Fungal infection in freshwater fishes of Andhra Pradesh , India

A total of 17 isolates of fungi were isolated from diseased fishes which belong to five species namely Saprolegnia diclina, Saprolegnia ferax, Saprolegnia hypogyana, Saprolegnia parasitica and Achlya americana. All these fungi were isolated from six different species of fresh water fishes viz. Channa stratius, Channa punctatus, Clarias batrachus, Labeo rohita, Heteropneuste fossilis and Mystus cavasius. The parasitic ability of all the fungal species were confirmed by conducting experimental infection under laboratory conditions using healthy fishes of the same species. All the species of fungi were found to be pathogenic to fish.


Collection of infected fish samples
Incidences of fungal infection were recorded during the winter months of 2008 to 2009 from unnamed water bodies of Akividu and Bhimavaram divisions of West Godavari Dist, A.P,.A total of 1,270 fishes were screened.The fungal infected fishes were brought to the laboratory in the living condition and kept in glass aquarium, of the size of 90 × 45 × 45 cm filled with clean fresh water.The dead as well as living fishes were examined grossly for lesions and ulcerations.
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Isolation of fungi from infected fishes
Isolation of fungi from infected fishes was carried out by taking small pieces of muscles about 2 mm in diameter from infected portions of the body.They were then washed thoroughly with sterilized distilled water to remove the unwanted micro organisms adhered on the surface.These tissues were then inoculated over the plates containing different agar media.Alternatively small pieces of mycelia taken out from infected parts of fish body were washed thoroughly with distilled water.They were placed in a Petri dish containing 20 to 30 ml distilled water and baited on different baits viz.Hemp seeds, and Mustered seeds.These Petri dishes were incubated at 15 to 22°C tem for a week.Pure and bacteria free cultures were prepared by using the methods of Coker (1923), Johnson (1956) and Scott (1961).Identification of fungi was done on the basis of their vegetative and reproductive characters using the monographs of Coker (1923) and Khulbe (1994).

Artificial infection trials
In order to demonstrate the pathogenicity of the isolates obtained from the naturally infected fishes, experimental infection trails were conducted in the laboratory.Isolated species of fungi viz.Saprolegnia diclina, Saprolegnia ferax, Saprolegnia hypogyana, Saprolegnia parasitica and Achlya americana were tested separately on the fingerlings of different species of fishes, having average size and weight 8.16 ± 0.13 cm and 12.5 ± 0.28 gm, respectively.The pathogenecity tests were carried out by employing the methodology of Scott and O' warren (1964).Covered glass troughs (12 x 9) wrapped in aluminum foils were sterilized in hot air oven at 120°C tem for 24 h.Filtered sterile lake water was filled aseptically in to each trough.An aerator was used to aerate the water throughout the experiment.Six fungal inoculated blocks (1.0 cm 2 ) of SPS agar/ Potato Dextrose Agar (PDA) medium were placed at different sites in the trough.Six uninoculated blocks of the same agar medium were placed in another trough which was used as control.After 48 h, when spores developed, experimentally injured fishes were placed in these troughs.Four fishes of each species were kept in each trough.All the experiments were conducted at 20.0 to 25.0°C temperature in triplicate sets.Water samples were collected from infected water bodies for analysis of various Physico-chemical parameters such as Temperature, Conductivity, PH, FCO2, Dissolved oxygen, Total alkalinity, Total hardness and Chloride as per methods of APHA (1995).

RESULTS AND DISCUSSION
In the present study, a total of 17 isolates of fungi were obtained from the fishes investigated.These isolates represent five species and belonged to two genera namely Saprolegnia and Achlya (Table 1   with fungi isolated from naturally infected fish to test their pathogenicity under laboratory conditions.Each isolate was tested on that particular species of fish from which it was originally isolated.The results are summarized in Table 2.All the isolates of genus Saprolegnia are found to be pathogenic to fish.Hyphal growth of fungi was clearly visible at the site of injured areas of experimental fishes within 8 to 48 h after inoculation.All the test fishes died within 24 to 96 h after catching infection (Table 2).
It is observed that although all the isolates has potentiality to parasitize the fish but S. parasitica is more vigorous showing infection within 8 h (Table 2).A wide range of fluctuations were noticed in various water quality parameters of affected water bodies (Table 3).Fungal infection in fish was first reported during mideighteen century (Arderon, 1748).Later, other workers reported several pathogenic fungi from different species of fish and fish eggs.(Sati, 1982;Fraser et al., 1992;Hatai and Hoshiai, 1992;Roberts et al., 1993;Chinnabut et al., 1995;Willoughby et al., 1995;Khulbe et al., 1995;Mastan, 2008;Rekha and Qureshi, 2012).
In India the mycological studies were initiated by Chidambaram (1942) who observed red patches on the body of Osphronemus gouramy due to Saprolegnia species.Tiffney (1939) was the first to demonstrate the ability of S. parasitica (Coker) to parasitize a wide range of fishes and amphibians and emphasized the fact that the injury greatly lowers the resistance of hosts to fungal infections.Vishniac and Nigrelli (1957) conducted laboratory experiments and demonstrated the parasitic ability of sixteen species of aquatic fungi belonging to seven genera of Saprolegniaceae.Scott (1964) demonstrated that S. parasitica, S. ferax, S. diclina, Saprolegnia monoica, Achyla bisexualis and some non fruiting isolates of Saprolegnia could parasitize wounded platy fish under controlled conditions.Sati and Khulbe (1983) carried out host range studies with S. diclina on nine species of coldwater fishes such as Barilius bendelisis, Carassius auratus, Cyprinus carpio, Nemachelius rupicola, Puntius conchonius, Puntius ticto, Schizothorax palgiostomus, Saprolegnia richardsoni and Tor tor.The experimental infection of Saprolegnia on different species of fishes has also been reported by Qureshi et al. (2002). Chinabut et al. (1995) and Hatai et al. (1994) reported the pathogenicity of Aphanomyces species on Dwarf gourami.Kitanchroen and Hatai (1996) have conducted experimental infection trials with Saprolegnia sp. on Rainbow trout eggs.
In the present study mycological examination of infected fishes revealed the presence of sixteen isolates of five species viz.S. diclina, S. ferax, S. hypogyana, S. parasitica, A. americana.All the species of Saprolegnia are found to be virulent for fishes.This observation is in agreement with the finding of Scott and O.Bier (1962) who have reported that the species of fungi, S. parasitica is found to be the most destructive.This finding confirms with the reports of Hatai and Hoshiai (1992) who have reported that the infection caused by S. parasitica in salmon resulted mass mortality.Both the scaly and nonscaly fishes were found to be equally susceptible to the species of fungi tested.S. hypogyana was isolated from and tried on C. straitus, also showed its wide range on fishes.The same is also reported by Chauhan and Qureshi (1994).Qureshi et al. (2000) have conducted pathogenicity studies with various species of Saprolegnia on different species of fishes of central India.

Table 1 .
Fungi isolated from infected fishes.

Isolate no. Name of fungi Host fish
). S. diclina was isolated twice from infected fishes.One isolate was collected from Channa gachua and one from Channa straitus.A total of three isolates of S. ferax were obtained, one was collected from Clarias batrachus, one from C. straitus and one from C. gachua.One isolate of S. hypogyana was collected from infected C. gachua.S. parasitica is the most frequently occurring parasite of fish.
in the month of December; 2008.While, the minimum percentage of infection was recorded to be 0.8 in the month of February, 2009.In case of fish species the highest percentage of infection (1.6) was reported in H. fossilis while lowest infection (0. 8) was reported in L. rohita.The experimental infection trails were conducted

Table 2 .
Experimental infection trails with various species of fungi isolated from diseased fishes.

Table 3 .
Showing Water quality parameters of affected water bodies during study period.