Identification and expression analysis of two HSP70 isoforms in mandarin fish Siniperca chuatsi

Heat shock protein 70 (HSP70) plays important roles in multiple cellular stress responses. Two HSP70 isoforms, ScHSP70a and ScHSP70b, were identified from Siniperca chuatsi in this study. ScHSP70a and ScHSP70b shared all but one of their 639 amino acids and showed a remarkable homology to HSP70s of other species. Expression profile analysis by qRT-PCR revealed that both isoforms were detected throughout embryonic development, and a striking feature of ScHSP70a was its significant up-regulation during the crystal stage. ScHSP70a and ScHSP70b were expressed ubiquitously and at a low level in tissues under non-stressed conditions. However, they were dramatically induced by heat shock at different levels, and their induction was positively correlated with the increasing rate of temperature. Although insensitive to hypoxia in the heart, both genes were greatly induced by hypoxia in the liver, and the induction was returned to the basal level after re-oxygenation for 24 h. Additionally, Aeromonas hydrophila infection also markedly augmented ScHSP70a and ScHSP70b expression in a time-dependent manner in the head kidney and spleen, and the ScHSP70a induction levels were much higher than those of ScHSP70b. These results suggest that ScHSP70a and ScHSP70b contribute differently to embryonic development and protection against damage from high temperature, hypoxia and bacterial infection.     

Recruitment and effects of Discocotyle sagittata (Monogenea) infection on farmed trout

Farmed rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta were monitored over 3 years for infection with the blood-feeding gill fluke Discocotyle sagittata. Parasite transmission is seasonal: new infections take place during summer/autumn, and transmission is generally negligible during winter/spring. There are 2 sources of infection for naïve fish-of-the-year: limited invasion when fish are in the raceways by riverborne larvae originating external to the farm; and internally, within the farm, when 0+ fish are transferred to ponds previously occupied by older cohorts of infected fish. Thereafter, infection levels continue to increase in rainbow trout primarily through transmission within the farm. Prevalence rose to 100% in 1+ fish by the end of their second summer. In O. mykiss, mean abundance reached 194 worms/host for 1+ fish (up to 489 worms/host) and 160 worms/host for 2+ fish. By contrast, in S. trutta, parasite prevalence never exceeded 85% and, after the first year's invasions, infection levels decreased over time: in 1+ and 2+ brown trout, parasite mean abundance was < 4 (maximum 15) worms/host. We present evidence of the detrimental effects of D. sagittata on the host: high burdens are associated with pale gills, decreased body condition and host mortality. Parasite burdens become overdispersed during the warmer part of the year, as prevalence and mean abundance increase. However, the degree of parasite overdispersion decreases over winter; we cannot distinguish whether decreased aggregation is due to parasite losses from infected fish (including immune-mediated parasite mortality) or parasite-induced host mortality.     

Perkinsus mediterraneus infection induces oxidative stress in the mollusc Mimachlamys varia

Perkinsus mediterraneus is a protozoan parasite that can cause marine mollusc diseases known as perkinsosis being a serious threat for clam cultures worldwide. The aim of the present study was first to determine the Perkinsus species infecting the variegated scallop Mimachlamys varia and then to evaluate the existence of oxidative stress in gills of M. varia according to different degrees of infection. DNA sequencing confirmed that P. mediterraneus was the species infecting M. varia. ROS production was progressively increasing with the degree of infection although the differences were only significant in the high-infected group. Low degree of infection significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and nitrite levels with respect to the control group. In the high-infected group, a significant increase was evidenced in all analysed enzymes, catalase, SOD, glutathione reductase and GST. Non-significant differences in MDA levels were observed between the control and low-infected groups; however, a significant increase in MDA levels was observed in the high-infected group. In conclusion, the infection by Perkinsus mediterraneus in M. varia induces oxidative stress and an antioxidant response directly related to the infection degree that can contribute to the pathogenicity of the infection.     

The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.     

Quantitative trait loci (QTL) associated with resistance of rainbow trout Oncorhynchus mykiss against the parasitic ciliate Ichthyophthirius multifiliis

The parasitic ciliate Ichthyophthirius multifiliis has a low host specificity eliciting white spot disease (WSD) in a wide range of freshwater fishes worldwide. The parasite multiplies rapidly whereby the infection may reach problematic levels in a host population within a few days. The parasite targets both wild and cultured fish but the huge economic impact of the protozoan is associated with mortality, morbidity and treatment in aquacultural enterprises. We have investigated the potential for genetic selection of WSD-resistant strains of rainbow trout. Applying the DNA typing system Affymetrix^® and characterizing the genome of the individual fish by use of 57,501 single nucleotide polymorphisms (SNP) and their location on the rainbow trout chromosomes, we have genetically characterized rainbow trout with different levels of natural resistance towards WSD. Quantitative trait loci (QTL) used for the selection of breeders with specific markers for resistance are reported. We found a significant association between resistance towards I. multifiliis infection and SNP markers located on the two specific rainbow trout chromosomes Omy 16 and Omy 17. Comparing the expression of immune-related genes in fish—with and without clinical signs—we recorded no significant difference. However, trout surviving the infection showed high expression levels of genes encoding IgT, T-cell receptor TCRβ, C3, cathelicidins 1 and 2 and SAA, suggesting these genes to be associated with protection.     

额尔齐斯河温氏指环虫的种群生态学研究

2009年4月至2010年1月对额尔齐斯河(中国段)东方欧鳊(Abramis brama orientalis Berg)鳃部寄生的温氏指环虫(Dactylogyrus wunderi Bychowsky)进行了取样调查,以期了解温氏指环虫的种群生态学特点。结果表明,温氏指环虫的总感染率为12.92%,平均感染强度为5.70(1～19)。感染率、感染强度在不同体长组的宿主中表现出不同的变化趋势,随着宿主体长(L)的增大,温氏指环虫的感染率在35cm以上的宿主中达到最大,为50%,而平均感染强度在20≤L<25的体长范围内最小,为1.75;温氏指环虫种群在不同体长组宿主中的分布类型主要为聚集分布,且各体长段宿主之间的感染强度差异不显著(P>0.05)。     

Preliminary characterization of Lactococcus garvieae bacteriophage isolated from wastewater as a potential agent for biological control of lactococcosis in aquaculture

Lactococcosis, a significant emerging disease of fish caused by Lactococcus garvieae, has become one of the devastating problems due to its serious economic damage in aquaculture. The aim of this study was to isolate and characterize a lytic phage infecting L. garvieae as a potential bioagent for the treatment of lactococcosis. In this regard, one strain of L. garvieae was isolated from diseased rainbow trout, and then, following biochemical and molecular identifications, its specific phage, WWP-1, which was able to destroy L. garvieae cells through the lytic cycle, was isolated from a municipal wastewater sample. Transmission electron microscope revealed that the isolated phage possesses an icosahedral head and a non-contractile short tail, resembled to members of the family Podoviridae. Moreover, phage WWP-1 represented optimal antibacterial activity at temperatures ranging from 15 to 30 °C, suggesting that it could be very effective at rainbow trout rearing temperature. Restriction profile analysis revealed that NdeI can digest WWP-1 genome while EcoRI, EcoRV, and BamHI were incapable of cutting its DNA. According to the in vivo experiment result, WWP-1 could decrease mortality rate of infected rainbow trout in aquaculture. The results suggest that this naturally occurring bacteriophage could be considered as a promising agent to control the disease caused by L. garvieae strains in rainbow trout rearing.     

Application of enzyme-linked immunosorbent assay (ELISA) for the study of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia): II. Impacts of Perkinsus olseni on clam reproduction

We investigated the effects of infection by the protozoan Perkinsus olseni on the reproduction of female Manila clams, Ruditapes philippinarum, from a population in Gomso Bay, Korea. The reproductive effort of the clams was assessed by ELISA using a clam egg-specific antibody and was expressed as a weight-based gonadosomatic index (GSI). The number of Perkinsus infecting each clam was estimated from the gills using Ray's fluid thioglycollate medium (RFTM) along with a NaOH digestion assay. We found that reproductive effort was negatively correlated with the intensity of the Perkinsus infection: more heavily infected clams produced fewer eggs during the spawning period from May to August. Frequency of spawning was also negatively correlated with the level of Perkinsus infection; heavily infected clams (HIC) exhibited a single spawning pulse in late July, whereas lightly infected clams (LIC) showed three spawning peaks in mid-May, late July, and late August. Egg production of HIC was only 30–75% of LIC during spawning. The level of total protein in LIC was also higher year round than in HIC. In conclusion, our investigation demonstrates that a high level of Perkinsus infection affects spawning frequency and reduces egg production, which may have long-term impacts on clam recruitment and population growth.     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

编码缺刻缘绿藻乙酰辅酶A羧化酶BCCP亚基的基因克隆与表达分析

为了探讨在氮饥饿对缺刻缘绿藻花生四烯酸(ArA)合成与积累的影响,通过cDNA末端快速扩增(RACE)和设计简并引物进行反转录(RT)-PCR扩增,克隆了编码该藻异质型乙酰辅酶A羧化酶(ACCase)的2个生物素羧基载体蛋白(BCCP)的基因序列。其中MiBCCP1基因的cDNA序列长1 267 bp,包含的5'-非翻译区(UTR)长44 bp,3'-UTR长524 bp,开放阅读框(ORF)长699 bp,编码232个氨基酸并含有45个氨基酸序列的叶绿体定位信号肽;预测成熟蛋白分子量约为20 ku。MiBCCP2基因的ORF长789 bp,编码263个氨基酸并含有49个氨基酸的叶绿体定位信号肽,推测成熟蛋白分子量约为22 ku,但其羧基端缺乏生物素酰基化位点。邻接法(NJ)构建的聚类图显示它们分属于2个不同的分支(靴带值为100)。采用半定量反转录(RT)-PCR技术分析这2个基因的表达量,结果显示,在氮饥饿光照培养条件下,它们的相对转录量都先短暂升高然后持续下调,从而表明缺刻缘绿藻脂肪酸的从头合成能力有下降的趋势。结合该藻的ArA含量在该培养条件下明显增加的结果,推测胞质中同质型ACCase对缺刻缘绿藻ArA合成与积累起着更重要的作用。     

坛紫菜两种Hsp90基因的克隆及表达特征分析

以坛紫菜转录组测序获得的unigene序列为基础,采用RACE(rapid amplification of cDNA ends)技术克隆获得了坛紫菜的两条Hsp90基因序列:PhHsp90-1和PhHsp90-2。序列分析结果表明,PhHsp90-1序列全长2 572 bp,包含一个2 427 bp的开放阅读框,所编码的多肽包含809个氨基酸,分子量为90.2 ku,等电点为4.79,属于Hsp90内质网亚家族(登录号:KF732651);PhHsp90-2序列全长2 510 bp,包含一个2 280 bp的开放阅读框,所编码的多肽包含760个氨基酸,分子量为86.2 ku,等电点为4.81,属于Hsp90细胞质亚家族(登录号:KF732652)。基因表达水平的定量分析结果表明,高温和失水胁迫对两条PhHsp90基因的表达水平均有显著影响,但在表达模式上存在明显差异。高温胁迫不同时间水平下,两条PhHsp90基因的表达均表现为先上调后下调的趋势;而在失水胁迫下,当失水率小于60%时,两条基因的表达水平均没有发生显著变化,但当失水率大于60%时,两条基因的表达水平均显著上调,说明两条PhHsp90基因均在应答高温胁迫和高度失水胁迫中发挥着重要作用。     

Effect of Bacteriophages on Vibrio alginolyticus Infection in the Sea Cucumber,Apostichopus japonicus (Selenka)

Vibrio alginolyticus is an important pathogen that causes a variety of diseases in marine animals including the sea cucumber, Apostichopus japonicus. Herein, we describe a two‐phage mixture which may have potential for use as an antibacterial agent to prevent V. alginolyticus infection in the sea cucumber. A Z1210 bacterial isolate was cultured from diseased sea cucumber suffering from skin ulcerations and viscera ejection. The isolate was identified as V. alginolyticus by morphology and sequence similarity analysis. Subsequently, two bacteriophages infecting isolate Z1210 were isolated from the drainpipe of an aquatic market. Morphologically, these two phages were classified as members of Podoviridae (PVA1) and Myoviridae (PVA2) and each phage showed high virulence in an in vitro experiment. Additionally, an experiment conducted in a marine environment showed that a mixture of the two phages increased the survival of sea cucumbers (10 ± 2 g) to 73, 50, and 47% when it was used with a multiplicity of infection of 10, 1, or 0.1, respectively. This result differed markedly from the control without phage (3% survival) while there was no significant difference between the 80 and 47% survival observed for two antibiotic treatments (5 mg/L doxycycline and 10 mg/L kanamycin, respectively).