The effects of green and blue-green algal diets on the reproductive rate of the rotifer Brachionus plicatilis

The reproductive rate of the rotifer Brachionus plicatilis cultured on a variety of diets was investigated. Unialgal and mixed diets of the green alga Chlorella and the blue-green alga Schizothriz were compared. Rotifer reproductive rate was found to be an average of 2.7 times higher on a mixed diet of Chlorella and Schizothrix than on either Chlorella or Schizothrix as a unialgal diet. Enhancement of rotifer reproductive rate was not observed on a mixed diet of Chlorella and Dunaliella compared with a unialgal diet of Chlorella. Ingestion of Schizothrix was shown not to be required for enhancement of rotifer reproduction. It was further shown that the enhancing factor is a heat labile substance.     

Disruption of quorum sensing in Vibrio harveyi by the AiiA protein of Bacillus thuringiensis

Quorum sensing is a mechanism in which bacteria coordinate the expression of certain genes in response to their population density by producing, releasing and detecting signal molecules called autoinducers. Quorum sensing is responsible for controlling a plethora of virulence genes in several bacterial pathogens. Disruption of the quorum sensing system of Vibrio harveyi has been proposed as a new anti-infective strategy. AiiA is a protein which can block the bacterial quorum sensing by hydrolyzing AHL-lactone, and could greatly attenuate the disease caused by many bacterial pathogens in which quorum sensing regulate the expression of virulence genes. In this study, primers were designed from the conserved sequences of aiiA gene in the genomes of several Bacillus strains, and the gene was amplified from Bacillus thuringiensis BF1 by PCR. AiiA gene was cloned to a cloning vector pUC and sequenced. The open reading frame of the aiiA gene was 753 bp, and the similarity of aiiA gene from B. thuringiensis BF1 to other sequences in the GenBank was as high as 99%. This gene was subsequently cloned into an expression vector pET-24d(+), and the recombinant AiiA protein was overexpressed in Escherichia coli overexpression strain BL21(DE3). The molecular weight of the expressed AiiA protein was estimated to be 28 kDa by SDS-PAGE. The optimized expression condition for the recombinant AiiA protein was at 25 °C for 6 h with 1 mM IPTG induction. The lysate of the recombinant E. coli could not only repress the pigment synthesize of the quorum sensing system reporter strain Chromobacterium violaceum ATCC 12472, but also attenuate the intensity of bioluminescence of V. harveyi VIB391 by 85%. This study was very important for further research on the disruption of infections caused by V. harveyi in aquaculture.     

西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测

2013年8-9月,四川雅安汉源湖养殖的西伯利亚鲟发生一种以体表溃疡、内脏出血和心外膜囊肿为特征的传染病.为明确其病因,本研究从自然发病鱼的肝、脾和肾进行了病原菌的分离、人工感染、分离菌的表型特征和分子生物学特征的检测.结果从患病鱼体内分离到一株G+链状球菌(Ab130920),人工感染实验证实了其病原性,生理生化特性与海豚链球菌(ATCC29178)基本一致;16S rDNA序列(GenBank登录号:KJ162337)与GenBank中S.iniae 16S rDNA序列同源性最高,在以16S rDNA序列及其GenBank中同源性较高的相关序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时,在基于S.iniae lctO基因的特异性PCR检测中,从分离株基因组DNA扩增出预期大小的870 bp条带,进而鉴定分离菌Ab130920为S.iniae.在对cpsD、simA、sagA、pdi和scpI等5种S.iniae毒力基因的多重PCR检测中,分离菌均扩增出相应大小的特异性片段,表明其为一毒力较强的菌株,与人工感染实验的高致病性结果相佐证;药物敏感性检测发现其对阿莫西林、强力霉素、氟苯尼考等抗菌药物敏感,但对新生霉素、利福平、氧氟沙星耐药.     

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。     

水族箱气单胞菌的鉴定及致病特性分析

为了确定南京市某渔场发病鱼感染的病原,本研究采集患鱼脏器,采用平板培养、生化实验和特异性gyrB基因扩增测序等方法进行细菌的分离鉴定;利用PCR技术检测分离株毒力基因的分布,分析其生物学特性,并进一步通过动物实验确定菌株致病力.结果分离到1株水族箱气单胞菌,命名为LK-25.该菌株携带5种主要毒力基因:气溶素(aer)、细胞毒性肠毒素(act)、细胞兴奋性肠毒素(alt)、温敏胞外蛋白酶(epr)和丝氨酸蛋白酶(ahp),其溶血性和溶蛋白能力较强,对斑马鱼的半数致死量为1.02×103 CFU/尾,确定为强毒株.进化树分析表明,水族箱气单胞菌与嗜水气单胞菌达卡亚种亲缘关系较近.本研究在国内首次报道发现水族箱气单胞菌,为进一步预防该菌所引起的相关疾病的发生和传播提供理论基础.     

Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.     

Survival and quality of halibut larvae (Hippoglossus hippoglossus L.) in intensive farming: Possible impact of the intestinal bacterial community

The high mortality commonly observed during the early life stages of intensively reared halibut (Hippoglossus hippoglossus L) is believed to be caused by e.g. opportunistic bacteria. However, the impact of particular bacterial species is poorly defined and still remains disputable. The study describes the bacterial diversity in the gastrointestinal tract of halibut larvae in a large number of incubators at a commercial production site. The overall success of larvae was found to be highly variable and analysis of the gut microbiota revealed high variation of the cultivable part as well as the bacterial community of surface sterilised larvae analysed by denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16s rDNA products. Analysis of the bacterial community of unfed yolk sac larvae revealed higher diversity than previously reported, with Marinomonas, Marinobacter, Aeromonas and Shewanella dominating the community structure. There are indications that Marinomonas is found only in the overall most successful first feeding larvae of the period where the Vibrio group dominated the bacterial community together with Shewanella. Vibrio wodanis was identified as a part of the bacterial community of feeding larvae that yielded the poorest overall success of the period. α-Proteobacteria, not previously reported in halibut, were also found as a part of the bacterial community of first feeding larvae. The diverse bacterial community was only partly reflected in the cultivable part which, however, may reflect the dominating bacterial groups of the highly heterogeneous bacterial community of larvae in the production system as a whole. The bacterial community of the Artemia was found to be highly variable in different samples collected through the period. Only a small part of the different groups observed in the bacterial community of surface sterilised larvae was reflected in the cultivable part which was dominated by highly variable groups in different samples of Artemia. Also, the numbers of cultivable bacteria were found to positively correlate with jaw deformation of unfed yolk sac larvae as well as incomplete metamorphosis of feeding larvae.     

Cost comparisons of rearing larvae of freshwater shrimp,Macrobrachium acanthurus and M. rosenbergii,to juveniles

Costs were determined for rearing larval Macrobrachium rosenbergii (De Man) and M. acanthurus (Wiegmann). Rearing was conducted in two phases. First-phase rearing (first 10 days) was conducted in conical fiberglass tanks with recirculated, filtered, brackish water; second-phase rearing (final 30 days) was done in aerated 1 000-l concrete tanks. To minimize water quality problems and debris accumulation in second-phase rearing, larvae were transferred every 4 to 5 days to a new tank of clean water. Larval food consisted of newly hatched Artemia and either freshly ground fish or freeze-fried fish.Mean survival in five M. rosenbergii rearing trials was 43% in 40 days at a final density of 12 juveniles per liter. Mean survival of four M. acanthurus trials was 25% in 44 days at a final density of 3 juveniles per liter. Average total cost of labor, operation (electricity), food and water per thousand juveniles produced was $3.56 for M. rosenbergii and $13.42 for M. acanthurus.Extrapolation of results to larger (3 000-l) second-phase rearing tanks showed M. rosenbergii rearing cost could be reduced to $\text{\$1.87}\text{1 000}$.     

Recruitment patterns and early growth of acroporid corals in Manado, Indonesia

A coral settlement device was used to examine recruitment patterns of Acropora at two stations (st.; A and B) in Bunaken Island, Manado. The recruitment timing of Acropora was not determined because of mixing with Isopora. A marine block (MB) was used to observe the growth of settled acroporid corals at st. A. Within a year of settlement (February–May of the following year), the corals which had settled on the MB had grown to a size of 13.4 ± 5.86 mm. They were dominated by Isopora, with a small proportion of Acropora. A supplementary experiment again using the MB was conducted at three other sites (st. C, D, E) where Isopora was not abundant. The size of the corals which settled on the MB was nearly the same as that of the corals at st. A. Juvenile Acropora corals (≤3 cm, encrusting form) were measured on the Manado coast (st. F) between February and May. At this site, Acropora corals were dominant and no Isopora corals were observed; the size of Acropora was 18.5 ± 5.01 mm. Acroporid corals in February and March at st. A appeared smaller than those at st. F and grew to nearly the same size in early April.     

Characterization and expression analysis of two distinct neuropeptide Ya paralogues in Jian carp (Cyprinus carpio var. Jian)

Two distinct neuropeptide Ya paralogues (jlNPYa1 and jlNPYa2) were cloned and characterized in Jian carp (Cyprinus carpio var. Jian), with a highly conserved organization encoded by four exons and three introns. The cDNAs for jlNPYa1 and jlNPYa2 were 693 and 730 bp in size, respectively. jlNPYa1 and jlNPYa2 both encoded a 96-amino acid protein, which shared 97.9 % identity. Phylogenetic tree showed that it has two NPYa genes, called jlNPYa1 and jlNPYa2, that presumably resulted from the tetraploidization event in the carp lineage. Analysis of expression profiles of jlNPYa1 and jlNPYa2 showed that the two NPY genes had a broad tissue distribution but expressed primarily in the forebrain, hypothalamus, testis and liver. The expression pattern was different in juvenile and adult (female and male) Jian carp. In juvenile, the highest expression level of jlNPYa1 and jlNPYa2 was detected in the testis. In adult, it was detected in the forebrain. In female hypothalamus, the expression level of jlNPYa1 was significantly higher than that of jlNPYa2. However, the opposite was true in male hypothalamus. The differing distribution patterns of the two NPY genes suggested that jlNPYa1 and jlNPYa2 might play different roles in Jian carp.     

New type of heat shock protein 70 homologue gene abounds in the genomic sequence of kuruma shrimp Marsupenaeus japonicus

Heat shock proteins (HSPs) are a group of highly conserved molecular chaperones. Shrimp HSPs have recently been a topic of increasing interest because of their roles in shrimp immunity and homeostasis. In penaeid shrimp, HSP70s and the cognate forms, heat shock cognate (HSC) 70s, have been reported, but their responses towards various stimulations are different. We found a novel type HSP70 (MjHSP70-2) from the hyperexpansion of the large segmental duplication that is present in kuruma shrimp Marsupenaeus japonicus, which shows about 60 % identity with reported shrimp HSP70s. In a phylogenetic tree, MjHSP70-2 formed a sister clade with eukaryote HSP70 family while MjHSP70 was located close to the shrimp HSP70 and HSC70 group. MjHSP70-2 gene expression was not significantly increased by heat shock or pathogen challenge by Vibrio penaeicida, but it was significantly increased by infection with white spot syndrome virus. In contrast, MjHSP70 gene expression was increased by heat shock but decreased by infection with V. penaeicida. The kuruma shrimp genome was found to have 400-fold more copies of the MjHSP70-2 gene than the putative single-copy gene transglutaminase. In conclusion, our results reveal the presence of a novel type HSP70 gene, HSP70-2, from kuruma shrimp. There are multiple forms of HSP70 in crustaceans, and these HSP70s behave differently under various stressors.     

三角帆蚌HcCUBDC基因cDNA的全长克隆与表达分析

利用cDNA末端快速扩增（Rapid Amplification of cDNA Ends,RACE）方法,获得了三角帆蚌HcCUBDC基因的全长cDNA序列,共5158bp,开放阅读框（ORF）1920bp,编码639个氨基酸,5′端非编码区576bp,3′端非编码区2662bp,GeneBank登陆号为KP067952。生物信息学分析表明,三角帆蚌HcCUBDC蛋白含有一个由19个氨基酸组成的信号肽和4个CUB结构域,但未能比对上已知的蛋白。经荧光定量PCR检测,HcCUBDC基因在紫色和白色蚌前端缘膜、后端缘膜、中央膜、鳃、斧足、肝胰腺、肠和肾8个组织中均有表达,并且都是肝胰腺表达量最低。在紫色蚌中,后端缘膜表达量极显著高于前端缘膜;在白色蚌中,前端和后端缘膜之间表达差异不显著。HcCUBDC基因在紫色蚌后端缘膜中的表达量极显著高于白色蚌,在紫色和白色蚌前端缘膜中的表达量差异不显著。外套膜原位杂交结果显示,HcCUBDC基因主要在缘膜外上皮细胞中表达。研究表明,HcCUBDC基因在三角帆蚌内壳色形成中发挥作用,可为进一步深入研究该基因在珍珠颜色形成过程中的功能及其调控机理提供基础资料。     

中华白海豚骨骼的描述

为详细了解中华白海豚骨骼的特征,将收藏于广东珠江口中华白海豚国家级自然保护区管理局的1副中华白海豚标本,经过解剖→取骨→煮制→酒精脱脂→汽油脱脂→漂白→串制骨骼等步骤制取骨骼标本,对其骨骼系统作较为全面的测量,并对测量资料进行相关统计分析。结果显示,中华白海豚V型骨最大高、最大宽和最大厚等尺度均呈现先逐渐变大,后逐渐变小的特点;胸椎质量与椎体长之间存在明显的二次函数关系(R2=0.864 1);尾椎质量与椎体高之间、质量与椎体长之间均存在明显的幂函数关系(R2=0.964 6,R2=0.912 8);尾椎质量与椎体宽度之间存在明显的指数关系(R2=0.970 9)。二次函数能很好地拟合V型骨质量与最大高之间的数学关系(R2=0.754 6);左、右肋骨完全对称;左、右胸肋完全对称。     

Comparative appetency for Azolla of Cichlasoma and Oreochromis (tilapia)

In a comparative study of the preferences of two species of fish for different Azolla species (aquatic fern), Cichlasoma fenestratum showed a clear preference for Azolla microphylla while tilapia (Oreochromis niloticus) preferred Azolla filiculoides. A. pinnata was neglected.     

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.     

中国明对虾MKK4基因克隆及其在氨氮胁迫下的表达分析

为初步研究中国明对虾MKK4的生物学功能,采用RACE技术克隆获得中国明对虾MKK4基因全长cDNA序列,并对该序列进行分析。结果显示,中国明对虾MKK4基因全长为2 064 bp,开放阅读框长1 221 bp,5'非编码区长214 bp,3'非翻译区长629 bp。将该基因命名为FcMKK4。推测该基因编码406个氨基酸,预测分子量为45.94 ku,理论等电点为8.50。同源性和系统进化分析发现FcMKK4与肩突硬蜱和印度跳蚁的同源性分别为80%和78%,与其他节肢动物MKK4聚为一支。荧光定量RT-PCR结果表明,FcMKK4基因在肌肉中的相对表达量最高,其次为肝胰腺。氨氮胁迫后该基因在中国明对虾肌肉、肝胰腺、血细胞、鳃、心脏、肠和胃中的表达量均显著增加,并有不同的时空表达谱式,表明FcMKK4可能参与中国明对虾非生物胁迫的应答反应。     

Comparison of growth and survival of white shrimp postlarvae (Litopenaeus vannamei) fed dried Artemia biomass versus four commercial feeds and three crustacean meals

The use of dried Artemia biomass meal as an exclusive feed for postlarval white shrimp (Litopenaeus vannamei) was compared with four commercial feeds and three crustacean meals in a series of trials. Postlarvae (PL1–PL6) were stocked at a density of 1.5–2.5/litres in 16 tanks (100 litres volume) and fed, ad libidum, five times a day, over 23–29 days. Feeding postlarval shrimp with dried Artemia biomass resulted in a significantly larger size than feeding with three of the commercial feeds, and the crustacean meals. There was no significant size difference observed in animals fed with Artemia biomass and the commercial 'Golden Pearls' feed for postlarvae, however the coefficient of variation among the size of the 'Golden Pearls' fed animals was significantly higher. The weight increase of animals fed with Artemia biomass was higher than in animals fed with all the tested feeds. The survival rate was not significantly different in animals fed with Artemia flakes from 'Salt Creek', 'Bio-Marine', 'Golden Pearls' and Artemia biomass, however the survival rate was significantly larger in animals fed with Artemia biomass than in animals fed with the crustacean meals and 'Artemac.' Results suggest that dried Artemia biomass is a well-suited feed for postlarval L. vannamei.     

Isolation of Pediococcus homari, etiological agent of gaffkemia in lobsters,from a California estuary

Virulent Pediococcus homari, the etiological agent of gaffkemia, a disease enzootic in Atlantic lobsters (genus Homarus), was isolated from two sites in Anaheim Bay, Calif. The concentration of P. homari was in the order of 1–10 bacteria per ml of sample. This is the first known report of free-living P. homari in the Pacific; the bacterium was previously reported to exist in infected decapod Crustaceae and sediments from the Atlantic. Introduction of P. homari may have occurred through transport of infected Homarus americanus (Atlantic lobster), or by discharge of water in contact with them.