Isolation and characterization of pathogenic Vibrio alginolyticus from diseased postlarval abalone, Haliotis diversicolor supertexta (Lischke)

Outbreaks of serious mortality among cultured abalone postlarvae have occurred across Southern China since July 2002. Five motile bacterial strains were isolated from diseased abalone postlarvae on tryptic soy agar supplemented with 1% NaCl (TSA1) and/or thiosulphate citrate bile salt (TCBS) sucrose agar plates during an outbreak in August 2003 in Shanwei, Guangdong province. All isolates were characterized and identified as Vibrio alginolyticus on the basis of biochemical characteristics and comparisons with those of the reference strain V. alginolyticus ATCC 17749. Strain 19 (a representative of five similar isolates) was virulent to abalone postlarvae with an LD₅₀ value of1.00 × 10⁴ colony‐forming units mL^?1. All abalone postlarvae exhibited the same signs as in natural outbreaks. The same bacterium could be re‐isolated from abalone postlarvae after bacterial challenge using TSA1 and TCBS plates. The results reveal that V. alginolyticus is an infectious agent of abalone postlarvae.     

Settling and raising postlarval abalone Haliotis diversicolor supertexta (Lischke) on microparticulate diets embedded in a layer of alginate

Two 21 day experiments were conducted to determine whether microparticulate diets could be used to substitute for diatoms for raising (Experiment 1) as well as settling and on‐growing (Experiment 2) Haliotis diversicolor supertexta postlarvae. The experiments were conducted in 10 L flow‐through tanks and each diet had three replicate tanks. In experiment 1, three experimental microparticulate diets supplied by a commercial feed company, labelled SF‐15, SF‐15 (T) and JFY‐13–1, were used, and in experiment 2, SF‐15 and SF‐15 (T). Artificial diets were compared to diatoms in terms of supporting growth and survival. The survival rate for experiment 1 was lowest for JFY‐13–1 (0%) and highest for diatoms (12.9±0.8%). The final size of postlarvae fed SF‐15 and SF‐15 (T) was significantly greater than that of postlarvae fed diatoms, 973±193 μm and 996±220 μm compared to 786±141 μm. In experiment 2, a total number of 501 larvae settled onto plates covered with SF‐15, 254 on SF‐15 (T) and 1085 on diatoms. The highest survival rate was 33.1±6.9% for SF‐15 and the lowest was 17.3±5.0% for diatoms. However, the final length of postlarvae fed diatoms was significantly greater than SF‐15 and SF‐15 (T); 1018±379 μm compared to 728±89 μm and 717±90 μm. The artificial micro diets used in this study were comparable to diatoms in terms of grow and survival of postlarval H. diversicolor supertexta.     

九孔鲍幼鲍生长性状的遗传参数估计

采用不平衡巢式设计方法和人工授精技术,1个父本配3个母本,建立九孔鲍(Haliotis diversicolor super-texta)12个半同胞家系和36个全同胞家系.在40日龄、120日龄、180日龄、240日龄时分别对所建36家系个体测量壳长,壳宽与体质量.采用单性状约束最大似然法(Restricted Maximum Likelihood Method, REML)估计九孔鲍(Haliotis diversicolor supertexta)各阶段体质量,壳宽与壳长性状的遗传参数.结果表明,九孔鲍幼鲍在40、120、180、240日龄时壳长遗传力估计值为(0.13±0.09)~(0.56±0.15),壳宽遗传力估计值为(0.13±0.06)~(0.62±0.13),体质量遗传力估计值为(0.18±0.15)~(0.26±0.08),均属于中等遗传力.另外,从性状的表型相关与遗传相关可以得出,九孔鲍稚鲍在不同日龄壳长、壳宽与体质量的表型相关和遗传相关均为正相关(P≤0.05).表型相关系数为0.66~0.94,遗传相关系数为0.85~0.98,研究结果表明,以3个主要生长性状之一作为选育目标,均能达到遗传改良的目的.     

CB抑制合浦珠母贝受精卵第一极体释放的染色体分离

对CB抑制合浦珠母贝受精卵 (3n♀× 2n ,2n× 2n)第一极体释放的染色体分离行为进行了细胞学观察。结果表明 ,CB抑制第一极体改变了正常的染色体分离行为 ,在第二次减数分裂过程中出现了多极分离 ,主要有二极分离 (2 8.6 7% )、三极分离 (40 .5 6 % )及四极分离 (2 3.78% )三种模式 ,不能确定的占 6 .99%。均等的二极分离可能导致四倍体的产生 ,而部分四极分离也能产生四倍体 ,三极分离主要产生非整倍体。未处理组的染色体仍按正常的二极形式进行分离 ,释放两个极体 ,但两极的染色体数目通常存在极大差异 ,导致大量的非整倍体产生。二倍体组 (抑制第一极体 )也存在同样的多极分离模式 ,二级分离占 2 5 .0 0 % ,三极分离占2 1.5 1% ,四极分离占 34.30 % ,不能确定的占 19.19%。另外 ,对各种染色体分离模式形成的机制进行了讨论     

Triploidy induction in Australian greenlip abalone Haliotis laevigata (Donovan) with cytochalasin B

Triploid induction in Australian greenlip abalone, Haliotis laevigata (Donovan), was conducted by blocking the formation of the second polar body using cytochalasin B (CB). Twenty minutes after fertilization, the zygotes of greenlip abalone were treated with four CB concentrations (0, 0.25, 0.5 and 0.75 mg L⁻¹) for 10, 15 and 20 min. The ploidy of resultant larvae was determined using flow cytometry at 72-h post fertilization. Our study showed that fertilization, hatching, survival and induced triploidy of abalone larvae were significantly affected by the CB concentration and treatment duration. The effective range of CB concentration for triploid induction on greenlip abalone was 0.5–0.75 mg L⁻¹ with an induction duration of 10–15 min. The results indicate that the most effective treatment combination for triploid induction in greenlip abalone is 0.5 mg CB L⁻¹ for 15 min starting at 20-min post fertilization.     

Evaluation of cryodamage in small abalone,Haliotis diversicolor,spermatozoa by using a flow cytometer

We used flow cytometry to determine the quality of small abalone, Haliotis diversicolor, sperm before freezing and after thawing. We investigated the effects of cryopreservation on the characteristics of small abalone semen and determined the motility and fertilization capacity of the pre-freezing and post-thawed sperm. The percentages of motility and fertility were 61 ± 2% and 67 ± 1%, respectively, for the post-thawed sperm and 90 ± 4% and 92 ± 0%, respectively, for the pre-freezing sperm. Sperm cells were stained with specific fluorescent dyes to measure plasma and acrosomal membrane integrity, mitochondrial status, oxidation level, DNA compaction, and viability through flow cytometry. The frozen–thawed sperm exhibited significantly higher mitopotential activity (p < 0.05, damaged mitochondria; 25.01 ± 1.18%) and oxidation value (p < 0.01, free radicals; 63.79 ± 3.93%) compared with the pre-freezing sperm. The oxidation level was the most sensitive signal of the cryopreservation-induced small abalone sperm damage. Flow cytometry is valuable for the objective, accurate, and rapid assessment of pre-freezing and post-thaw small abalone sperm quality.     

Molecular cloning,expression and functional analysis of a predicted HdiQM gene in small abalone,Haliotis diversicolor

The small abalone (Haliotis diversicolor) is a mollusc and was cultured in south of china. Problems such as the decreasing pathogen‐resistance and their mass mortality during the summer. The increased immunity of small abalone populations is a key factor in resolving these problems. Thus, the study of immunity‐related genes in small abalone has become important. In this study, three bacterial species were initially isolated from small abalone carcasses. The regression of infection was analysed, which revealed that the bacteria species could cause rapid morbidity in small abalone. A QM‐like gene (HdiQM) was found and bacterial challenge tests showed that HdiQM gene expression was induced by the bacterial isolates from small abalone carcasses. Therefore, our results implied that HdiQM was found to be an inflammatory stress‐inducible gene associated with pathogen infection, with important functions in small abalone immunity.     

Molecular analysis of the intestinal bacterial flora in cage‐cultured adult small abalone,Haliotis diversicolor

To characterize the composition of the intestinal bacterial flora in cage‐cultured adult small abalone, Haliotis diversicolor, we used both culture‐dependent and culture‐independent approaches. In addition, in order to investigate the presence of probable pathogens, the intestinal bacterial community structures of healthy and diseased adult abalones were compared using the denaturing gradient gel electrophoresis (DGGE) fingerprinting technique. Bacteria isolated from the healthy abalone intestine belonged to the genera Vibrio, Flammeovirga, Shewanella, Persicobacter and Paraferrimonas. From the 16S rRNA gene clone library analysis, the intestinal bacterial community comprised two groups: the Proteobacteria and Tenericutes. Betaproteobacteria (18.48%), Deltaproteobacteria (30.43%) and Mollicutes (35.87%) were the most dominant components in the clones and the Mollicutes were mainly Mycoplasma‐affiliated clones. The DGGE band pattern demonstrated that more bands appeared in the healthy abalones (H group) than in the diseased abalones (D group). Although few potential pathogens were detected from the D group, a clustering dendrogram indicated that individuals of the H and D groups (except for D6) formed two separate clusters. An unidentified Deltaproteobacteria species and Mycoplasma sp. were the dominant components in the intestine of both the H and the D group.     

九孔鲍养殖水体及消化道细菌学的研究

2002年对汕尾健生鲍鱼养殖场养殖水体和鲍消化道中异养细菌及弧菌的数量和类群组成进行了研究。研究结果表明:养殖水体中异养细菌数量的四季变化从2 4×104～1 3×105cfu ml,平均为7 6×104cfu ml;消化道中异养细菌数量四季变化则从1 6×107～5 4×107cfu g(湿重),平均为3 3×107cfu g。水体中弧菌数量四季变化从1 2×104～5 1×104cfu ml,平均为2 5×104cfu ml;而消化道中弧菌数量四季变化则从2 8×105～3 8×105cfu g,平均为3 2×105cfu g。水体中细菌类群由Sphingomonas,Vibrio,Pasteurella,Moraxella,Pseudomonas,Aeromonas,Flavobacterium,Klebsiella,Weeksella和Alcall组成,消化道中细菌类群由Sphingomonas,Vibrio,Aeromonas,Flavobacterium,Shewanella,Sphingobacterium,Pseudomonas组成,水体和消化道弧菌种类主要为Vibriofluvialis,Vibrioparahaemolyticus,Vibriovulnificus,Vibriocholerae,Vibriominicus,Vibrioalginolyticus。并对不同养殖场水源弧菌数量也进行了比较。     

Vibrio parahaemolyticus Associated with Mass Mortality of Postlarval Abalone,Haliotis diversicolor supertexta (L.), in Sanya,China

Outbreaks of mass mortality in postlarval abalone, Haliotis diversicolor supertexta (L.), have swept across south China since 2002 and in turn have resulted in many abalone farms closing. Twenty‐five representative bacterial isolates were isolated from a sample of five diseased postlarval abalone, taken 15 d postfertilization during an outbreak of postlarval disease in Sanya, Hainan Province, China in October 2004. A dominant isolate, referred to as Strain 6, was found to be highly virulent to postlarvae in an experimental challenge test, with a 50% lethal dose (LD₅₀) value of 3.2 × 10⁴ colony forming units (CFU)/mL, while six of the other isolates were weakly virulent with LD₅₀ values ranging from 1 × 10⁶ to 1 × 10⁷ CFU/mL, and the remaining 18 isolates were classified as avirulent with LD₅₀ values greater than 1 × 10⁸ CFU/mL. Using both an API 20E kit and 16S recombinant DNA sequence analysis, Strain 6 was shown to be Vibrio parahaemolyticus. It was sensitive to 4 and intermediately sensitive to 5 of the 16 antibiotics used when screening the antibiotic sensitivities of the bacterium. Extracellular products (ECPs) prepared from the bacterium were lethal to postlarvae when used in a toxicity test at a concentration of 3.77 mg protein/mL, and complete liquefaction of postlarvae tissues occurred within 24 h postexposure. Results from this study implicate V. parahaemolyticus as the pathogen involved in the disease outbreaks in postlarval abalone in Sanya and show that the ECPs may be involved in the pathogenesis of the disease.     

Evaluation of cytochalasin B (CB) treatments for triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814)

The effectiveness of cytochalasin B (CB) treatments for inducing triploidy was evaluated in the blacklip abalone Haliotis rubra (Leach, 1814) in two orthogonal design experiments. The first experiment employed three dosages (DSs) of 0.25, 0.5 and 1.0 mg CB L^?1, three starting times (STs) of 5, 15 and25 min post fertilization and three treatment durations (TDs) of 10, 20 and 40 min, for a total of 27 treatments. The second experiment comprised of two DSs of 0.25 and 0.5 mg CB L^?1, five STs of 5, 15, 20, 25and 30 min post fertilization, and three TDs of 10, 20 and 40 min, for a total of 30 treatments. Water temperature was held at 17.5–18.5°C. Day 3 larvae were sampled for triploidy using flow cytometry (FCM) and survival. Optimal inductions were treatments starting at 15 or 20 min post fertilization and continuing for 40 min, and those initiated 25 or 30 min post fertilization for 20 or 40 min, using 0.5 mg CB L^?1. These treatments were all targeted at inhibition of the second polar body (PB2) formation and yielded triploidy rates of 84.8–89.5% coupled with (relative) survival rates of 20.1–52.1% in the first experiment, and corresponding rates of 86.5–96.5% and 33.0–74.1%, respectively, in the second experiment. A common and essential feature of these optimal conditions is that treatment must fully span the period of time for most of the eggs to extrude PB2. Treatments that resulted in suppression of the first polar body (PB1) formation induced triploidy levels below 71.5% and 57.6% in experiments 1 and 2 respectively. Treatments that had overlapping effects on both PB1 and PB2 extrusion led to triploidy rates above 80% but very low survival rates of 1.8% and 5.4% in experiments 1 and 2 respectively.     

Identification of a microsatellite marker that is completely linked to shell colour in small abalone,Haliotis diversicolor

A novel orange shell colour has been discovered in small abalone, Haliotis diversicolor (Reeve, 1846). To facilitate and accelerate a selective breeding programme for orange shell colour, a microsatellite marker that was completely linked to the shell colour gene was identified using a selective DNA pool approach and was verified in 448 individuals. Two DNA pools were constructed by pooling equal amounts of genomic DNA from 74 individuals with dark‐brown shells and 87 individuals with orange shells. Approximately 12 of 308 microsatellite markers that were linked to the shell colour gene were isolated using the selective DNA pooling method. After verification in 32 individuals with dark shells and 32 individuals with orange shells, only the Hdi3‐119 locus remained tightly linked to the shell colour gene. Finally, four families were used to verify the accuracy of the Hdi3‐119 locus. All of the individuals with orange shells in the four families harboured a unique 381‐bp allele in marker Hdi3‐119. This marker could be used for selective breeding of orange small abalone.     

Studies on the Development of Small Abalone (Haliotis diversicolor supertexta) Essence and its Antioxidative Properties

Essences of small abalone (Haliotis diversicolor supertexta) were prepared by two methods, steaming and boiling. The chemical constituents and antioxidative properties of these essences were determined and compared to those of commercial freshwater clam essence (CFCE). Boiled small abalone essence (BSAE) contained more total soluble solids than steamed small abalone essence (SSAE). Total soluble solids and peptide content of both essences increased with longer extraction time. Glycogen contents of SSAE, BSAE, and CFCE were 14.01, 13.95, and 10.74 mg/mL, respectively. The concentrations of free amino acids in SSAE and BSAE (725.05 and 818.56 mg/100 g, respectively) were significantly higher than in CFCE (33.57 mg/100 g). The antioxidative properties of SSAE and BSAE were higher than that of CFCE. The reducing power and ferrous iron (Fe²⁺) chelating activity of 3-h SSAE were significantly higher than those of 10-min BSAE. The predominant free amino acid of both SSAE and BSAE was taurine (227.14–273.64 mg/100 g), followed by proline and arginine. Both essences also showed high correlations between peptide content and Fe²⁺ chelating activity and reducing power. Sensory evaluation trials suggested that 3-h SSAE is more favorable than BSAE in terms of appearance and overall acceptability. In conclusion, SSAE had better antioxidative properties and sensory scores than BSAE.     

Growth of the abalone Haliotis diversicolor (Reeve) fed with macroalgae in floating net cage and plastic tank

The abalone Haliotis diversicolor (Reeve) or ‘tokobushi’ is being cultured in Kagoshima, southern Japan for stock enhancement of fishing grounds. However, with decreasing catches from the wild some fishermen's group may eventually resort to rearing tokobushi until of marketable size. Growth experiments of tokobushi fed macroalgae in floating net cage and plastic tanks were conducted to evaluate the influence of culture system, macroalgal food, age and water temperature on their culture. In floating net cage, marketable sizes were obtained at the age of 457–822‐day old (15–27‐month old) with highest growth rate of 2.36% g day^?1 and 0.21 mm day^?1. One advantage of the plastic tank culture system was that the tokobushi shell remained clean throughout the culture period. Macroalgal food affected the growth rate; Sargassum fusiforme (Setchell)‐ and Ulva pertusa (Kjellman)‐fed tokobushi were better than Meristotheca papulosa (Montagne)‐fed. Further, 1‐year‐old tokobushi had better growth performance and condition index than the 2‐year‐old cohort, and growth rates were higher at 17°C than at 12°C. These findings should help tokobushi farmers design their mariculture scheme considering both economic and environmental aspects.     

杂色鲍幼苗“急性死亡脱落症”病原菌分析

从“急性死亡脱落症”发病期的杂色鲍幼苗体内和病苗附着的薄膜上，共分离到4株优势菌，即NA0301、NA0302、NA0303和NA0304。经人工浸泡感染实验证明，从病幼苗体内和苗附着的薄膜上分离到的优势菌株NA0301和NA0302是杂色鲍幼苗的致病菌，它们均为革兰氏阴性、短杆状、极生鞭毛，能运动。常规生理生化和Biolog细菌鉴定系统测试结果表明，菌株NA0301与溶珊瑚弧菌（Vibrio coralliilyticus）的亲缘关系最近，而菌株NA0302与溶藻弧菌（Vibrio alginolyticus）的亲缘关系最近。为了进一步确定它们的分类学地位，分别测定了溶珊瑚弧菌和溶藻弧菌的16S rDNA序列，分析相关细菌相应序列的同源性，并构建系统进化树。结果表明，菌株NA0301与溶珊瑚弧菌的亲缘关系最近，菌株NA0302与溶藻弧菌的亲缘关系最近。因此，菌株NA0301和菌株NA0302分别鉴定为：溶珊瑚弧菌和溶藻弧菌。[中国水产科学，2006，13（4）：655—661]     

Effects of salinity on the growth and mucous cells of the abalone Haliotis diversicolor Reeve, 1846

International Aquatic Research - This study was conducted to determine the influence of salinity on the growth of abalone Haliotis diversicolor Reeve, including the density and size of mucous...     

Optimum temperature for growth of Australian abalone: preferred temperature and critical thermal maximum for blacklip abalone, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Leach)

The preferred temperature and critical thermal maximum of Australian blacklip abalone, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Leach), were found to differ only slightly; the blacklip abalone exhibited lower temperature tolerance and preference, as expected from its habitat distribution. Preferred temperatures were 16.9 and 18.9°C, and 50% critical thermal maxima were 26.9 and 2 7.5°C for blacklip and greenlip abalone, respectively. The optimum temperatures for growth calculated from each of these indices and averaged were 17.0 and 18.3°C, respectively.     

Changes in growth characteristics of the small abalone Haliotis diversicolor (Reeve, 1846) after one decade in a closed culture system: a comparison with wild populations

Small abalone Haliotis diversicolor (Reeve, 1846) is one of the smallest commercial abalone in the world. The successful application of artificial propagation and mass seed production techniques since the 1980s have resulted in the establishment of well-developed culture systems for small abalone in Taiwan. In the study reported here, we estimated the growth of a population of small abalone after a decade in a closed culture system and its growth characteristics with those of wild populations reported in previous studies. The von Bertalanffy growth equations of the shell length (L) and body weight (W) of cultured abalone were L _t  = 71.73 (1 ? e^{?0.84 (t?0.16)}) and W _t  = 47.70 (1 ? e^{?0.84 (t?0.16)})^3.180, respectively. The instantaneous rate of change for weight had an inflection point at the age of 1.54 years, indicating that cultured abalones reach their apex of body growth around this age. Compared with the wild populations, the cultured population exhibits a significantly smaller maximal shell length (L _∞) and a significantly larger growth coefficient (k). Based on our results, it appears that the artificial culture of generations of small albalone for one decade or more in a closed system could be one of the major factors causing the observed minimization of size in the cultured abalone; this may be an adaptation in which growth is traded off for the larger k.     

杂色鲍野生群体与养殖群体自交和杂交子一代的RAPD分析

杂色鲍野生群体和养殖群体分别自交和杂交产生野生鲍自繁F1代(WW)、养殖鲍自繁子代(CC)、正交子一代(W♀×C♂,简称WC)和反交子一代(C♀×W♂,简称CW)4个群体,采用RAPD分析4个群体遗传特性。就多态位点比例、Nei基因多样性值和Shannon信息指数而言,WW群体比CC群体高;杂交群体WC的值分别为88.76%、0.3246和0.4811,比其余群体都要高,与群体WW最接近;另一杂交子代群体CW除多态位点比例比群体CC高外,其余参数均为最低。群体间的遗传距离分析表明,WW与各群体的遗传距离都较大,其中与CC群体的遗传距离最大,为0.0849,与两个杂交群体间的遗传距离分别为0.0772(CW)和0.0671(WC);CC群体与两个杂交群体间的遗传距离都较小,分别为0.0610(与CW)和0.0597(与WC);杂交子代间的遗传距离为0.0600;杂交群体WC与WW的遗传距离大于与CC的遗传距离,CW与WW的遗传距离也大于与CC间的遗传距离,两个杂交群体与亲本的遗传距离不对等,偏向养殖亲本。