Isolation And Identification Of Ichthyobodo Sp. From Scophthalmus Maximus And Screening Of Sensitive Drugs In China

Ichthyobodo necator is one of the member of Order Kinetoplastida, Classflagellates and one of important freshwater fish ectoparasite with a broad host andecological range. Ichthyobodiosis always seriously harm to fish worldwide. In recentyears, findings of similar flagellates in strictly marine fishes have indicated thatIchthyobodiosis may be caused by more than1flagellate species. The reports aboutIchthyobodiosis in China were focus on freshwater cultured fish and remedialmeasures. There were few research on Ichthyobodiosis in domestic mariculturefishes.This is the first report of Ichthyobodiosis in cultured Scophthalmus maximusin Wei Fang, China. Identificating and researching on the pathogeny ofIchthyobodiosis in cultured Scophthalmus maximus will supply more basicinformation about domestic researches of Ichthyobodiosis in mariculture fishes andoffer certain theoretical reference for protection from Ichthyobodiosis.Four main research area were carried on this research.(Ⅰ) Primary diagnosisfor moribund Scophthalmus maximus and morphological features of the pathogeny ofIchthyobodiosis were obtained by serial study including investigation of water quality,microscopic and scanning electron microscope observation.(Ⅱ) Isolation andartificial culture of Ichthyobodo sp. in laboratory.(Ⅲ) The screening of sensitivitychemical for Ichthyobodo sp.(Ⅳ) The phylogenetic trees were constructed by PCRand sequence alignment.The main results in details as following:(1) The first report and record with regard to symptoms and signs of sickScophthalmus maximus infected by Ichthyobodo sp. are obtained. The micrographiaby optical microscopy and scanning electron microscopy shows that the pathogenyhas morphological features of Ichthyobodo sp. for example ovoid shape and twoflagella et al. From the above, it is certain that the the pathogeny of thisIchthyobodiosis infected Scophthalmus maximus in China is Ichthyobodo sp..At thesame time, there are other there marine fish parasites in Shandong province:Paralembusdigitiformiskahl, Amyloodiniumocellatum and a species of ciliate.(2) We successfully isolate the Ichthyobodo sp. from the gill of diseased Scophthalmus maximus with protozoan micro-separation method and screened thebest medium, the optimal temperature and salinity for Ichthyobodo sp.. The resultsindicate that Ichthyobodo sp. can reach the exponential growth period quickly but notkeep too long time in0.3g/L beef extract medium(BE). However, in1%fish gillhomogenate supernatant medium (FG medium), the population density ofIchthyobodo sp. reaches maximum(450000ind/mL) in all experimental groups after96h cultured. Besides, this prosperous state could last4-10days and the populationnatural increase rate is significantly higher than BE groups. Therefore the FG mediumwas regarded as one of perfect medium for Ichthyobodo sp. In the same time, we findIchthyobodo sp. can survive in the4-30℃and22℃is the best culture temperature.The effect of salinity for Ichthyobodo sp. is not significant, but Ichthyobodo sp. in ourresearch cannot survive in the freshwater.(3) Four common chemicals include copper sulfate, copper sulfate-ferroussulfate coalition, potassium permanganate and formalin all can kill Ichthyobodo sp. invarying degrees. Integrated some factors, potassium permanganate should beconsidered preferentially because of its low prices, no residues, no side effect andconvenience.(4) According to the identification results of pathogenic infected Scophthalmusmaximus in China, the18s RNA part sequence of Ichthyobodo sp. isolated fromScophthalmus maximus in China is highly similar with other countries’. Aftersequence alignment between the18s RNA part sequence of Ichthyobodo sp. isolatedfrom Scophthalmus maximus in China with other1418s RNA part sequence ofIchthyobodo sp. from different countries and hosts and obtained phylogenetic trees,the results reveal that the Ichthyobodo sp. isolated from Scophthalmus maximus inChina is more close genetic relationship with Ichthyobodo sp. isolated fromApistogramma sp in Brazil and relatively close genetic relationship with Ichthyobodosp. isolated from Morone hybrid and Xiphophorus helleri in USA. However, theIchthyobodo sp. of China also exist certain difference with other Ichthyobodo sp., Soit is conjectured that the Ichthyobodo sp. of China should be a new species ofIchthyobodo necator.