9%的拟杆菌门(Bacteroidetes)、各0.7%的浮霉状菌门(Planctomycetes)、绿屈挠菌门(Chloroflexi)、绿菌门(Chlorobi)细菌和OP11类群。本研究结果阐明了网箱养殖大黄鱼水体细菌的群落结构, 为大黄鱼养殖区病害防治、环境监测以及探讨大黄鱼健康养殖与养殖水体细菌间的关系奠定了基础。     

Dietary lysine requirement of large yellow croaker (Pseudosciaena crocea,Richardson 1846) larvae

A 30‐day feeding experiment was conducted to estimate the lysine requirement of large yellow croaker larvae (2.75 ± 0.11 mg). Six isonitrogenous (509.5–519.7 g kg^?1 crude protein) and isoenergetic (22.3–22.5 kJ g^?1 energy) microdiets containing graded levels of lysine·HCl ranging from 24.8 to 41.0 g kg^?1 diet in placement of glycine and glutamic acid were formulated. Mixture of crystalline amino acids (MAA) was supplemented to simulate the amino acid (AA) profiles of whole body of this larva, except for lysine. The MAA and supplemented lysine for each diet were coated with tripalmitin. Triplicate groups of 3000 fish were fed to apparent satiation by hand eight times per day. The results showed that specific growth rate (SGR), survival, body composition and the specific activity of digestive enzymes were significantly affected by dietary lysine levels (P<0.05). The optimal dietary lysine requirements estimated by second‐order polynomial model based on SGR and survival were 33.7 (65.5 g kg^?1 dietary protein) and 33.4 (64.9 g kg^?1 dietary protein) g kg^?1 dry diet respectively. The estimated requirements for the other essential AAs were calculated by A/E ratios of whole body AA profile of this larva based on lysine requirement.     

突变高温胁迫对大黄鱼血清生理指标的影响

鱼类作为变温动物,在水温突变时体内会发生一系列生理变化。本研究分析了突变高温胁迫下大黄鱼幼鱼血清皮质醇(COR)、血糖(GLU)以及乳酸(BLA)含量的变化规律。实验选取8月龄平均体重为(118.8±6.05)g的大黄鱼作为实验对象,对照组设定温度为(23±0.3)℃,实验组设定温度为(33℃±0.3)℃,变温过程为突变,观察记录实验现象。结果表明:大黄鱼幼鱼在33℃高温胁迫下有强烈的应激反应,表现为呼吸加快、极度不安、游动剧烈。经33℃高温胁迫2 h后,实验组幼鱼血清中皮质醇、血糖、乳酸含量与对照组相比均有显著的升高(P0.05或0.01),陆续出现死亡,最终全部死亡。研究认为,33℃高温胁迫对大黄鱼有致命的影响,在养殖生产过程中要将水温严格控制在33℃以下。     

Effects of dietary tea polyphenols on growth,biochemical and antioxidant responses,fatty acid composition and expression of lipid metabolism related genes of large yellow croaker (Larimichthys crocea)

The study was conducted to investigate the effects of dietary tea polyphenols (TP) on growth performance, biochemical and antioxidants responses, fatty acid composition, and lipid metabolism‐related gene expressions of large yellow croaker (Larimichthys crocea). Four diets were formulated with different levels of TP (0.00%, 0.01%, 0.02% and 0.05%). Results showed that growth performance of L. crocea were not different among dietary treatments. Compared with the control group, fish in 0.02% TP group had lower body and hepatic lipid content and lower total cholesterol content. The minimum content of triglycerides and low‐density lipoprotein‐cholesterol were found in 0.05% TP group. Hepatic n‐6 PUFA and n‐3 PUFA were significantly higher in TP supplementation groups. Malondialdehyde content was lower in TP supplementation groups, and superoxide dismutase activity was higher in 0.01% TP group than the control group. The mRNA expressions of carnitine palmitoyltransferase1, acyl‐CoA oxidase and peroxisome proliferators‐activated receptor α were up‐regulated in 0.01% and 0.02% TP groups, while lipoprotein lipase expression was down‐regulated in TP supplementation groups than the control group. Results suggested that 0.01%–0.02% TP supplementation could reduce the deposition of liver lipid of L. crocea caused by high‐lipid diet, which might be due to the increase in lipid oxidation related gene expressions.     

大黄鱼选育子二代生长性状研究

通过对选育和未选育2个群体大黄鱼（Larimichthys crocea）繁育得到的子代的卵径、油球径和口径进行测量,以及对其子代养殖过程中的生长性状进行测量比较,分析选育效果。结果显示,选育大黄鱼子代（XY-F2）和未经选育大黄鱼子代（HB-F1）平均卵径、平均油球径和平均口径之间差异不显著（P〉0.05）。拟合1~10月龄XY-F2和HB-F1的体长（x）、体质量（y）的生长曲线及体长（x）与体质量（y）的关系表明,两群体间体长（x）与体质量（y）的关系存在差异;XY-F2在体长（x）和体质量（y）的生长在后期高于HB-F1。分析其数量性状,选育子一代（XY-F1）2龄鱼体长、体质量现实遗传力分别为0.26、0.18,而XY-F2的体长、体质量现实遗传力分别为0.029、0.134。结果说明选育具有一定的作用,今后选育应该结合家系选择和家系内选择进行。     

Dietary fishmeal levels affect the volatile compounds in cooked muscle of farmed large yellow croaker Larimichthys crocea

A comparative study on the volatile compounds in cooked muscle of wild and farmed large yellow croaker (LYC) was conducted. The two farmed LYC groups were fed with diets containing 44% (CF) and 25% (LF) of fish meal (FM) respectively. Results showed that 48 volatiles, including aldehydes, alcohols, ketones, hydrocarbons, aromatics, acids, esters, furans and miscellaneous compound, were detected in cooked fillets. The LF group had significantly lower amounts of total aldehydes and ketones, higher content of miscellaneous compound in cooked fillets than that in the CF and wild groups (p < .05). Compared with the wild group, the LF group had significantly lower amounts of total alcohols, acids and esters, while the CF group had significantly lower amounts of total aldehydes, higher content of total ketones in cooked muscle (p < .05). According to the principal component analysis (PCA), some volatiles (propanal, nonanal, etc.) could be considered as sensitive indicators to classify cooked muscle samples. In conclusion, differences in the volatiles in the cooked muscle between the wild and farmed LYC have been found. Low level of dietary FM (25%) changed the volatile profiles in cooked fillets of farmed LYC. A PCA may be useful to screen potential volatiles to classify cooked muscle samples in this study.     

大黄鱼UBXN1基因鉴定及其过表达后的转录组分析

为探究UBXN1在大黄鱼抗盾纤毛虫感染中的作用及其可能涉及的免疫信号通路,本实验采用RT-PCR鉴定了大黄鱼UBXN1基因,并利用在线软件对其序列特征进行生物信息学分析; 采用实时荧光定量 PCR(qPCR)探究UBXN1在多个组织的表达情况,及盾纤毛虫感染下的表达变化;免疫荧光分析UBXN1的亚细胞定位;转录组筛选UBXN1过表达后的差异表达基因。结果显示：UBXN1基因cDNA全长为915 bp,编码304 个氨基酸。蛋白序列同源比对和结构预测表明UBXN1是一个进化保守的蛋白,包含UBA和UBX结构域。qPCR分析表明UBXN1呈组成型分布表达,脑中表达量最高,其次是肝脏、心脏和肾脏,在肌肉中表达量最低;盾纤毛虫感染大黄鱼,UBXN1在脾脏、脑、肝脏和肾脏表达量早期显著升高,后期逐步恢复至正常水平。亚细胞定位分析表明,UBXN1在大黄鱼肾脏细胞质和细胞核均有表达。在293T细胞过表达UBXN1,转录组分析筛选到12个上调基因,4个下调基因,其中RPL41/RPL39/XIST/RNA45SN4表达量显著增加,而ATP8/ND4L表达量显著减少。这些研究结果表明UBXN1在大黄鱼抗寄生虫免疫应答中发挥重要作用, 并为进一步深入研究UBXN1的免疫调控信号通路奠定基础, 这是目前在鱼类首次报道UBXN1。     

Effects of dietary vitamin E and astaxanthin on growth,skin colour and antioxidative capacity of large yellow croaker Larimichthys crocea

A 10‐week feeding trial was conducted to evaluate the effect of dietary vitamin E and astaxanthin on growth performance, skin colour and antioxidative capacity of large yellow croaker Larimichthys crocea. Six practical diets were formulated in a 2 × 3 factorial design to supplement with two levels of astaxanthin (25 and 50 mg/kg) and three levels of vitamin E (0, 120 and 800 mg/kg). The results showed that both the highest final body weight and specific growth rate were found in fish fed diets with 120 mg/kg vitamin E supplementation. No significant differences were found in survival rate, feed conversion ratio and protein efficiency ratio among all the treatments (p > .05). Skin lightness (L*) was not significantly affected by dietary treatments (p > .05). Ventral skin redness (a*) of fish fed diet with 25 mg/kg astaxanthin and 0 mg/kg vitamin E supplementation was significantly lower than that of fish fed with other diets. Yellowness (b*) and carotenoid contents both in the dorsal and in the ventral skin were found to be significantly increased with increasing dietary astaxanthin or vitamin E (p < .05), but no significant interactions were found (p > .05). The vitamin E content in liver reflected the dietary vitamin E content. Level of vitamin E content in fish fed diets with 800 mg/kg vitamin E supplementation was significantly higher than that in fish fed with the other diets (p < .05). Liver superoxide dismutase activity and thiobarbituric acid reactive substance levels were found to be decreased with increasing dietary astaxanthin and vitamin E levels, respectively. Levels of reduced glutathione in the liver were found to be increased with increasing dietary vitamin E contents. The total antioxidative capacity in the liver was found to be decreased with increasing dietary vitamin E or astaxanthin contents. In conclusion, adequate dietary vitamin E can improve the growth of large yellow croaker, and the supplementation of astaxanthin and vitamin E benefited the skin coloration and antioxidative capacity of large yellow croaker.     

Influence of dietary biotin levels on growth and non‐specific immune response in large yellow croaker,Larimichthys crocea R

A 9‐week feeding experiment was conducted to determine the effect of dietary biotin levels on growth performance and non‐specific immune response of large yellow croaker. Fish (6.16 ± 0.09 g) were fed twice daily to apparent satiation with diets containing 0.00 (as the basal diet), 0.01, 0.05, 0.25, 1.24 and 6.22 mg biotin kg^?1 diet. Results showed that fish fed the basal diet had the lowest survival rate, and fish fed 0.05 mg kg^?1 dietary biotin achieved significantly higher final weight and weight gain. Dietary biotin levels had no significant influence on carcass crude lipid, moisture and ash content, but significantly influenced the carcass crude protein. Liver biotin concentration significantly increased with the supplementation of biotin, but no tissue saturation was found within the supplementation scope of biotin. Broken‐line regression analysis of weight gain showed that juvenile large yellow croaker requires a minimum dietary biotin of 0.039 mg kg^?1 for maximal growth. The analyses of serum parameters showed that the moderate‐ (0.05 mg kg^?1) and high‐dose (6.22 mg kg^?1) dietary biotin significantly improved both lysozyme and alternative complement pathway activities, indicating dietary biotin within a certain range could improve the non‐specific immune response of large yellow croaker.     

Evaluation of Enteromorpha prolifera as a feed component in large yellow croaker (Pseudosciaena crocea,Richardson, 1846) diets

A 9‐week feeding trial was conducted to investigate the effects of dietary Enteromorpha prolifera on the growth performance and body composition of juvenile large yellow croaker (Pseudosciaena crocea) (Richardson, 1846) (11.41 ± 1.59 g) in floating sea cages (1.5 × 1.5 × 2.0 m). Four isonitrogenous and isoenergetic diets were formulated to contain graded levels (0%, 5%, 10% and 15%) of E. prolifera. Survival ranged from 98.7% to 99.7%, and was independent of dietary treatment (P>0.05). There were no significant differences in the feeding rate among dietary treatments (P>0.05). The specific growth rate (SGR) increased with increasing levels of E. prolifera. When the supplementation of E. prolifera was >5%, SGR was significantly higher compared with the control group (0%). The feed efficiency ratio (FER) in fish fed the diet with 5%E. prolifera (diet 2) was higher than that of the other groups, while in fish fed the diet with 10%E. prolifera (diet 3), it was the lowest (P<0.05). The protein retention (PR) decreased as the level of E. prolifera increased in diets (5%, 10% and 15%). The protein body content displayed a trend similar to that of PR. No significant difference was observed in body moisture and ash among the dietary treatments. An increase in minerals of potassium, magnesium and sodium in body was observed with an increase in dietary seaweed concentrations. On basis of the SGR and FER, supplementation levels of E. prolifera can reach at least 15% without affecting the growth and still maintain a high survival rate for juvenile large yellow croaker.     

Effects of dietary carbohydrate and lipid levels on growth performance,feed utilization,body composition and non‐specific immunity of large yellow croaker (Larimichthys crocea)

The present study was conducted to investigate the effects of dietary lipid, carbohydrate and their interactions on growth performance, feed utilization, body composition and non‐specific immunity of large yellow croaker (Larimichthys crocea). The experimental fish were fed the diets with graded levels of dietary crude lipid (80, 110 and 140 g/kg diet) and dietary carbohydrate (60, 90 and 120 g/kg diet) for 7 weeks. Results showed that the specific growth rate decreased significantly as dietary carbohydrate increased, which partly resulted from the progressively reduced feed intake and protein efficiency ratio. Meanwhile, the result of postprandial glucose content confirmed glucose intolerance of the croaker. However, the specific growth rate was not significantly altered by dietary lipid, but the feed intake was remarkably reduced. The different growth performance and feed utilization of croaker in response to dietary lipid and carbohydrate resulted in the variation of body composition. In addition, the activity of lysozyme and classical complement pathway was significantly improved in croaker fed the diet with 110 g/kg lipid. In above, dietary carbohydrate and lipid of large yellow croaker should be maintained at 60 and 110 g/kg diet, respectively, based on the above indices.