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.     

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。     

Occurrence of domoic acid in tropical bivalves

Domoic acid was found in bivalves belonging to the genus Spondylus collected randomly from the Philippines, Vietnam, Thailand and Ishigaki Island, Japan. This is the first report of domoic acid accumulating in bivalves from tropical waters. Large differences in domoic acid levels were observed among the Spondylus specimens collected from different areas. The Spondylus specimens from the Philippines and Vietnam showed higher levels of domoic acid whereas the level in Spondylus spp. from Thailand and Japan was low. The highest level of domoic acid—more than four times the safe consumption level of 20 μg/g whole tissue—was found in a specimen from the Philippines. On the other hand, bivalves other than Spondylus collected from the same area at the same time in the Philippines showed very low levels of domoic acid, showing that bivalves belonging to the genus Spondylus accumulate specifically high levels of domoic acid. These results suggest the wide distribution of causative phytoplankton in tropical waters.     

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。     

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.     

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。     

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。     

Morphological studies of peripheral blood cells of the Chinese sturgeon, <Emphasis Type="Italic">Acipenser sinensis</Emphasis>

The peripheral blood cells of one-year-old Chinese sturgeon (Acipenser sinensis) have been studied by light microscopy and transmission electron microscopy. The erythrocyte count was 84.86 × 10⁴ cell mm⁻³ in the peripheral blood of the fish and that of leukocytes was 2.24 × 10⁴ cell mm⁻³. The erythrocytes and four main types of leucocyte—thrombocytes, lymphocytes, granulocytes (including neutrophils and eosinophils), and monocytes, were identified in the peripheral blood. In addition to normal erythrocytes, reticulocytes and division of erythrocytes were observed. Thrombocytes were the most numerous among the leukocytes, and the number of neutrophils with lobated nuclei was larger than for other fish. The structures of the erythrocytes, lymphocytes, monocytes, granulocytes, and thrombocytes of the fish were studied. The erythrocytes were almost completely devoid of organelles, except for some mitochondria and granules. A large number of vacuoles and a few organelles were observed in cytoplasm of the monocytes. There were many microvilli on the membrane and pseudopodia-like cytoplasm bulge in the lymphocytes. The neutrophils were round or oval in shape with bilobed, trilobed, or multilobed nuclei whereas the eosinophils had big special granules, dark stained. There were many vesicles in some thrombocytes, which were related to its phagocytosis; some thrombocytes had almost no cytoplasm or organelles.     

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.     

Movement of the fluvial form of masu salmon, <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis>, in a mountain stream in Kyushu,Japan

Using mark-recapture methods, the movements of the fluvial form of masu salmon (Oncorhynchus masou masou) in a mountain stream on the island of Kyushu, Japan, were studied. Most (78%) of the masu salmon were recaptured in the pool in which they had been originally caught and tagged. Of those that moved between pools, the proportion of individuals that moved during the breeding period was not significantly higher than the proportion that moved during the non-breeding period. However, during the breeding period, a higher proportion of larger salmon moved than did smaller fish. The proportion of mobile large males during breeding period was higher than that for small males. Also, it was found that a few individuals showed long-range movement in the autumn. As a long-term movement, 78 individual fish (65%) that were recaptured more than three times showed high sedentary tendencies. Sixteen individual mobile fish (13%) moved and returned to the original pool. Fluvial form of masu salmon in Kyushu show a high sedentary nature; however, large mature males seem to actively move in search of female during breeding period.     

The effect of synthetic and natural pigments on the colour of the cichlids (<Emphasis Type="Italic">Cichlasoma severum</Emphasis> sp., Heckel 1840)

In this study, we have investigated the effects of Porphyridium cruentum (Rodophyta) as a natural pigment source and astaxanthin and β-carotene as synthetic pigment sources on the skin colour of cichlid fish (Cichlasoma severum sp., Heckel 1840), which are generally light orange with white patches and becomes shiny orange in the reproductive phase. The fish were fed diets containing 50 mg kg⁻¹ astaxanthin and β-carotene, and P. cruentum powder. The amount of both natural and synthetic pigment sources given as feed was 50 mg kg⁻¹, and the experiment was continued for 50 days. Total carotenoid content of the fish was determined spectrophotometrically at the end of the experiment. As a result, while a visible change of colour in the skin of the fish fed on the feed containing astaxanthin was observed with 0.34 ± 0.2 mg g⁻¹ of pigment accumulation, a relatively small change of colour was observed in the skin of other fish that were fed on the feed containing P. cruentum and β-carotene with 0.22 ± 0.2 mg g⁻¹ and 0.26 ± 0.1 mg g⁻¹ of pigment accumulations, respectively. Therefore, it was determined that these pigment sources have an effect on the colour of cichlid fish.     

Growth of wild pearl oysters <Emphasis Type="Italic">Pinctada fucata,Pinctada margaritifera</Emphasis> and <Emphasis Type="Italic">Pinctada sugillata</Emphasis> (Bivalvia: Pteriidae) in Taiwan

In order to understand growth features of pearl oysters in the genus Pinctada, i.e. Pinctada fucata, Pinctada margaritifera, and Pinctada sugillata in Taiwan, a total of 3062 wild individuals of these species from juvenile to adult were collected monthyly from March 2001 to April 2002 in Jukeng, Pingtung County, south-west Taiwan. Quantitative measurements of live oysters were conducted for shell height (SH), shell length (SL), shell width (SW), hinge length (HL), and wet weight (WW). Different cohorts were identified through multiple length frequency analysis on SH of P. fucata and P. margaritifera, and growth curves with seasonal variation were estimated for these species. Pinctada fucata in Taiwan had a different seasonal growth pattern from the Japanese population, but had similar growth rates during the high growth period. The growth rate of P. margaritifera in Taiwan was slower than in French Polynesia, the Solomon Islands, and the Red Sea. Comparisons of morphological growth features among the three species show large differences in the SW-related features. Pinctada fucata in Taiwan had larger SW than in Japan and Korea. The differences in growth rates and morphological features suggested that the wild Taiwanese oysters may retain genetically pristine characteristics, thus genetic conservation might be urgently needed.     

南海北部海域大眼鲷资源的时空异质性

研究经济鱼类的资源密度重心及其时空异质性有助于深入了解其渔场形成过程,并为其渔业资源的可持续利用和科学管理提供参考依据。本研究基于2009—2014年南海捕捞信息网络中的南海北部海域底拖网渔业生产数据,运用重心分布、自举法(Bootstrap)和地统计等方法研究了大眼鲷资源密度重心分布和时空异质性。结果发现,2009—2014年大眼鲷资源密度总体上呈逐年降低的趋势,且差异极显著,最高值为2012年的(26.69±7.34) g/(h·kW·nets)。不同年份大眼鲷资源密度重心均分布在南海北部50～100 m等深线间,资源密度重心年际分布差异不显著,主要集中于调查海域中部。大眼鲷资源密度在70～90 km的尺度范围内呈强烈的空间自相关格局及良好的空间结构性(块金系数25%),主要受环境因素的作用;在90 km以上的尺度范围,空间自相关性较弱,受随机成分影响较大。     

海月水母螅状体无性繁殖方式观察及一种新发现报道

无性繁殖是水母螅状体有效补充种群数量及扩展栖息地的重要方式,为正确掌握海月水母的无性繁殖方式进而深入了解其生活史,在显微镜下常年观察并详细描述了海月水母螅状体进行匍匐茎生殖、芽体生殖、足囊生殖、繁殖体生殖、纵向分裂生殖和破裂生殖等复杂无性繁殖方式的过程,发现繁殖体生殖可产生浮游态繁殖体芽,在水中漂浮并生长发育成螅状体,新发现一种破裂生殖方式,仅发生在螅状体极度饱食的状态下。对6种无性繁殖方式的行为特征和功能作用进行了对比分析,认为匍匐茎生殖和破裂生殖有利于螅状体种群快速增加个体数量,芽体生殖和纵向分裂生殖有利于子代幼体生长,足囊生殖有利于种群抵御不良的环境条件,繁殖体生殖有利于种群随水流进行长距离迁移,以扩展或转移栖息地。研究表明,不同的繁殖方式是海月水母应对不同环境条件的适应性繁殖对策。     

Substrate specificity of brain acetylcholinesterase and its sensitivity to carbamate insecticides in <Emphasis Type="Italic">Carassius auratus</Emphasis>

The substrate specificity of brain acetylcholinesterase (AChE) in adult Carassius auratus fish and its sensitivity to carbamate insecticides were investigated in vitro. The results showed that the order of four substrates hydrolyzed by brain AChE in C. auratus was acetylthiocholine iodide > β-methylthiocholine iodide > propionylthiocholine iodide > butyrylthiocholine iodide, and the maximum velocity (V _max) of AChE hydrolyzing acetylthiocholine iodide (ATCh) was the highest among the four substrates, and the V _max values were 0.067 and 0.082 mmol min⁻¹ mg⁻¹ for male and female fish respectively. But their Michaelis–Menten constants (K _m) were the lowest, only 0.071 and 0.072 mmol/l respectively. Compared with other carbamate insecticides, the sensitivity of brain AChE to carbofuran was the highest and the IC₅₀ values were 1.04 × 10⁻⁶ mol/l for females and 1.17 × 10⁻⁶ mol/l for males. The inhibitory tendencies of eserine, methomyl, and aldicarb to brain AChE were very similar, and the percentage inhibition increased with time at the concentration of 1 × 10⁻⁶ mol/l. The order of inhibition potential of the three inhibitors from the highest to the lowest was eserine, aldicarb, and methomyl.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Effect of ration size on growth,body composition,and energy and protein maintenance requirement of fingerling Indian major carp, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton)

An eight-week feeding trial has been conducted to determine the optimum ration for Indian major carp, Labeo rohita, fingerling (4.10 ± 0.30 cm, 0.55 ± 0.16 g) by feeding a purified diet (40% CP; 3.61 kcal g⁻¹ GE) at six levels, 2, 4, 6, 8, 10, and 12% of body weight per day, at 0800 and 1600 h, in triplicate, to 20 fish per trough fitted with a water flow-through system. Highest weight gain, best feed conversion ratio (FCR), best specific growth rate (SGR%), and highest protein efficiency ratio (PER) were evident for rations of 6–8% body weight. Second-degree polynomial regression analysis for FCR, PER, protein, and energy retention data indicated the break-points occurred at 6.55, 6.75, 6.80, and 6.95% bw per day, respectively. Significant (P < 0.05) differences between body composition were observed for fish fed different rations. Maximum body protein content was recorded for 6% and 8% rations. A linear increase in body fat content was evident with increasing ration. Body moisture and ash content remained non-significantly (P > 0.05) low for higher rations, however. On the basis of these results it is recommended that feeding in the range 6.5–7.0% bw per day corresponding to 2.6–2.8 g protein and 23.49–25.31 kcal energy per 100 g of the diet per day is optimum for growth and efficient feed utilization of Labeo rohita. Results for 2–4% rations (0.8–1.6 g protein and 7.23–14.46 kcal energy) suggest these amounts approximate to the maintenance requirement of this fish.     

Masculinization of Convict Cichlid (<Emphasis Type="Italic">Cichlasoma nigrofasciatum</Emphasis>) by immersion in <Emphasis Type="Italic">Tribulus terrestris</Emphasis> extract

We have examined the effects of Tribulus terrestris (TT), a non-toxic herb, on sex reversal in the Convict Cichlid Cichlasoma nigrofasciatum with the aim of introducing a new environmentally friendly method for masculinization in C. nigrofasciatum. TT is a natural plant product that elevates the testosterone levels in humans and animals. Different concentrations (0.0, 0.10, 0.20 and 0.30 g/l) of TT extract were tested for their effect on sex reversal in C. nigrofasciatum by immersing newly hatched offspring once weekly for 2 months in TT extract. Of the dosages used in the present study, 0.30 g/l TT was the most effective in terms of masculinization, resulting in a maximum male ratio of 87.23% (P < 0.001). Sex ratios of 79 and 85% at 0.10 and 0.20 g/l TT, respectively, were also significantly different from the expected 1:1 ratio (P < 0.001). Histological examinations revealed that the testes of fish treated with TT extract contained all stages of spermatogenesis, clearly demonstrating that the administration of TT extract to C. nigrofasciatum stimulated spermatogenesis. Total survival rates in all treatments and the control were uniformly high, ranging from 88.57 to 90% (P > 0.05). We concluded that TT had no negative effect on the survival rate of C. nigrofasciatum. In addition, all groups of TT-treated fish exhibited successful growth acceleration compared to the control group, but only the 0.30 g/l TT treatment significantly improved the growth rate of C. nigrofasciatum. (P < 0.01). Sex reversal in C. nigrofasciatum demonstrated that TT-treated 0-day-old larvae showed successful sex reversal, spermatogenesis and a better growth rate than untreated progenies.     

Plasma biochemical responses of the omnivorous crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 μg MC-LR equiv kg⁻¹ BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.