Incidence of Brown-gill (fungal) disease in three Penaeus species grow out ponds of Vellapallam, Nagapattinam district of Tamil Nadu, India

Transcription

1 Indian Journal of Geo-Marine Sciences Vol. 43 (8), August 2014, pp Incidence of Brown-gill (fungal) disease in three Penaeus species grow out ponds of Vellapallam, Nagapattinam district of Tamil Nadu, India V Karthikeyan*, P Gnanamoorthy & A Gopalakrishnan Center of Advance Study in Marine Biology, Faculty of Marine Sciences, Annamalai University Parangipettai Tamil Nadu, India. [ karthikeyancansee@gmail.com] Received 19 March 2013; revised 25 February 2014 Genus Penaeus are the most common shrimp species cultured in India with very susceptible to microbial diseases. Mycofloral brown-gill disease was identified from P. monodon, P. indicus and P. vannamei in shrimp pond. A totally 6 genera of 20 species from 427 colonies were isolated from the samples. Maximum growth of colonies was occurred at 80 th day of sampling. Microscopical and pathological studies revealed that the fungal infection on gill. Present investigation revealed the poor water quality conditions are a budding problem and due to this factor diseased shrimps are to be frail for consumption. [Keywords: Penaeus, Fungi, Brown gill disease, Histopathology] Introduction Shrimp diseases caused by microbes, culture environments and feed, influence the success of shrimp culture 1. Microbiological contamination of species may arise from sources, such as indigenous microflora of plants and animals, microorganisms present in processing plant, air, post harvest contamination from dust, use of contaminated water and by human contact. Microbial communities are of primary importance in all the biological activities 2. Fungi are major decomposers of woody and herbaceous substrates in the marine ecosystems and it grows on a wide variety of substrate ranging from wood to sediments, sand, algae, coral, calcareous tubes of mollusks, decaying leaves of mangroves, intertidal grasses and living animals 3. The distribution of fungi in shrimp diseases has not been studied well as compared to the studies on fungi in freshwater and terrestrial ecosystems. While the fungi occur widely on dead organic matter in fresh and marine environment they occur in the diseases of shrimp and fishes as well. Hence the present study aims to isolate, culture and document the fungal distribution from three different shrimp species of affected with brown gill disease. Materials and Methods Penaied shrimp (21-30 g) sample was collected during August 2011 to July 2012 by trawl net from ( N E) Vellapallam shrimp pond, Nagapattinam district, Tamil Nadu, India (Fig. 1). For each collection, 8-20 number of fungal brown-gill infected Penaeus sp., were collected for further processing (Fig. 2). Penaeus spp infected gills were washed three times in sterile physiological saline (0.85% NaCl) and inoculated on plates with Potato Dextrose Agar (PDA) medium (Potato infusion-200 g, Dextrose-20 g, Agar-20 g, Distilled water-1000 ml, ph 5.6). Ampicillin and streptomycin sulphate (25µg/mL) were added to the medium to inhibit bacterial growth. Plates were incubated for 2 4 days at 25 C and then sub-cultured on to fresh PDA plates. The single spore culture method was applied to obtain pure isolates. The isolates were maintained at 25 C on PDA slants for subsequent experiments. Fungal identification was done by lacto phenol cotton blue mount method to observe fungal morphology under microscopy. Routine histological techniques were used. Davidson s fixative was injected into the hepatopancreas and muscle of live fungal fouling infected shrimp in order to avoid autolysis of those organs. After injection, the whole shrimp was immersed in the same fixative for 48 h before further processing. The infected portion of brown-gill tissue were dissected and immersed in Davidson s fixative for 48 h and it was transferred to 70% ethanol for further processing. Sections were stained with usual Ehrlich s hematoxylin and eosin stains.

2 KARTHIKEYAN et al.: INCIDENCE OF BROWN-GILL (FUNGAL) DISEASE IN THREE PENAEUS SPECIES 1595 Fig. 1 Study area Fig. 2 A, Penaeus monodon infected brown gill disease; B-Penaeus indicus infected brown gill disease; C and D-Penaeus vannamei infected brown gill disease; D-Stacked Penaeus vannamei showing Brown-gill infection

3 1596 INDIAN J MAR SCI, VOL. 43, NO. 8, AUGUST 2014 Results and Discussion Totally 427 colonies were isolated from the three brown-gill diseased shrimps such as P. monodon, P.indicus and P.vannamei collected from grow out pond from Vellapallam. Twenty fungal species were isolated from diseased samples and identified six genera viz Mucor hiemalis, M. racemosus, Rhizopus nigricans, R. oryzae, R. stolonifer, Aspergillus fumigatus, A. japonicus, A. niger, A. terreus, A. versicolor, Fusarium aquaeductum, F. oxysporum, F. solani, Pencillum chrysogenum, P. grisofulvum, P. implicatum, P. oxalicum, P. rubrum, Trichoderma harzianum and T. viridae (Table 1). Out of those 20 fungal species, 16 were isolated from P. indicus and P. vannamei. 15 fungal species were from P. indicus alone. Genus Aspergillus was the predominant species and occurred in all the samples. It is important to accentuate that this is the first report of the isolation of conidial fungi from shrimp 4, demonstrated a significant diversity of cultivable fungi from adult shrimp Litopenaeus vannamei. Calorni 5 reported a fungal infection in Penaeus semisulcatus cultivated in Israel and Khoa 6 isolated Fusarium incarnatum from gill lesions of the black tiger shrimp P. monodon grown in Vietnam and found these shrimps to present high mortality. In the present investigation, the isolated fungal species has been previously reported as human mycotic agents which are responsible for causing otomycosis, keratitis, dermatitis, lung infections and even systemic infections. These are opportunistic fungi, which affect humans and other animals when they are immune-compromised 7. Safety of products for consumption is prime concern from the view of managing fish culture systems ensuring public health as well 8. Fungal strains Table 1 Fungal infection on Penaeus species collected from the grow out pond Penaeus monodon Penaeus indicus Penaeus vannamei Days of Sampling Mucor hiemalis Mucor racemosus Rhizopus nigricans Rhizopus oryzae Rhizopus stolonifer Aspergillus flavus Aspergillus fumigatus Aspergillus Japonicus Aspergillus niger Aspergillus terreus Aspergillus versicolor Fusarium aquaeductum Fusarium oxysporum Fusarium solani Pencillium chrysogenum Pencillium grisofulvum Pencillium implicatum Pencillium oxalicum Pencillium rubrum Trichoderma harzianum Trichoderma viridae Abbreviation (-)= No growth (+)= Average growth (++)= Over growth.

4 KARTHIKEYAN et al.: INCIDENCE OF BROWN-GILL (FUNGAL) DISEASE IN THREE PENAEUS SPECIES 1597 The Fig. 3 shows the occurrence of mycoflora on the three different infected shrimp species. The maximum growth of colonies occurred at 80 th day of sampling of three different infected shrimp species. Present study has observed the percentage composition of mycoflora on three different shrimp (P. monodon, P. indicus and P. vannamei) species at different (20, 40, 60, 80, 100 and 120) days of sampling from the aquaculture pond. In Penaeus monodon, the average of log value CFU/g and the maximum of species (14 species) were identified at 80 th day. Minimum of species (3 species) were identified at 120 th day. The maximum growth of Mucor racemosus (28.57%), Rhizopus nigricans (28.57%) and R. stolonifer (28.57%) was cultured on Penaeus monodon at 40 th day. While the P. indicus and P. vannamei the average log value was CFU/g and CFU/g as well as the maximum of species (14 species from 6 genera and 13 species from 5 genera) respectively. Mucor racemosus (20%), Rhizopus nigricans (20%), Fusarium oxysporum (20%) and Pencillum chrysogenum (20%) were isolated from the P.indicus at 120 th day of sampling and the Trichoderma harzianum (33.33%) was isolated from the P. vannamei at 120 th day of sampling (Figs. 4, 5 & 6). Bintvihok 9 analyzed 150 samples of black tiger shrimp Penaeus monodon in Thailand fed with feed contaminated with aflatoxin B and concluded that this type of system may reduce production, causing economic losses. The fungal population was found to be higher density in the infected shrimp of Penaeus monodon, Penaeus indicus and Penaeus vannamei collected from aquaculture pond located at Vellapalam. The external clinical symptoms of a Penaeus shrimps with fungal infection were brown or black staining due to melanization and gill fading due to Fig. 4 Percentage composition of fungal infection on Penaeus monodon at different days. Fig. 3 Number of species identified during the period of days on three shrimp species. Fig. 5 Percentage composition of fungal infection on Penaeus indicus at different days.

5 1598 INDIAN J MAR SCI, VOL. 43, NO. 8, AUGUST 2014 Fig. 6 Percentage composition of fungal infection on Penaeus vannamei at different days. deliquescence. Infected gill with pathogenic fungal were observed in under a ( 40 view) microscope (Fig. 7 A, C & E). Histopathologic examination of gill tissues from diseased shrimps has offered invaluable insights into the pathogenesis of brown gill diseased. Large foci of gill necrosis appeared all among the infected gill tissues, more usually the necrosis involved a larger area of gill. Here the necrotic cell with fungal strain was observed in the underlying connective tissues and erythrocytes had begun affected in gill tissues. Major histopathologic findings of the disease were observed in gill tissues which were completely defined in Fig. 7 B, D & F. Based on histopathological observations, the fungi were regarded as pathogenic and the cause of disease in the Penaeus shrimps. In the pact with the report of Bian and Egusa 10, melanized cuticular lesions seemed as granulomatous nodules, deceptively due to the encapsulation of fungal hyphae by host haemocytes. In addition, the present result was in agreement with those studies on another anamorphic fungus infecting the gills of the kuruma prawn, P. japonicus 11. Hence the results showed that the infected shrimps species is considered to be unfit for human consumption. So the poor water quality conditions are a budding problem of Fig. 7A, C & E Microcopical view of adhered fungi on infected gill of P.monodon, P. indicus and P. vannamei respectively ( 40 view); B, D & F- Pathological view of fungal adherence on infected gill of P.monodon, P. indicus and P. vannamei respectively (hematoxylin and eosin, 40). cultural pond. Acknowledgements Authors wish to thank authorities of Annamalai University and this work was supported by DST. References 1 Lightner, D.V., Diseases of cultured Penaeid shrimp. 5. In: CRC Handbook of maricultur: Crust Aqua (ed.by JP.Mc Vey)., CRC Press, boca raton FL., (1993) pp White, C., Sayer, J.A., Gadd, G.M., Microbial immobilization of solubilization and toxic metals: Key biogeochemical processes for treatment of contamination. FEMS Microbio Rev., 20 (1997) Kohlmeyer, J., Kohlmeyer, E., Mari Myco : The Higher Fungi. London: Academic Press., (1979). 4 Silva, L.R.C., Souza1, O.C., Fernandes, M.J.S., Lima, D.M.M., Coelh, R.R.R., Mottal, C.M.S., Culturable fungal diversity of Shrimp Litopenaeus vannamei boone from breeding farms in Brazil. Brazil Jour of Microbio., 42 (2011) Calorni, A., Campos, A.A.B., Maia, E.P., Costa W.M. and Brito. Fusariosis in the shrimp Penaeus semisulcatus cultured in Israel. Mycophatolo., 108 (1989) Khoa, L.V., Hatai, K., Aoki, T., Fusarium incarnatum isolated from black tiger shrimp, Penaeus monodon Fabricius, with black gill disease cultured in Vietnam. J. Fish. Dis., 27 (2004) Hoog, G.S., Guarro, v., Gene, J., Atlas of clini fun.

6 KARTHIKEYAN et al.: INCIDENCE OF BROWN-GILL (FUNGAL) DISEASE IN THREE PENAEUS SPECIES 1599 Centraalbureua voor Schimmelcultures, 2 nd Edition., Utrecht, The Netherlands., (2000). 8 Schotissek, C., and Naylor, E.R. Fish farming and influenza pandemic Nature., Bintvihok, A., Ponpornpisit, A., Tangtrongpiros, J., Panichkriangkrai, W., Rattanapanee, R., Doi., K., Kumagai S., Aflatoxin contamination in shrimp feed and effects of aflatoxin addition to feed on shrimp production. J. Food Prot., 66 (2003) (5) Bian, B.Z., Egusa, S., Histopathology of black gill disease caused by Fusarium solani (Martius) infection in the kuruma prawn, Penaeus japonicus Bate. J. Fish, Dis., 4 (1981) Khoa, L.V. and Hatai, K. First case of Fusarium oxysporum infection in culture kuruma prawn, Penaeus japonicus in Japan. Fish. Pathol., 40 (2005)