Studies On Amyloodiniosis And Epitheliocystis Of Spotted Knifejaw (Oplegnathus Puncatus)

Spotted knifejaw（Oplegnathus puncatus） is high valuable marine fish in subtropical and temperate water, but its nature population is relatively small. The artificial breeding of spotted knifejaw was successful in 2014 in China. However, repeated outbreaks of infestation by parasite in hatchery-reared spotted knifejaw caused 100% mortality in three days. Diseased fish are anorexia, swimming near the water surface with labored breathing. Examined under cover slips using light microscope, the gill filaments are attached by many ovoid parasites. The morphological characteristics and the life cycle of the parasite were recorded by light microscope and scanning electron microscope（SEM）. A specific PCR product（²50 bp） corresponding to dinoflagellate was amplified and sequenced from extracted DNA of the parasite. We get a 4737 bp length gene sequence according to specific primers. The BLAST result of the sequence confirmed that the parasite was Amyloodinium ocellatum. To our knowledge, this is the first report of the occurrence of Amyloodinium ocellatum in Oplegnathus puncatus.However, Amyloodiniosis of spotted knifejaw has not reported up to now. Mass mortality of hatchery-reared spotted knifejaw juveniles occurred at a fish hatchery in July 2014 and May 2015. The causal agent was then characterized as Amyloodinium ocellatum and it means that Amyloodiniosis becomes a serious parasitic disease in juveniles of spotted knifejaw. Copper sulfate（Cu SO₄） is legal and efficient fishery drug to cure this disease. However, the safe concentration of copper sulfate to spotted knifejaw juveniles was unknown. Furthermore, the eradication effect of copper sulfate to A. ocellatum has not been documented up to now. In this study, healthy spotted knifejaw juveniles（ 4 cm in total length） were used to investigate the acute toxicity of copper sulfate. Total 180 of juveniles were divided randomly into 6 groups（3 parallels of each group） and kept in 18 of tanks with 20 L of seawater. Each tank contained 10 fish. The water temperature was 23-25 °C and the salinity was 30. The mass concentration of Cu SO₄·5H₂O was 0.00, 1.56, 3.13, 4.69, 6.26, 9.39 mg/L in each group, respectively. Fish were observed at 15, 30, 60 min and 2, 4, 6, 8, 14±2, 24, 48, 72, 96 h. The activity and mortality of fish was recorded. The results showed that the safe concentration of Cu SO₄·5H₂O to spotted knifejaw juveniles is 2.22 mg/L, which corresponding to 1.42 mg/L of copper sulfate. Furthermore, 0.40 mg/L of Cu SO₄·5H₂O can kill dinospores of A. ocellatum quickly and 0.70 mg/L of Cu SO₄·5H₂O can entirely inhibit the division of tomonts. We concluded that the safe and effective concentration of Cu SO₄·5H₂O used to cure Amyloodiniosis of spotted knifejaw juveniles is 0.70-2.22 mg/L. The study provided important basis for the treatment of Amyloodiniosis in spotted knifejaw juveniles.Epitheliocystis is a kind of fish disease which was caused by bacteria in the order of Chlamydiales. Its typical characterization is epithelial hypertrophy in gills and skin of diseased fish. No case was reported on fish epitheliocystis in China. As a newly cultured marine fish species, spotted knifejaw（Oplegnathus puncatus） has high economic value. Spotted knifejaw juveniles at 15 cm in total length died continuously in a hatchery located in Shandong province in 2015. And the cumulative mortality was more than 40% in half month. The field investigation recorded that the water temperature was 21℃ and the water salinity was 30 during epidemics. Diseased spotted knifejaws usually scattered in the tank. They stagnated in the water with little vitality and/or lose balance even drifted with the water flow. Clinical signs of diseased fish contained debility, anorexia and dyspnea. Diseased fish kept mouth open with operculum opening and closing at a high rate. They did not response to feeding. Clinical examination and necropsy revealed that gills of diseased fish were covered by a lot of mucus. The gill filaments were damaged and the intestinal tract was empty. Many cysts with 30⁷0 μm in diameter, round or ovoid in shape and light yellow-brown in color were observed on wet mounts of gill filaments from diseased fish by the light microscope. The histopathology by hematoxylin and eosin（H&E） staining indicated that the tops of secondary lamellae adhered each other and many epithelial cells enlarged and formed cysts. These cysts were basophilia and homogeneous. The examination of scanning electron microscope showed that the gill filaments of diseased fish were clavate. The secondary lamellae were covered by a lot of mucus and embedded by many cysts with smooth surface. Based on the field investigation, clinical examination and necropsy of diseased fish and microscopic observation of gills, this disease was considered as epitheliocystis of spotted knifejaw. This is the first report of epitheliocystis in cultured spotted knifejaw in China.