Seasonal variation of domoic acid in a bivalve Spondylus versicolor in association with that in plankton samples in Nha Phu Bay,Khanh Hoa,Vietnam

Previously we reported that a significant level of domoic acid, a causative toxin for amnesic shellfish poisoning, was detected in bivalves belonging to a genus Spondylus from various tropical Asian countries. These findings indicate that causative plankton species for domoic acid widely distribute in these areas. In the present study, we monitored seasonal change of domoic acid level of Spondylus versicolor in association with that of plankton samples in Nha Phu Bay, Khanh Hoa, Vietnam from December 2004 to October 2005. The toxicity of S. versicolor showed distinct seasonal variation. During the period when domoic acid level of S. versicolor was increasing, a significant level of domoic acid was detected in the plankton samples, showing the correlation between these two parameters. These findings show that plankton causative for domoic acid occurred in the bay, and S. versicolor accumulate domoic acid during a bloom of the toxic plankton by food web transfer.     

软骨藻酸对海湾扇贝(Argopecten irradians)血淋巴免疫力和抗氧化力的影响研究

软骨藻酸是一种神经性贝类毒素,大多数研究只报道了对陆生动物神经系统的毒性和在贝类组织内累计的软骨藻酸的浓度,而对软骨藻酸对贝类自身的免疫及抗氧化系统的毒性作用相关的研究不足。扇贝作为无脊椎动物,缺乏适应性免疫,主要依靠先天免疫系统进行防御。因此,本研究以海湾扇贝（Argopecten irradian）为研究对象,在0、10 ng/mL、50 ng/mL和100 ng/mL软骨藻酸浸泡6 h、12 h和24 h后通过测定扇贝血淋巴中超氧化物歧化酶（SOD）、溶菌酶（LZM）活性和谷胱甘肽（GSH）含量以及免疫、抗氧化相关基因（Cu/ZnSOD、MnSOD、GST和ACP）相对表达量来研究其对扇贝免疫力及抗氧化系统的影响。结果发现,LZM活性在10 ng/mL和50 ng/mL软骨藻酸处理后显著提高;ACP基因表达量在6 h和12 h内表达量受到上调,而在24 h表达量显著下调;10 ng/mL、50 ng/mL和100 ng/mL软骨藻酸浸泡扇贝6~24 h后SOD活性受到抑制,并且Cu/ZnSOD和MnSOD基因表达受到调控;GSH含量显著提高,并且GST基因表达量显著上调。以上结果表明软骨藻酸处理虽然会抑制抗氧化酶SOD活性,并且高浓度长时间处理会造成免疫疲劳现象,但机体可通过提高血淋巴GSH水平和GST基因的表达量来抵抗软骨藻酸的毒性。因此,本研究初步揭示了软骨藻酸对扇贝等双壳类免疫力、抗氧化力和解毒力的影响。     

Grow-out culture of tropical abalone, Haliotis asinina (Linnaeus) in suspended mesh cages with different shelter surface areas

Thisstudy investigated the effects of shelter surface area (SSA) on the feeding,growth and survival of the donkey-ear abalone, Haliotisasinina reared in mesh cages (0.38×0.38×0.28m) suspended in flow-through tanks (water volume = 6m³). Cages had sections of polyvinylchloride (PVC) thatprovided shelters with surface area of 0.22 m², 0.44m² and 0.66 m².Hatchery-produced abalone with initial shell length of 32 ± 1mm and wet weight of 7.5 g were stocked at 50individuals cage^–1 that corresponded to stocking densities ofca. 227, 113 and 75 abalone m^–2 of SSA. The ratios of sheltersurface area to cage volume (SSA:CV) were 5.5, 11 and 16.5. Abalones wereprovided an excess red seaweed Gracilariopsis bailinae(= Gracilaria heteroclada) at weekly intervals overa 270-day culture period. Feeding rates (18–20% of wet weight), foodconversion ratio (26–27) and percent survival (88–92%) did notdiffer significantly among treatments (p > 0.05). Body size at harvest rangedfrom 56 to 59 mm SL and 52 to 57 g wet body weightwith significant differences between abalone reared at SSA 0.22m² and 0.66 m² (p < 0.05).Abalone reared in cages with 0.66 m² SSA grewsignificantly faster at average daily growth rates of 132 m and188 mg day^–1. Stocking densities of 75–113m^–2 SSA in mesh cages suspended in flow-throughtanks resulted in better growth of abalone fed red seaweed.     

Direct contact between Pseudo-nitzschia?multiseries and bacteria is necessary for the diatom to produce a high level of domoic acid

Pseudo-nitzschia multiseries loses most of its ability to produce domoic acid when it is cultured axenically. However, domoic acid production recovers when bacteria from the original culture are reintroduced to axenic cultures, indicating a bacterial association with domoic acid production in this species. In order to determine the role of bacteria in domoic acid production, an axenic strain prepared from a nonaxenic strain of P. multiseries was inoculated into media in cellophane tubes, which were then placed in a bottle containing the original nonaxenic culture. Both strains showed characteristic domoic acid production similar to that previously observed. Domoic acid level in the cells of the axenic strain in the tubes was much smaller than that of the cells outside the tubes. These results show that direct contact between living bacteria and P. multiseries is necessary for producing high levels of domoic acid in P. multiseries.     

藻-菌单一及共生系统对海水养殖尾水的净化作用

为探索高效可行的海水养殖尾水处理技术,在净化尾水污染物的同时达到微藻生物量积累,本文对比了普通小球藻(Chlorellavulgaris)和芽孢杆菌(Bacillusspp.)在游离和固定形态下,单一和共生系统的细胞生长及其对尾水中氨氮(NH_4~+-N),正磷酸盐(PO_4~(3–)-P),总磷(TP),化学需氧量(COD_(Mn))的去除效果。实验结果表明:游离状态下,藻–菌共生系统促进小球藻细胞生长及尾水中PO_4~(3–)-P和COD_(Mn)的去除。固定态藻–菌共生系统对NH4~+-N, PO43–-P和TP的处理效果优于单一固定态小球藻和单一固定态芽孢杆菌。对比实验设计的所有处理(游离或固定态下的单一藻、单一菌、藻–菌共生),藻–菌共固定系统的处理效果相对最优,对NH_4~+-N, PO_4~(3–)-P, TP和COD_(Mn)的去除率分别达到96.57%, 98.62%, 89.89%和39.09%,出水NH_4~+-N, PO_4~(3–)-P分别达到中国《渔业水质标准》和《海水养殖水排放要求》二级标准, COD_(Mn)达到《污水综合排放标准》二级标准。     

中国东南沿海青蟹线粒体COI基因部分序列分析

对我国东南沿海5个地区的72个青蟹个体的线粒体细胞色素氧化酶亚基I (COI)部分序列序列进行了测定和分析。获得的72个细胞色素氧化酶亚基I (COI)序列可分为12个单倍型,与GenBank中已知的Scylla paramamosain COI序列的相似性达到98%以上,与其它3种 青蟹的差异为7.36%～15.54%。这些序列与S. paramamosain的遗传距离仅为0.00783,但是与S. serrata、S. olivacea和S. tranquebarica〗的遗传距离却分别达到0.11659、0.17812和0.08423。序列特征、遗传距离和系统进化等分析结果都表明本文研究的青蟹为S. paramamosain。结果提示,在进行青蟹属相关研究应当仔细鉴别采集样本的种类。     

怀头鲇体表溃烂症病原鉴定及致病性分析

为探讨黑龙江流域怀头鲇体表溃烂症的病因及防控措施,本研究采用常规方法从患病鱼的肝脏、脾脏和肾脏等部位分离病原菌,通过人工感染试验确定分离菌株的致病性,并对菌株的基本形态、理化特性、分子特征、毒力基因携带情况及耐药性等进行了系统研究。结果显示,从患病鱼体内分离得到3株病原菌,分别命名为NY-8、NY-9和NY-12;人工感染试验发现,NY-8和NY-9株对试验鱼有较强的致病力,NY-12株毒力较弱;3株细菌混合感染后,鱼体发病症状与临床自然发病症状一致,试验鱼死亡率高达到100%。综合理化特征和16S r RNA基因序列分析结果,确定NY-8、NY-9和NY-12株分别为气单胞菌属的维氏气单胞菌、杀鲑气单胞菌和嗜水气单胞菌。5种毒力基因在3株气单胞菌中的分布表现为两种基因型,h l y+/a e r+/a c t+/a l t+/G C A T+和h l y+/a e r-/act+/alt+/GCAT+,同时携带5种毒力基因的NY-8和NY-9分离株致病性显著高于NY-12株。3株细菌在耐药谱上有一定差异性,NY-8和NY-9株均对4种氟喹诺酮类药物敏感,对氨基糖苷类、呋喃类等药物耐药;NY-12株仅对左氧氟沙星和氟苯尼考等2种药物敏感。     

Resolving species identification problems in the genus Sebastes using nuclear genetic markers

The identification of North Atlantic redfish has been controversial and remains a difficult task due to overlapping of meristic and morphological characters. Here we used nine microsatellite loci to assess the level of genetic differentiation among these species and assess the resolution power of these microsatellite loci for individual assignment-based analyses. Conventional analyses as well as individual Bayesian assignment methods clearly separated the four species of North Atlantic redfish as well as the giant form of Sebastes marinus and the so-called “oceanic” and “deep-sea” types of Sebastes mentella. Locus-by-locus analyses revealed that only five microsatellite loci out of nine used could discriminate the concerned species. The advantage of the Bayesian methods relies in the individual information retrieved. It therefore gave additional information on the interrelationship among species. Indeed, we provide evidence of potential hybridization among species as well as individual misclassification based on morphological identification. We provide a powerful tool to discriminate North Atlantic redfish species, which might be useful for legal issues such as poaching, unintentional harvesting and control label.     

Experimental effects of salinity on gonad growth and maintenance in small tropical freshwater ostariophysans

A selection of cyprinids of the genera Rasbora and Puntius, various neotropical characins and a species of kuhli loach, Pangio semicincta, were exposed to different salinity regimes (in the conductivity range 0--2000 S cm–1), to determine whether there was any effect on gonadal development. There was no conclusive evidence for such an effect in any species, although there was a trend for ovarian development to be somewhat reduced at high conductivities in some species. These results appear to refute any suggestion that, in the wild, changes in environmental salinity may play any considerable role in the regulation of gonadal cycles of small non-electroreceptive tropical freshwater teleosts     

三种水族馆珍贵鱼类的营养成分分析及脂肪酸营养评价

睛斑鳗狼鱼、巨巴西骨舌鱼和锥齿鲨是水族馆中珍贵的展示鱼类,但是有关这3种鱼类成体的营养生理研究却十分缺乏。本研究对从水族馆获得的成年睛斑鳗狼鱼、巨巴西骨舌鱼和锥齿鲨的肌肉和肝脏组织进行了常规营养成分(水分、灰分、粗蛋白和粗脂肪含量)和脂肪酸组成测定分析,并进一步将3种鱼类所食饵料与其肝脏和肌肉中的脂肪酸相对含量进行了比较。结果显示,在3种鱼中,锥齿鲨肌肉和肝脏中的蛋白质含量最高,分别为26.84%和24.03%;睛斑鳗狼鱼的肌肉和肝脏中的脂肪含量最高,分别为41.96%和32.27%;巨巴西骨舌鱼的肌肉和肝脏中的灰分含量最高,分别为4.95%和5.51%。锥齿鲨肌肉和肝脏中饱和脂肪酸(SFA)含量高于睛斑鳗狼鱼,睛斑鳗狼鱼肌肉和肝脏中单不饱和脂肪酸(MUFA)含量高于巨巴西骨舌鱼和锥齿鲨,巨巴西骨舌鱼肌肉和肝脏中多不饱和脂肪酸(PUFA)含量及其肌肉中UFA/SFA值高于睛斑鳗狼鱼和锥齿鲨。研究表明,锥齿鲨所食饵料与其体内脂肪酸组成最为相似,其肌肉和肝脏与饵料的相关系数分别为0.972和0.861,其次是巨巴西骨舌鱼(0.912和0.846),而睛斑鳗狼鱼肌肉和肝脏中脂肪酸组成与所食饵料的相关系数仅分别为0.760和0.681。从脂肪酸营养的角度分析,在当前水族馆养殖条件下,锥齿鲨饵料较为合适,巨巴西骨舌鱼的饵料中应注意添加EPA和DHA,而睛斑鳗狼鱼饵料中则需提高MUFA比例。本研究首次阐明了3种珍贵的水族馆鱼类成体的营养组成,并为水族馆相应的饲料配制提供了重要的参考依据。     

Experimental approach on the selection and persistence of anti-microbial-resistant Aeromonads in faecal matter of rainbow trout during and after an oxolinic acid treatment

The presence of anti-microbial-resistant bacteria in the aquatic environment remains a matter of concern for environmental, animal and human health risks. Many published data have reported increased anti-microbial resistance among the aquatic microbiota from fish farm environments which often coincides with the use of anti-microbial agents. It can be assumed that the selection of anti-microbial-resistant bacteria first occurs in the fish farms themselves, at locations where anti-microbial concentrations are active. As anti-microbial agents are frequently administered through feed pellets, faecal matter excreted could favour conditions for such selection. In this study, OA (oxolinic acid) concentrations and OA resistance of Aeromonads were surveyed in output water and in faecal matter from OA medicated (test) and non-medicated (control) fish tanks during and after an OA oral treatment. Because of the low counts of Aeromonads in the output water, it was not possible to obtain reliable data on the proportion of anti-microbial resistance in this compartment. On the other hand, a time-limited effect on the abundance of Aeromonads isolated from faecal matter was only observed in test tanks. In these tanks, high proportion (80-100%) of OA-resistant Aeromonads was present in deposited faecal matter until at least one week after the end of the anti-microbial treatment. At that time OA was no longer detected within the faecal matter, after having reached concentrations between 100 and 190 μg/g during the treatment. We discuss how the leaching of OA from faecal pellets, from the intestine to their deposit, may favour the selection of OA-resistant Aeromonads. Further investigations in real conditions on microbiota associated with faecal matter in transit through the fish farm system are required. Particular attention should be paid to the wastes collected by the filter treatments of fish farm effluents.     

鲫肠道乳酸菌的分离及益生特性

为获得鱼源益生乳酸菌,本研究从健康鲫肠道内分离鉴定得到38株乳酸菌,并选择其中8株乳酸菌进行体外益生特性分析。结果显示,8株乳酸菌均能在p H=4.5和10%鲫胆汁环境中存活,对嗜水气单胞菌、豚鼠气单胞菌、无乳链球菌和副溶血弧菌均有较强的抑菌能力,但菌体表面疏水性和自凝集能力具有菌株特异性。选择5株疏水性较高(63%~89%)、自凝集能力强(37%~45%)的乳酸乳球菌进行体外黏附能力测定,结果显示,黏附能力在菌株间具有差异性(4.5%~7.9%),但均能显著降低嗜水气单胞菌NJ-35的体外黏附率,黏附抑制率达30%~35%;最后对筛选出的3株高黏附力的乳酸乳球菌进行安全性评价,发现3株菌对鲫均无致病力。鱼源乳酸菌的筛选,为鲫养殖生产中益生菌的实际应用提供了理论依据。     

基于16SrDNA分析的浒苔外生细菌多样性研究

为研究浙江近海浒苔Ulva spp.(Enteromorpha spp.)外生细菌多样性,采用传统的形态学和16S rDNA测序分析的方法,从舟山朱家尖、宁海国华电厂和奉化南沙3个地区分离到可培养的浒苔外生细菌及其周围海水细菌65株。根据细菌菌落特征和革兰氏染色结果等将分离到的细菌分为26种表型。16S rDNA序列测序比对发现:菌株与不动杆菌属(Acinetobacter)、交替单胞菌属(Alteromonas)、弧菌属(Vibrio)、假交替单胞菌属(Pseudoalteromonas)、微小杆菌属(Exiguobacterium)、芽孢杆菌属(Bacillus)、赤细菌属(Erythrobacter)、微球菌属(Micrococcus)、假单胞菌属(Pseudomonas)、深海杆菌属(Idiomarina)、Phaeobacter、Roseivirgaj和Silicibacter等23个属相应菌株具有较高同源性。对不同地区浒苔外生细菌进行了Shannon-Wiener多样性指数分析。研究表明:(1)宁海国华电厂的浒苔外生细菌多样性最丰富,多样性指数为93.98%;(2)浒苔外生细菌与其生活地区密切相关,其群落组成具有地域差异,其优势类群也不尽相同,但均归属于变形菌门。     

黄颡鱼早期发育阶段受精卵和鱼体脂肪酸组成变化

研究了黄颡鱼受精卵孵化期间和仔鱼发育阶段脂肪含量和脂肪酸的组成变化规律.采用常规化学分析方法和气相色谱法对黄颡鱼从鱼卵受精开始至仔鱼孵化后未投饵的7 d内的脂肪含量和脂肪酸组成进行测定.结果表明,受精卵在整个孵化期间脂肪含量有下降趋势.受精卵中不饱和脂肪酸含量大于饱和脂肪酸含量.受精卵在整个孵化期间各种脂肪酸含量无明显变化.仔鱼孵化后,鱼体总脂肪含量急剧下降,总脂含量从0日龄的4.57%降低到7日龄的0.75%.仔鱼在饥饿期间鱼体脂肪酸组成发生明显变化,单不饱和脂肪酸含量下降最为明显,尤其是C18:1.仔鱼在饥饿期间,脂肪酸按n-9>n-6>n-3顺序被先后利用,黄颡鱼仔鱼发育阶段主要以单不饱和脂肪酸作为能量代谢基质,而C20:4n6(AA)和C22:6n3(DHA)优先于C20:5n3(EPA)被保存下来.     

Genetic variation in mitochondrial genes and intergenic spacer region in harmful algae <Emphasis Type="Italic">Chattonella</Emphasis> species

In this study, nuclear ribosomal RNA gene internal transcribed spacer regions and the cox2-cox1 fragment of the mitochondrial (mt) genome were sequenced in 24 strains of Chattonella spp. Variability in both regions showed that the mt genome sequences of Chattonella spp. have a higher evolutionary rate than the nuclear rRNA gene sequences. A maximum likelihood tree based on the mt sequence grouped the Japanese Chattonella strains into two groups (Groups A and B), although no correlation was observed amongst the phylogenetic groups, their morphologies, or the isolated areas. Groups A and B were clearly identified by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using Fokl, without the need for a sequencing experiment. The PCR-RFLP assay revealed that Chattonella cells obtained from sea water in Oita, Japan, in 2004 and 2005 belonged to Group B. This is the first report showing the genetic variation in Chattonella spp. using a PCR-RFLP identification protocol.     

Antifreeze proteins in the urine of marine fish

Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6–33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each of these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.     

Reinvestigation of positional distribution of tetracosahexaenoic acid in triacyl-sn-glycerols of flathead flounder flesh

Positional distribution of fatty acids in triacyl-sn-glycerols (TAG) of the flathead flounder Hippoglossoides dubius has been reinvestigated in order to accurately determine the contents of tetracosahexaenoic acid (24:6n-3) in the sn-1, sn-2, and sn-3 positions. Flesh TAG obtained from three flounders were subjected to stereospecific analysis using a suitable procedure for fish TAG analysis. The 24:6n-3 acid was found in the three positions at the concentrations of 0.3–5.5 mole% (the sn-1 position), 1.6–23.3 mole% (the sn-2 position), and 0.6–8.9 mole% (the sn-3 position). In contrast to a previous analysis, the present analysis revealed that 24:6n-3 is preferentially esterified in the sn-2 position followed by the sn-3 and sn-1 positions. Other polyunsaturated fatty acids, docosahexaenoic acid (22:6n-3; DHA) and docosapentaenoic acid (22:5n-3; DPA), showed a similar distribution pattern. These results indicate that the general tendency observed for long-chain polyunsaturated fatty acids in marine fish TAG can be extended to the distribution of 24:6n-3 in flathead flounder TAG. Because the use of flathead flounders is entirely for human food, we thus intake 24:6n-3 concentrated in the sn-2 position of their TAG.     

饲料中花生四烯酸含量对刺参生长性能、抗氧化能力及脂肪酸代谢的影响

为探究饲料中添加花生四烯酸(arachidonic acid,ARA)对刺参(Apostichopus japonicus)生长性能、抗氧化能力及脂肪酸代谢的影响,选用初始体重为(10.78±0.06)g的刺参为研究对象,以鱼粉和发酵豆粕为主要蛋白质源,小麦粉为主要糖源制作基础饲料,通过在基础饲料中添加不同比例的ARA-纯化油,制成ARA含量分别为0.02%(对照组)、0.17%、0.36%、0.51%、0.59%和0.98%(占饲料干重)的6组等氮等脂的实验饲料,在室内循环水养殖系统进行为期56 d的养殖实验。结果表明,随着饲料中ARA含量的升高,刺参增重率(weight gain rate,WGR)呈先上升后降低的趋势,0.36%和0.51%ARA饲料组刺参WGR显著高于其他处理组(P0.05),刺参的特定生长率(specific growth rate,SGR)和饲料效率(feed efficiency,FE)与WGR具有相同的变化趋势;刺参体壁粗脂肪含量随饲料ARA含量升高呈先降低后升高的趋势,在0.51%ARA饲料组含量最低,且显著低于对照组与0.98%ARA饲料组(P0.05);同时,随饲料中ARA含量的提高,刺参体壁中ARA和n-6多不饱和脂肪酸(n-6 polyunsaturated fatty acids,n-6 PUFA)含量呈显著上升趋势,而二十碳五烯酸(eicosapentaenioc acid,EPA)、二十二碳六烯酸(docosahexaenoic acid,DHA)和n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids,n-3 PUFA)含量显著降低(P0.05);抗氧化能力方面,0.36%和0.51%ARA饲料组刺参肠道中超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)和总抗氧化能力酶(total antioxidant capacity enzyme,T-AOC)活性均显著高于对照组与0.98%ARA饲料组(P0.05),而肠道丙二醛(malondialdehyde,MDA)含量呈相反的变化趋势(P0.05);刺参肠道中脂肪酸合成酶(fatty acid synthase,FAS)和乙酰辅酶A羧化酶(acetyl-Co A carboxylase,ACC)活性随饲料ARA含量的升高呈显著降低趋势(P0.05);刺参肠道中肉毒碱棕榈酰转移酶-1(carnitine palmitoyltransferase-1,CPT-1)活性随饲料ARA含量升高呈先升高后降低的趋势(P0.05)。研究表明,在本实验条件下,饲料中添加适量ARA(0.36%~0.51%)能够对刺参生长、抗氧化能力起到一定的促进作用,同时结果显示,饲料ARA含量会对刺参肠道内脂肪酸代谢产生一定的影响。     

Effect of ascorbic acid supplement in vitro onrainbow trout sperm viability

We evaluated motility and fertilizing ability of rainbow trout Oncorhynchus mykiss semen obtained from fish fed diets without ascorbic acid and a diet supplemented with 870 mg kg^{minus 1} of ascorbyl monophosphate. Semen was stored in vitro on ice (0 °C) during 14 days. The spermatozoa from the supplemented group had the highest motility and lowest decline in fertilizing ability after storage. Lack of a positive effect of exogenous vitamin C on semen in fish deficient in ascorbic acid (milt was supplemented with 50 mg l^–1 of ascorbic acid) suggests that the positive effect of ascorbic acid on semen quality is related to its long-term effects during spermatogenesis.