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.     

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.     

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.     

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.     

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.     

温度对红鳍东方鲀早期生长和性别分化的影响

研究了全同胞家系的红鳍东方鲀（Takifugu rubripes）早期幼苗在4个不同温度（15℃、17℃、19℃和正常养殖温度21～25℃）条件下,水温对其生长性状和雌雄性别组合的影响。结果表明,培育温度越低,鱼苗生长速度越慢。对照组（正常养殖温度21～25℃）的幼苗生长最快,其次依次为19℃、17℃和15℃。30～90日龄,不同温度处理组绝对增长率和生长指标具有相似变化趋势。70日龄前,正常养殖温度组的绝对增长率和生长指标最高;80～90日龄,19℃组最高。统计180日龄各温度组雄性和雌性个体比例,利用组织学切片法辨别雄雌。15℃组雄性和雌性比例为86.67%和13.33%,17℃组为70.00%和30.00%,19℃组为56.67%和43.33%,正常养殖温度组为53.33%和46.67%。低温能够有效提高红鳍东方鲀家系雄性个体比例,15℃组为最佳诱导温度处理组。     

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.     

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。     

Dietary taurine impacts the growth,amino acid profile and resistance against heat stress of tiger puffer (Takifugu rubripes)

A 56‐day feeding trail was conducted to evaluate the effects of taurine (Tau) supplementation on growth performance, amino acid profile and heat stress resistance capacity in juvenile Takifugu rubripes (initial body weight: 32.28 ± 0.20 g). Tiger puffer were distributed into five groups and each received one of the following diets: each diet was supplemented with 0 (T1), 5 (T2), 10 (T3), 20 (T4) or 50 (T5) g Tau/kg, respectively. Growth performances were significantly ameliorated with fed diet T3 (p < .05). The Tau content of muscle increased significantly as dietary Tau increased (p < .05). Compared to the control group, fish fed diets T4 and T5 showed significantly higher concentration of triglyceride and urea, respectively (p < .05), while taurine transporter (TauT) gene and IGF‐1 gene expression increased significantly in fish fed diets T3 and T4, respectively (p < .05). After acute heat stress, HSP70 gene expression levels were significantly greater in the livers and spleens of T. rubripes (p < .05) fed diet supplemented with Tau. Levels of complement C4 were highest in fish fed 10 g/kg Tau (p < .05). These data suggested that dietary supplemented with taurine indicated positive effects on growth performances, amino acid profile and resistance against heat stress for T. rubripes.     

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.     

Efficacy of feeding tiger puffer Takifugu rubripes on moon jellyfish with respect to nutritional composition and behavioural traits

Although jellyfish blooms are serious nuisances for fisheries and other industries, the utilization and nutritional value of by‐catch jellyfish has drawn public attention. Here, we evaluated the efficacy of feeding tiger puffer Takifugu rubripes with moon jellyfish Aurelia sp. using 20‐day rearing experiments. Feeding on jellyfish had no positive effect on growth performance, although it significantly reduced neutral lipids and increased proportions of polar lipids, n?3 and n?6 highly unsaturated fatty acids (HUFAs), especially arachidonic and docosahexaenoic acids, and taurine. Supplemental feeding on jellyfish plus pellets elevated activity and responsiveness in the fish, with no effect on burst swimming speed or prevention of aggressive biting by siblings. The present research suggests that feeding on jellyfish improves body composition and some behaviours in tiger puffers. As cultured fish tend to accumulate excessive amounts of lipids from lipid‐rich pellets that can induce an unhealthy condition, and behavioural deficits of hatchery‐reared fish reduce survival after release in the wild, we propose the use of jellyfish as a supplemental diet in the nursery production of tiger puffer. Moreover, feeding on jellyfish can overcome deficiencies in alternative soy‐material feeds by supplying phospholipid, HUFAs, or taurine.     

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...     

江蓠、浒苔、藻渣和菌渣替代鱼粉对红鳍东方鲀幼鱼生长性能、相关生化指标的影响

本实验旨在研究江蓠(Gracilaria verrucosa)、浒苔(Enteromorpha prolifera)、藻渣和菌渣替代鱼粉对红鳍东方鲀(Takifugu rubripes)幼鱼生长性能、血清和肝脏生化指标和体组成的影响。实验共制成6种等氮、等脂的饲料,以含鱼粉60%为对照组1和45%鱼粉的为对照组2,分别将10%的江蓠、浒苔、藻渣、菌渣与植物蛋白质配合后替代对照组1饲料中30%的鱼粉,投喂初始体重为(17.33±0.55) g的红鳍东方鲀幼鱼56 d。结果显示,1)浒苔组特定生长率(SGR)显著高于其他5组(P<0.05),藻渣组SGR显著高于对照组2和江蓠组(P<0.05),与对照组1和菌渣组无显著差异(P>0.05);对照组1和浒苔组饲料效率(FER)显著高于对照组2和菌渣(P<0.05),与江蓠和藻渣组无显著差异(P>0.05);对照组1的蛋白质沉积率(PPV)和蛋白质效率(PER)显著高于对照组2和菌渣组(P<0.05),与江蓠、浒苔和藻渣组无显著差异(P>0.05)。2)各组血清和肝脏中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性均无显著差异(P>0.05)。各组血清丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性均无显著差异(P>0.05)。3)菌渣组鱼体粗蛋白质含量显著高于藻渣组(P<0.05),与对照组1、对照组2、江蓠和浒苔无显著差异(P>0.05);浒苔和藻渣组鱼体粗脂肪含量显著高于对照组2 (P<0.05),与对照组1、江蓠和菌渣组无显著差异(P>0.05)。综合来看,将10%江蓠、浒苔、藻渣和菌渣与植物蛋白质配合后替代饲料中30%的鱼粉对红鳍东方鲀幼鱼的生长性能无不良影响,并且浒苔可以显著提高红鳍东方鲀的生长性能。     

饲料中蛋白质含量及养殖密度对红鳍东方鲀幼鱼生长性能、氮排泄及相关生化指标的影响

本实验以平均初始体重为15.60 g的红鳍东方鲀(Takifugu rubripes)幼鱼为研究对象,研究饲料中蛋白含量及养殖密度对红鳍东方鲀幼鱼生长性能、氮排泄及相关生理生化指标的影响。设计两因素三水平(2×3)实验,配制3种不同蛋白梯度(38.87%、45.55%和51.00%,干重)的等脂实验饲料,设置3个密度梯度为1.53 kg/m^3(0.196 m3体积的实验桶,每桶20尾鱼)、2.30 kg/m^3(每桶30尾鱼)和3.06 kg/m^3(每桶40尾鱼)。每组饲料设3个重复,养殖实验为期56 d,在室内流水系统内进行。结果显示,增重率在高、中蛋白组显著高于低蛋白组(P<0.05),但当饲料蛋白含量一定时,养殖密度对增重率没有显著性影响。饲料蛋白含量和养殖密度对鱼体常规成分没有显著性影响。当饲料蛋白一定时,高密度组的血清总蛋白和胆固醇含量显著高于中密度组(P<0.05)。血清总蛋白含量在低蛋白组显著高于中蛋白组(P<0.05)。血清碱性磷酸酶含量在低蛋白组显著高于高蛋白组(P<0.05)。饲料蛋白含量和养殖密度对红鳍东方鲀幼鱼的生长、氨氮排泄没有显著性交互作用。静水投喂3 h后,氨氮排泄率在高密度组显著高于低密度组(P<0.05)。研究表明,45.55%饲料蛋白质含量已经能够满足红鳍东方鲀幼鱼正常生长的需求。饲料蛋白含量和养殖密度对红鳍东方鲀幼鱼的生长性能和氨氮排泄没有显著性交互作用。