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Research Article Egypt. J. Exp. Biol. (Bot.), 7(2): 285 – 291 (2011) © The Egyptian Society of Experimental Biology RESEARCH ARTICLE El-Kassas H. Yassin Abdel Aziz N. El-Said CONTROL OF FISH FUNGAL PATHOGEN SAPROLEGNIA PARASITICA USING ROTIFER BRACHIONUS PLICATILIS AND SODIUM CHLORIDE ABSTRACT: This investigation was designed to compare INTRODUCTION: between the inhibiting effect of sodium There is a steady increase in chloride and the rotifer Brachionus plicatilis constructing Fish Farms in Egypt especially on the growth of a fish pathogen, Saprolegnia during the last fifty years as a source of parasitica as safe remediation techniques in alternative protein due to the rapid increase aquacultures. The results showed that the in population and also the high prices of addition of 100 cells of B. plicatilis, caused animal protein. Outbreaks of waterborne decrease in spores count of S. parasitica, by fungal infections (saprolegniasis) on fish, 34% after 2 hours, and 79% after 6 hours, fish eggs and water continue to cause respectively. Increasing the number of B. problems among cultured fish (Ali, 2009; plicatilis to 150 cells decreased the spores Refai et al., 2010). count of S. parasitica by 80.00%. The two way Members of the genus Saprolegnia analysis of variance showed that the tested cause saprolegniasis; a devastating disease, rotifer species exhibited a great efficiency in with symptoms of visible white or grey grazing fungal zoospores (P<0.001). On the patches of filamentous mycelium on the body other hand, NaCl solution reported stronger or fins of freshwater fish. Great losses in effect on the growth of the tested fish weight are sometimes observed due to the pathogen as compared to that occurred by the infection by this genus, as well as water rotifer species. After 14 days of incubation, S. mould (Lartseva, 1986) and loss of total parasitica was markedly suppressed incubated eggs (Dudkai et al., 1989; Sati and (P<0.001) at different concentrations of NaCl Khulbe, 1981). Kitancharoen and Hatai solution. (1997) stated that surrounding hyphal growth could reduce the water circulation around the KEY WORDS: eggs and block oxygen transportation, thus Saprolegnia parasitica, Sodium chloride, contributing to the death of the eggs. Brachionus plicatilis, Pathogenic inhibitors, Disease also reduces hatchery efficiency and Lake Edku. production. Several aquatic fungi, including chytrids and soil Hyphomycetes have been reported from two lakes on Soignée Island (Ellis Evans, 1985). CORRESPONDENCE: Prevention and control of saprolegniasis are especially difficult, even Hala Yassin El-Kassas under fish-farming conditions, owing to the Hydrobiology Laboratory, Marine Environment ubiquitous nature and the rapid spreading of Division, National Institute of Oceanography and such fungi (Alderman, 1994). Fisheries, Alexandria, Egypt. E-mail: [email protected] Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green (an organic dye) that is very efficient at killing the pathogen. However, the Nagwa El-Said Abdel Aziz use of malachite green has been banned worldwide due to its carcinogenic and Hydrobiology Laboratory, Marine Environment toxicological effects and this had resulted in Division, National Institute of Oceanography and a dramatic re-emergence of Saprolegnia Fisheries, Alexandria, Egypt. infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new ARTICLE CODE: 35.02.11 alternative control strategies (West, 2006). ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org 286 Egypt. J. Exp. Biol. (Bot.), 7(2): 285 – 291 (2011) Magaraggia et al. (2006) studied the treatment of microbiologically polluted aquaculture waters by a novel photochemical technique of potentially low environmental impact. Antifungal agents are essential for the maintenance of healthy stocks of fish and their eggs in intensive aquaculture operations. Ali (2009) reported the antifungal activity of sodium chloride on Aphanomyces sp. and Saprolegnia diclina. The rotifer Brachionus plicatilis is a euryhaline species that has been most widely used as essential food source for various types of small larvae, in rearing fish and shrimp, as well as crab larvae due to its tolerance to the marine environment (Walkes, 1981; Watanabe et al., 1983; Fig.1. Map showing Lake Edku and the study Lubzens, 1987; Dhert and Sorgeloos, 1994). Farms, Alexandria, Egypt Kagami et al. (2004) reported that the cladocera Daphnia galeata hyalina can Isolation of Saprolegnia parasitica: protect diatoms from fungal parasitism through reducing the abundance of fungal Previously prepared glass bottles each containing eight germinated and sterilized zoospores. sesame seeds was used to capture and Accordingly, this investigation was recovery of the resident zoosporic fungi designed, for studying the occurrence of a (Khallil, 1984). Water samples in these bottles zoosporic fungus in some fish farms and fish were poured under aseptic conditions into in Lake Edku, Egypt. A comparison between equivalent numbers of sterilized Petri-dishes, the Pathogenic inhibitors of sodium chloride and then kept at 22°C for 24 hours for the and Brachionus plicatilis as safe remediation colonization of sesame seeds by fungal techniques in aquacultures were carried out. propagules. Colonized sesame seeds were then transferred into other clean and sterile Salinity in Lake Edku and in Boughaz Petri-dishes containing sterile distilled water El-Maadiya fluctuated between a minimum of to which crystalline penicillin (2000 units per 0.48 ppt and a maximum of 35.69 ppt (Abdel- liter of water) was added (Roberts, 1963). Aziz and Dorgham, 1999). Therefore, These dishes were incubated at 22°C for suppression tests using sodium chloride on twenty days during which the fungal growth the two tested pathogens were performed was daily examined. The water surrounding using sodium chloride concentrations ranged the seeds contained numerous motile from 5 ppt to 30 ppt. secondary zoospores. Zoospore suspensions As far as the authors knowledge, this were then obtained by filtering this water could be considered as the first trial to follow through double layers of Whatman 541 filter up the grazing effect of Brachionus plicatilis paper (Carballo and Munoz, 1991). on Saprolegnia parasitica. Identification of the recovered fungus: The isolated fungus was identified to the MATERIAL AND METHODS: species level at the Regional Center for Mycology and Biotechnology (RCMB), Al- Sampling and collection: Azhar University, Cairo, Egypt (Khulbe, 2001; Surface water samples and about 10 random Watanabe, 2002). The most dominating fungal fishes (Tillapia spp. and Mugil spp.) were species identified was Saprolegnia parasitica. collected. Each water sample (three Media: replicates) was collected during March 2011 Glucose peptone (GP) agar medium was from eight basins from fish farms at Lake used for purification of S. parasitica and in a Edku (Fig. 1) and put in brown and sterilized broth form for estimation of the zoospores glass bottles (400 ml capacity each). Some counts as affected by the different treatments fungal isolates were collected from skin, gills of the rotifer: Brachionus plicatilis. The fungal and stomachs of the collected fish samples. isolate namely S. parasitica which dominated The rotifer Brachionus plicatilis was kindly in fish farms and fish hatcheries were used for provided by Dr. Heba Saad, Aquaculture further studies. S. parasitica was preserved in division, National Institute of Oceanography pure culture (water sesame seeds culture). and Fisheries, Alexandria. ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Yassin & El-Said, Control of Fish Fungal Pathogen Saprolegnia parasitica Using Rotifer Brachionus plicatilis and NaCl 287 Growth of Saprolegnia parasitica in and the result was expressed as the mean of presence of Brachionus plicatilis: two perpendicular radii. Grazing experiments: b. Dry weight (DW) method: Each experiment was triplicates and the Glucose peptone broth (100 ml) amended average result was recorded. At the end of with different NaCl concentrations (0, 5, 10, 15, each experiment the rotifers counts were 20, 25, & 30 ppt), in 250 ml Erlenmeyer flasks recorded to detect if there were mortality then inoculated with 5 mm discs of each fungus among the rotifers during the experiments. separately. The flasks were incubated at 25 ± Experiment 1: 2°C for 7 days. The fungal mats and the spores were removed by filtration and dried at 60 ± 3°C For estimating the grazing efficiency of to constant weight. Fungal dry weights were B. plicatilis on S. parasitica, a set of six 100- expressed as g/l. ml flasks were inoculated with 20 ml of fungal spores suspension (known initial count) c. Germinating spores count: and100 individuals of B. plicatilis were added The tested fungus was grown on glucose to each flask. Flasks were incubated for 2, 4 peptone broth amended with different & 6 hrs under florescent light 40 µmol quanta concentrations of NaCl (0, 5, 10, 15, 20, 25, & m-2 s-1 then zoospore's counts were recorded 30 ppt) and incubated at 25 ± 2°C for 10 days. after each time interval (Fig. 2). The germinating spores were counted at 800x magnification with a compound microscope (CARLZEISS) according to (Guzman and Axtell, 1986). Statistical analyses: Each single treatment was triplicates and the mean value ± standard
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