Cultured Tiger Puffer Takifugu Rubripes Research Articles

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

A first for Southern Africa: description of a new Heterobothrium (Monogenea: Diclidophoridae) parasitizing the evileye pufferfish Amblyrhynchotes honckenii (Tetraodontiformes: Tetraodontidae).

The monogenean genus Heterobothrium (Monogenea: Diclidophoridae) currently consists of 12 accepted species described globally from various pufferfishes (Tetraodontidae). This includes the economically important Heterobothrium okamotoi Ogawa 1991 that causes severe disease in the cultured tiger puffer Takifugu rubripes in Japan. During parasitological surveys targeting near shore marine fishes of South Africa, a new Heterobothrium was discovered on the gills of five evileye pufferfish Amblyrhynchotes honckenii with a prevalence of 100% and mean intensity of 23 (4-72). Specimens were morphologically studied using both light and scanning electron microscopy and molecularly characterized by sequencing the partial ribosomal gene 28S. Heterobothrium victorwepeneri n. sp. differs from its 12 congeners by a combination of morphological characters, such as fourth clamp pair (anteriormost, 180° inverted) as the smallest with some of the sclerites differing in shape from the other pairs, 8 to 9 genital hooks, number of testes 40-50, and absent isthmus. The 28S tree depicted two main branches, one clustering together species of mazocraeidean families while the other clustered together only species of the Diclidophoridae. Heterobothrium victorwepeneri n. sp. is the first species of its genus to be recorded and described from South Africa and from the tetraodontid A. honckenii. This study also provides for the first time 28S sequence for a species of this highly host-specific genus.

Influence of Diluted Seawater Rearing on Flesh Quality of Cultured Tiger Puffer Takifugu rubripes

Influence of Diluted Seawater Rearing on Flesh Quality of Cultured Tiger Puffer Takifugu rubripes

Field Trials of Febantel against Gill Fluke Disease Caused by the Monogenean Heterobothrium okamotoi in Cultured Tiger Puffer Takifugu rubripes

We conducted six field trials on the efficacy of febantel against the monogenean Heterobothrium okamotoi infecting cultured tiger puffer Takifugu rubripes at four sites in Kumamoto and Ehime, Japan. From 2,500 to 4,500 one-year-old tiger puffers per experiment group were used in these trials, in which tiger puffer were orally administrated febantel at 12.5 or 25 mg/kg fish/day for 5 days. The maximum rate of worm eradication was 80.9%. However, a large difference was observed in the eradication rate between the experiment groups. We observed no adverse effect of febantel such as decreased appetite or increased mortality on the experimental fish.

Concentration-time Profile of Febantel in Cultured Tiger Puffer Takifugu rubripes

Concentration-time Profile of Febantel in Cultured Tiger Puffer Takifugu rubripes

Pathophysiology of Cultured Tiger Puffer Takifugu rubripes Suffering from the Myxosporean Emaciation Disease

The present study suggests that the myxosporean emaciation disease by enteric infection of Enteromyxum leei disrupts intestinal water uptake of tiger puffer Takifugu rubripes. This idea is based on a significant negative correlation between plasma chloride concentration and condition factor in diseased fish, and significantly higher osmolarity of plasma and major ion con- centrations of the intestinal fluid in infected fish than in healthy control fish. Additionally, surgical ligation of the junction between the stomach and the intestine of healthy fish resulted in a 24% drop of body weight within 50 h, and significantly lower water content of the white muscle (operated fish 74 ± 0.5%; sham-operated fish 82 ± 1%). However, in vitro water uptake by isolated intestine sacs was not significantly different between the control and infected fish. Meanwhile, hepatic function appeared to be impaired as evidenced by the significantly lower hepato-somatic index (control fish 8.7 ± 0.8%; infected fish 2.7 ± 0.8%). Plasma activities of LDH, AST and ALT were all significantly lower in the infected fish. We propose that rapid loss of body weight of infected tiger puffer is mainly due to osmoregulatory failure but probably malnutrition is also involved in the pathogenesis.

Efficacy of Orally Administered Febantel against Monogenean Heterobothrium okamotoi infection of Cultured Tiger Puffer Takifugu rubripes

We examined the efficacy of febantel against the infection of the diclidophorid monogenean Heterobothrium okamotoi in cultured tiger puffer Takifugu rubripes. Febantel is a prodrug of fenbendazole, a benzimidazole drug, which is widely used in the field of veterinary medicine. Febantel was metabolized to fenbendazole in tiger puffer just like veterinary animals for oral administration. At the doses of 12.5 mg/kg and 25 mg/kg for 5 days, and 50 mg/kg for 3 days, 88%, 96% and 94%, respectively, of mature worms, and 97%, 95% and 97%, respectively, of immature worms were eradicated at 22-23°C. These results suggest that febantel has high anti-H. okamotoi activity.

Development of a PCR-based Method for the Detection of Enteric Myxozoans Causing the Emaciation Disease of Cultured Tiger Puffer

A polymerase chain reaction (PCR) -based method was developed to detect enteric myxozoans which cause the emaciation disease of cultured tiger puffer Takifugu rubripes. Three primer sets were designed based on the sequences of small subunit ribosomal DNA of Enteromyxum leei, Enteromyxum fugu and Leptotheca fugu. All of the primer sets specifically amplified the target DNA of each species. The PCR successfully detected parasite DNA not only from the intestinal mucosa of killed fish but also from gut contents collected from live fish using a swab inserted into the anus. These PCR analyses were more sensitive to detection of parasites than microscopic observation on the intestinal imprints. The present PCR method is useful for rapid diagnosis of the myxosporean emaciation disease.

Molecular and Morphological Redescriptions of Enteric Myxozoans, Enteromyxum leei (formerly Myxidium sp. TP) and Enteromyxum fugu comb. n. (syn. Myxidium fugu) from Cultured Tiger Puffer

The phylogenies of two enteric myxozoans, Myxidium fugu and Myxidium sp. TP, from cultured tiger puffer Takifugu rubripes were re-evaluated based on their small subunit ribosomal RNA (SSU rRNA) gene sequence and spore morphology. Phylogenetic analysis shows that M. fugu and Myxidium sp. TP are grouped with Enteromyxum species as a monophyletic clade, not with other Myxidium spp. It was also illustrated that spores of M. fugu satisfy the criteria for the genus Enteromyxum for spore morphology. SSU rRNA gene sequence of Myxidium sp. TP shared 99.6% identity to that of Enteromyxum leei. Experimental transmission of Myxidium sp. TP to red sea bream Pagrus major induced the formation of mature spores, which were indistinguishable from those of E. leei in morphology and morphometrics. Myxidium sp. TP is identified as E. leei, and M. fugu is reassigned as Enteromyxum fugu comb. n.

Myxosporeans and Their Hyperparasitic Microsporeans in the Intestine of Emaciated Tiger Puffer.

Three myxosporeans and 2 hyperparasitic microsporeans were found in the intestine of emaciated cultured tiger puffer Takifugu rubripes in Kyushu district of Japan. Morphology of the 3 myxosporeans were examined and described as Myxidium fugu n. sp., Leptotheca fugu n. sp. and unidentified Myxidium sp. Spores of M. fugu n. sp. having 2 club-shaped polar capsules were bean-shaped, 14.0 (13.5-15.5) μm in length and 9.0 (8.0-10.0) μm in width. Spores of L. fugu n.sp. having 2 spherical polar capsules were trapezium-shaped, 9.0 (8.3-9.5) μm in length and 14.0 (13.0-15.0) μm in thickness. Their piscine intestinal developmental stages were illustrated and arranged in a hypothetical developmental sequence. Plasmodia of M.fugu n. sp. were located on the epithelium of intestine, whereas those of L. fugu n. sp. and Myxidium sp. were found intercellularly in the intestinal epithelium. Only a few mature spores could be observed in these myxosporeans, but the differences in stainability by Diff-Quik and Uvitex 2B and in location of development made it possible to differentiate the species. Two hyperparasitic microsporeans were often found in the plasmodia of M. fugu n. sp. and L. fugu n. sp, but their pathogenic effects to host myxosporeans were unclarified.

Induction of Ovarian Maturation and Ovulation in Cultured Tiger Puffer Takifugu rubripes by Different Hormonal Treatments

Induction of Ovarian Maturation and Ovulation in Cultured Tiger Puffer Takifugu rubripes by Different Hormonal Treatments

トラフグ養成親魚からの採卵技法の開発

We developed an effective method for the induction of oocyte maturation and ovulation in the cultured tiger puffer Takifugu rubripes by hormonal treatments, in order to supply the fertilized eggs for seedling production. Ovarian biopsy just prior to the hormonal treatments revealed the oocyte diameter and developmental stages of the oocytes.A luteinizing hormone-releasing hormone analogue (LHRH-a) cholesterol pellet (400μg/kg) implantation successfully induced oocyte maturation for three-year-old cultured tiger puffer which had yolk accumulated ovaries. Thereafter, single injection of HCG (500IU/kg) combined with chum salmon pituitary (SP) homogenate (7mg/kg) was applied to the fish to synchronize ovulation among fish. Ovulation mainly occurred between 96 and 120 hours after HCG+SP injection. Thus, a technique for obtaining eggs of high quality from the cultured tiger puffer was developed.

Copulation and Egg Production of the Monogenean Heterobothrium okamotoi, a Gill Parasite of Cultured Tiger Puffer(Takifugu rubripes).

The monogenean Heterobothrium okamotoi, parasitizing, in clusters, the branchial cavity wall of cultured tiger puffer Takifugu rubripes, were collected together with some host tissue, in which the posterior part of the parasite body was embedded, to make in vitro observations of copulation and egg production under the stereomicroscope. Some specimens copulated with other worms within the same cluster by attaching their male copulatory organ to the ventro-lateral or dorso-lateral body of their partners. Self-fertilization was also observed. Eggs were produced at about 2-min intervals. After egg assembly in the ootype, the egg was transferred to the uterus and large numbers of eggs accumulated there. Eggs were connected, at both ends, with previous and successive ones through a continuous filament, forming a long egg string. The length of the filament connecting the eggs corresponded with the length of the duct between the ootype and the uterus. Eggs were extracted from the uterus of freshly collected worms and counted. The highest number of eggs per worm exceeded 1, 500. Based on the measurements of the egg and filament lengths and the number of eggs recorded, the longest egg string was calculated to be over 2.8 m long.

養殖トラフグの“口白症”に関する研究　Ｉ　　養殖トラフグの“口白症”自然発生例の病理組織学的所見

養殖トラフグの“口白症”に関する研究　Ｉ　　養殖トラフグの“口白症”自然発生例の病理組織学的所見