Temperature effects on the development of Enteromyxum spp. (Myxozoa) in experimentally infected tiger puffer, Takifugu rubripes (Temminck & Schlegel)

The effects of water temperature on the development of the enteric myxosporeans, Enteromyxum fugu and Enteromyxum leei, were investigated in experimentally infected tiger puffer, Takifugu rubripes. After naïve tiger puffer were fed gut tissue infected with both E. fugu and E. leei, they were divided into separate tanks and kept at different constant temperature regimes between 10 and 25 °C. Regardless of the water temperature tested, E. fugu was consistently detected with a high prevalence of infection (60–100%), although no sporulation occurred at 10 and 15 °C. Development of E. leei and the onset of disease were suppressed by low water temperatures (<15 °C). However, a temperature increase to 20 °C promoted the development of E. leei, followed by an increase of disease rate in the fish. The present study demonstrates that water temperatures below 15 °C have an inhibitory effect on the development of E. fugu and E. leei, resulting in suppression of enteromyxosis at low temperatures.     

Histopathological studies on Vibrio harveyi– infected tiger puffer,Takifugu rubripes (Temminck et Schlegel), cultured in Japan

Vibrio harveyi infection occurred with a moderate mortality in tiger puffer, Takifugu rubripes (Temminck et Schlegel), in autumn 2007, at a mariculture farm in western Japan. The diseased fish showed nodular lesions in the branchial chamber and the inner surface of the operculum. Histopathologically, the lesions comprised granulation tissue containing many suppurative foci allowing propagation of the bacteria and granuloma encapsulating abscesses with a decrease in bacteria. The bacteria were disseminated in visceral organs including the spleen, kidney, liver, and myocardium, resulting in the formation of granulomatous lesions. Two groups of tiger puffer juveniles were artificially infected by an intramuscular injection with an isolate (1.0_10⁸ CFU/fish). During the experimental period, 20% mortality occurred within 4–6 days post‐infection (d.p.i). The fish sampled on 4 d.p.i showed abscesses in the lateral musculature at the injection site. The fish sampled 5 d.p.i. displayed the production of granulation tissue containing many suppurative foci, which replaced the necrotic dermis and lateral musculature. Surviving fish (15 d.p.i.) had granulomatous lesions in the lateral musculature at the injection site. Pyogranulomatosis is pathognomonic in V. harveyi infection of tiger puffer.     

Pathological changes induced by three myxosporeans in the intestine of cultured tiger puffer, Takifugu rubripes (Temminck and Schlegel)

Pathological changes in cultured tiger puffer, Takifugu rubripes , with emaciation disease in Kyushu, Japan were studied histologically. In most cases, diseased fish were heavily infected with at least one of three myxosporeans ( Myxidium fugu , Myxidium sp. and Leptotheca fugu ) and two unidentified hyperparasitic microsporeans, attached to, or in, the intestinal epithelium. Myxidium fugu attached to the surface of the epithelium, caused no noticeable effects on the host tissue, irrespective of its infection with the hyperparasite. Myxidium sp., which proliferated in the epithelium, induced severe pathological changes including accumulation of cell debris between the epithelium and lamina propria and resultant detachment of the epithelium. Leptotheca fugu, another histozoic myxosporean, induced degeneration of the epithelium, associated with massive infiltration of macrophages into the epithelium to encapsulate parasites. When L. fugu was infected with its hyperparasitic microsporean, shortened villi were also observed. This is probably because passage of macrophage-parasite aggregates through the basement membrane of the epithelium severely damaged the epithelial structure. It is evident histologically that, unlike epicellular M. fugu , histozoic Myxidium sp. and L. fugu with or without hyperparasitic microsporeans, were highly pathogenic to host fish. This strongly suggests that they are causative agents of the emaciation disease.     

Growth of tiger puffer Takifugu rubripes in closed recirculating culture system

ABSTRACT:   Two feeding experiments were conducted to elucidate growth performance of tiger puffer in a 10 m³ water volume closed system. In experiment 1, 1000 fish of 3.5 g average body weight were fed tiger puffer commercial feed twice daily to apparent satiation, 6 days a week for 224 days. Sand-filtered sea water was used and no water was exchanged during the rearing period. Immediately after cutting of lower teeth at day 112, daily feed consumption decreased greatly and 60 fish died in few days. Feeding rates recovered and then decreased gradually as nitrate levels increased from 600–1048 mg N/L. Fish grew to 343 g with 91% survival rate and 87% feed efficiency. Rearing conditions of experiment 2 were similar to experiment 1, except that culture water was exchanged to maintain the nitrate level less than 600 mg N/L during the 224-day experiment. Mortality and reduction of feed consumption occurred immediately after teeth cutting as was observed in experiment 1. Significant reduction of feed intake was not found during other rearing periods. Fish of 3 g grew to 303 g with 91% survival rate and 72% feed efficiency.     

Cytogenetic analysis of hybrids and hybrid triploids between the river puffer,Takifugu obscurus,and the tiger puffer,Takifugu rubripes

Cytogenetic analyses of the river puffer, Takifugu obscurus, the tiger puffer, Takifugu rubripes and hybrids produced between female T. obscurus and male T. rubripes and their hybrid triploids (produced by cold shock treatment at 4°C) were performed. T. obscurus had 2n = 44 chromosomes and 1.84 ± 0.019 pg DNA/cell, T. rubripes had 2n = 44 and 2.64 ± 0.015, the hybrids had 2n = 44 and 2.15 ± 0.010 and the hybrid triploids had 3n = 66 and 3.22 ± 0.010. The erythrocyte values of the hybrids were more similar to those for T. obscurus, whereas the hepatocyte, midgut and proximal tubule kidney cell values of the hybrids fell down between those for the parental species (p < .05). The erythrocyte, proximal tubule, hepatocyte and midgut epithelial cell sizes for the hybrid triploids were 1.5‐fold larger than those for the hybrids (p < .05). The thickness of retina and each layer in the hybrid triploids were 1.1‐fold larger than those of the hybrids (p < .05) and did not differ significantly among T. obscurus, T. rubripes and the hybrids (p > .05); however, the hybrid triploids had fewer cell nucleus outer layers than the hybrids (p < .05). Gonad development in the hybrids and hybrid triploids was less matured than in T. obscurus and T. rubripes. The metaphase nucleolus organizer regions (NORs) and the gill cells of T. obscurus, T. rubripes and the hybrids contained two satellite telocentrics, whereas the hybrid triploids contained three satellite telocentrics.     

Isolation of microsatellite markers by in silico screening implicated for genetic linkage mapping in Japanese pufferfish Takifugu rubripes

ABSTRACT:   The search for dinucleotide repeat microsatellites within scaffolds 1–25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.     

饲料中淀粉和糊精对红鳍东方鲀生长和低氧耐受性的影响

在水产养殖鱼粉鱼油供应相对紧缺的背景下,充分挖掘碳水化合物(糖类)的营养功能具有重要意义。低氧是水产养殖中一种常见的环境应激。大量研究表明,鱼类在低氧条件下主要利用糖来提供能量,但饲料糖源对红鳍东方鲀(Takifugu rubripes)的生长和急性低氧耐受性尚未见报道。本研究分别在饲料中添加玉米淀粉或糊精(玉米淀粉水解物)作为糖源,养殖8周后,测定生长、急性低氧存活率、代谢产物和低氧诱导因子(HIF)信号通路等指标。结果显示,与淀粉组相比,糊精虽然不会影响红鳍东方鲀的生长性能和鱼体成分,但显著提高了其在急性低氧条件下的存活率。在常氧条件下,淀粉组和糊精组肝脏的糖原和乳酸含量均无显著差异(P>0.05),但在低氧条件下,糊精组肝脏的乳酸含量和乳酸脱氢酶基因表达量均显著高于淀粉组(P<0.05)。同时,糊精组肝脏hif-1α (hypoxia inducible factor 1 subunit alpha)和hif-3α (hypoxia inducible factor 1 subunit alpha, like)、肌肉hif-1α和vegfa (vascular endothelial growth factor A)的表达量均显著高于淀粉组(P<0.05)。在低氧条件下,糊精组血清甘油三酯(TG)含量显著升高,但淀粉组血清和肝脏的TG含量在低氧后却显著下降(P<0.05)。与常氧组相比,糊精组在低氧后的肌肉总蛋白含量、肝脏蛋白激酶B (v-akt murine thymoma viral oncogene homolog 1, akt1)和哺乳动物雷帕霉素靶蛋白(mechanistic target of rapamycin kinase, mtor)基因表达量均显著降低,但淀粉组肝脏的mtor基因表达在低氧后却显著升高(P<0.05)。综上所述,用易消化的糊精替代玉米淀粉激活了HIF信号通路介导的无氧糖酵解来为鱼体提供更多的能量,同时还可以抑制脂肪的分解和蛋白质的合成,减少了O2的消耗,从而提高了红鳍东方鲀对急性低氧的耐受性。本研究结果为鱼类耐低氧饲料的设计和健康养殖提供了重要参考。     

Metabolomic investigation of pathogenesis of myxosporean emaciation disease of tiger puffer fish Takifugu rubripes

Serum biochemical analysis was undertaken to study the pathophysiological details of emaciation disease of the tiger puffer fish Takifugu rubripes (Temminck and Schlegel). Serum parameters were measured by biochemical analysis using automated dry chemistry and gas chromatography/mass spectrometry (GC/MS). Serum concentrations of albumin, amylase, calcium, creatinine, glucose and total protein were significantly lower in the emaciated fish when compared with those of normal fish. Regression analyses found close correlation between concentrations of total protein, albumin, amylase, glucose and progress of the disease. In contrast, serum alanine aminotransferase increased significantly in emaciated fish indicating liver function disorder. Further, GC/MS metabolic profiling of the puffer serum showed that the profile of the emaciated fish was distinct to that of non‐infected control. The serum content of amino acids including glycine, 5‐oxo‐proline and proline, and ascorbic acid, fumaric acid and glycerol increased significantly in serum in moderately emaciated fish. The serum glucose, linolenic acid and tyrosine level decreased significantly in the late phase of the disease. Our results clearly show that prolonged intestinal damage caused by myxosporean infection impairs absorption of nutrients, resulting in extreme emaciation.     

饲料蛋白能量比对红鳍东方鲀幼鱼的生长性能、饲料利用和体组成的影响

为探求红鳍东方鲀(Takifugu rubripes)幼鱼饲料的最适蛋白质能量比,以鱼粉和豆粕作为主要蛋白源,鱼油和豆油作为主要脂肪源,配制粗蛋白含量分别为36％、42％和48％,粗脂肪含量分别为8％、12％和16％,蛋能比为17.06～24.20 mg/kJ的9组饲料.投喂初始体重为14.95 g的红鳍东方鲀幼鱼56...     

Dietary calcium/phosphorus ratio influences the efficacy of microbial phytase on growth,mineral digestibility and vertebral mineralization in juvenile tiger puffer,Takifugu rubripes

A 2 × 3 factorial feeding trial was conducted to determine effects of dietary Ca/P ratio and dietary microbial phytase on growth, mineral digestibility and vertebral mineralization in tiger puffer. The treatments consisted of three levels of Ca/P ratios (0.5, 1.0 and 1.5) combined either with phytase (2000 FTU kg^?1 diet) or without supplementation, respectively. The Ca/P ratios were achieved by supplementing calcium at 0, 6 and 12 g kg^?1 combined with the same level of inorganic P at 5 g kg^?1. After a 50‐day feeding trial, puffer fish fed the diet at low Ca/P ratio (0.5) together with phytase had significantly higher growth rate and feed intake (FI) than other groups. Both dietary Ca/P ratio and phytase supplement were independent effects on plasma minerals and alkaline phosphatase. Interactive effect between both dietary treatments was observed on P and Zn contents in vertebrae and whole body. P and Zn digestibilities tended to increase with increased Ca/P ratio from 0.5 to 1.0, especially when phytase was supplemented. In conclusion, fish fed a diet with highest Ca/P ratio (1.5) showed the poorest growth performance and nutrients utilization. Dietary Ca/P ratio of 0.5 (without Ca supplement) with 2000 FTU phytase per kg would be the optimum combination in the diet of tiger puffer.     

Inhibitory modes of Kunitz and Bowman-Birk soybean trypsin inhibitors to tryptic and chymotryptic proteinases of tiger puffer and yellowtail

SUMMARY: An inhibitory effect of Kunitz and Bowman-Birk soybean trypsin inhibitors (K- and BB-SBTI) was investigated on tryptic and chymotryptic proteinases of a brood of tiger puffer Takifugu rubripes and yellowtail Seriola quinqueradiata . These assist to comprehend the differences in dietary soybean meal (SBM) utility by fish species and to develop low cost SBM diets with low contents of fish meal. The K-SBTI strongly inhibited tryptic proteinase and weakly inhibited chymotryptic proteinase from the hepatopancreas and intestine including pyloric ceca of the fishes. The BB-SBTI strongly inhibited both proteinases of the fishes. The K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for tryptic and chymotryptic proteases were relatively constant in tiger puffer with growth of 12–199 g bodyweight. In yellowtail with growth of 7–672 g bodyweight, the K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for chymotryptic protease were also relatively constant. The BB-SBTI inhibition for yellowtail tryptic protease typically fell in the stage from 7 g to 57 g bodyweight, and then decreased slightly or was maintained constantly. These results indicate that there are different inhibitory modes of K- and BB-SBTI and there is inter- and intraspecific diversity of tryptic protease conformation between the two species.     

Availability of environmental and dietary calcium in tiger puffer

Tiger puffer, Takifugu rubripes, juveniles were fed with four semi-purified experimental diets containing 0.2% Ca from Ca-lactate (diet 1), no supplemental Ca (diet 2) and 0.2% and 2.5% Ca from tricalcium phosphate, TCP (diets 3 and 4), respectively. After a 10 week rearing period, growth and feed utilization were significantly lower in the fish group fed on diet 2 than in the fish group fed on diet 1. Fish groups fed on diets 3 and 4 also showed poor growth performances compared with group 1. It appears that Ca intake from seawater is not sufficient for the normal growth of tiger puffer. Furthermore, Ca in dietary TCP appeared to be unavailable to this species. Dietary TCP strongly inhibited the bone mineralization of Zn and Mn. The findings indicate that easily digestible Ca supplementation is indispensable in a diet of tiger puffer for normal growth, feed utilization and bone mineralization. © Rapid Science Ltd. 1998     

微卫星标记对红鳍东方鲀繁殖的指导应用及遗传分析

利用已有设计的20个具有多态性的微卫星标记对16尾红鳍东方鲀亲鱼(10尾♂,6尾♀)的有效等位基因数、观望杂合度、期望杂合度、多态信息含量、遗传相似性系数、遗传距离等进行了检测.据此设计了10对亲鱼配组,获得10个子代家系.其中母本中16和8号的平均有效等位基因数和平均期望杂合度较其他亲本高,将其分别与3个父本配组,其他则参照遗传距离进行配组.于次年,对F110个家系的体质量、体长均值进行了比对,发现16♀与11♂配组得到的子代体质量、体长均值为最大.8♀和10♂配组得到的子代体质量、体长均值最小.结果表明,红鳍东方鲀子代杂种优势与亲本个体差异有关.     

Dietary astaxanthin differentially affected the lipid accumulation in the liver and muscle of the marine teleost,tiger puffer Takifugu rubripes

The lipid‐regulating effects of astaxanthin (ASTX) have been widely reported in terrestrial animals. However, little relevant information has been available in fish although ASTX has been used as exogenous pigment and antioxidant in fish feed. A 74‐day feeding study was conducted to investigate the effects of dietary ASTX on lipid accumulation in the marine teleost tiger puffer. Four experimental diets differing only in ASTX supplementation, that is, 0 (control), 50 (ASTX50), 100 (ASTX100) and 500 (ASTX500) mg kg^‐1, were randomly assigned to 12 tanks of juvenile tiger puffer. Compared to control, the liver lipid content in group ASTX50 was significantly higher, while those in groups ASTX100 and ASTX500 were lower. The muscle lipid contents in group ASTX500 were significantly higher compared to control. Group ASTX50 had the best growth performances, while diet ASTX500 seemed to have adverse effects. In the liver, compared to control, groups ASTX50 and ASTX100 showed significantly lower mRNA expressions of genes related to triglycerol synthesis and fatty acid synthesis, transport and uptake, but higher expressions of genes related to β‐oxidation and monoglycerol hydrolysis. In the muscle, compared to control, ASTX100 showed higher expressions of genes related to β‐oxidation. ASTX50 resulted in higher contents of saturated and monounsaturated fatty acids but lower contents of n‐3 polyunsaturated fatty acids in fish. In conclusion, astaxanthin in diets for tiger puffer differentially regulated the lipid accumulation in the liver and muscle, both in dose‐dependent manners. Excess dietary astaxanthin (500 mg/kg) had adverse effects on tiger puffer.     

Effects of estradiol‐17β on survival,growth performance,gonadal structure and sex ratio of the tiger puffer,Takifugu rubripes (Temminck & Schlegel, 1850), fingerlings

Systematic research was performed to assess the survival, growth performance, gonadal structure and sex ratio during and after sex reversal induced by estradiol‐17β in tiger puffers. The fish of 20 days after hatching (dah) were immersed in different doses (0.2, 2, 20, 100 ppb) for 60 days. There were significant differences in survival rates among groups (p < .05). With the increase in the estradiol‐17β dose, the survival rates of tiger puffers decreased, revealing a linear regression relationship with log₁₀ (estradiol + 1; p < .05). The growth tendency of fish in the low‐dose group was similar to those in the control group over the same period. The growth in the 20 ppb dose group was significantly slower than in the low‐dose and control groups in fish of same age (p < .05), but was significantly faster than in the 100 ppb dose group (p < .05) after 60 days. Intersex fish were categorized into intersex I, II and III types, according to the feminization degree from low to high. Phenotypic intersex puffer fish were found in the 2, 20 and 100 ppb dose groups. Intersex ratios in 2, 20 and 100 ppb were 10%, 43.3% and 46.7%, respectively, the ratios except the ones in 2 ppb group being significantly higher than those in the control group (p < .05). Moreover, 100.0% male induction for intersex II and III types occurred in 20 and 100 ppb groups. Together, these results indicated that estradiol‐17β at a dose of 20 ppb was most effective for pseudo‐female production.     

Seasonal variations of free amino acids and nucleotide-related compounds in the muscle of cultured Taiwanese puffer Takifugu rubripes

SUMMARY: Seasonal variation in the level of extractive nitrogenous components was investigated in the muscle of puffer Takifugu rubripes cultured in two different areas of Taiwan. There were no seasonal and regional variations in the proximate composition of the fish. Of the free amino acids (FAA) in the muscle of puffer, the predominant one was taurine, followed by glycine, lysine, and alanine. Among nucleotide-related compounds, inosine monophosphate (IMP) and guanosine monophosphate (GMP) were the most prominent compounds. The total nucleotide-related compounds in the muscle of puffer were higher in July and November than those in the other months. The level of total taste-active components including glycine, alanine, arginine, GMP, IMP, and adenosine 5'-monophosphate was much higher in the muscle of puffer collected from July to January. Therefore, the puffer is probably more palatable in these periods.     

在循环水养殖系统中养殖密度对红鳍东方鲀应激反应和抗氧化状态的影响

养殖密度是影响养殖水体水质和鱼类生长性能的重要因素。通过红鳍东方鲀(Takifugu rubripes)循环水养殖试验和硝酸盐氮急性处理试验,分别研究不同养殖密度(12.5~20.0 kg/m^3)和不同硝酸盐氮质量浓度(1.0、25.0、100.0和150.0 mg/L)对养殖水体水质、红鳍东方鲀生长性能、应激反应和抗氧化状态的影响。结果显示:不同养殖密度对红鳍东方鲀养殖水体的pH、氨氮和亚硝酸盐氮质量浓度无显著性影响,但较高密度会导致硝酸盐氮质量浓度显著升高,最高升至24.5 mg/L。在较高养殖密度条件下,红鳍东方鲀的生长性能(终体质量、特定增长率和饲料转化率)和抗氧化能力(总抗氧化能力、超氧化物歧化酶、谷胱甘肽过氧化物酶)较弱,但其脂质过氧化(丙二醛)和应激反应(葡萄糖、乳酸和皮质醇)较强。此外,在硝酸盐氮急性处理试验中,红鳍东方鲀未出现死亡。当硝酸盐氮质量浓度为100.0和150.0 mg/L时,红鳍东方鲀抗氧化能力较弱,而应激反应较强。综上,当养殖密度为16.5 kg/m^3时,红鳍东方鲀的生长和抗氧化状态较好,且应激压力较小,这表明在较高密度条件下,养殖水体硝酸盐氮质量浓度的升高不是引起鱼类生长抑制的主要原因。     

红鳍东方鲀低温胁迫应答主效QTL候选基因的表达特征分析

红鳍东方鲀(Takifugu rubripes)为暖温性、广温性鱼类,低温的冬季海水会对其生存造成很大影响,因此,选育具有抗寒性状的品系对其产业发展具有重要意义。本研究在利用QTL (quantitative trait locus)定位分析筛选出6个与红鳍东方鲀耐低温相关的候选基因(tacc2、fsip1、exoc4、arhgap44a、pde10a和unc5b)的基础上,通过Real-time PCR检测这6个基因在低温胁迫下在肝脏、心脏和肾脏中表达量的变化。实验用鱼为课题组建立的同一家系的8月龄鱼,共设置3个温度梯度(8 ℃、13 ℃和18 ℃),8 ℃和13 ℃为低温组,18 ℃为对照组。结果显示,6个基因在不同温度下的3个组织中均有不同程度的表达。其中,pde10a基因在3个组织中的表达均呈先升高后下降的趋势;tacc2和exoc4基因在8 ℃组肝脏、肾脏以及心脏中的表达分别呈先下降再趋于稳定、先升高再趋于稳定和先上升后下降的趋势;unc5b基因在肝脏和心脏中的表达量较低,在低温组实验前期的肾脏中呈现高表达;arhgap44a基因在肝脏中的表达呈上升趋势,在心脏和肾脏中整体表达无明显变化;fsip1基因在肝脏中的表达呈下降趋势,在心脏和肾脏中的表达呈先上升后下降的趋势。这6个基因在红鳍东方鲀组织中均随着时间和温度变化具有差异性表达,在低温胁迫下呈现积极响应,表明这6个QTL候选基因在红鳍东方鲀低温适应中具有潜在的重要作用。本研究可为红鳍东方鲀耐低温相关信号通路研究以及耐低温品种选育提供理论依据。