Microsoft Word - 11 BIOTROPIA No. 20, 2003: 11 - 23 POTENCY OF VIBRIO ISOLATES FOR BIOCONTROL OF VIBRIOSIS IN TIGER SHRIMP (PENAEUS MONODON) LARVAE WIDANARNI1'2. A. SUWANTOu',SUKENDA2,and B. W. LAY4 'Department of Biology, Faculty of Science and Mathematics, Bogor Agricultural University, Bogor 16144, Indonesia 2 Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Bogor 16680, Indonesia 3SEAMEO-BIOTROP, Jl. Raya Tajur Km 6, Bogor 16720. Indonesia ^Faculty of Veterinary Science, Bogor Agricultural University, Bogor 16680, Indonesia ABSTRACT This study was carried out to obtain Vibrio isolates able to function as biocontrol of vibriosis in shrimp hatchery. Thirty one Vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i.e. Labuan, Pangandaran, and Lampung, Indonesia. Pathogenic V. harveyi MR5339 was obtained from Maros, South-Sulawesi and was made as a rifampicin resistant mutant (RFR) to screen for those 31 Vibrio isolates in in vitro assays and to allow us to monitor their presence in shrimp larvae and larval rearing water. Almost all Vibrio isolates could inhibit the growth of pathogenic V. harveyi MR5339 RFR. SKT-b isolate from Skeletonema was the most effective to inhibit the growth of V. harveyi MR5339 Rf* and significantly reduced larval mortality in pathogen challenge assays. These prospective biocontrol bacteria, at concentration of 10" CFU/ml, did not show pathogenicity to shrimp larvae. SKT-b was Gram negative, short rod-shape, exhibited yellow colonies on TCBS and swarming on SWC-agar media, motile, utilized glucose and sucrose but not lactose: produced extra- cellular protease and amylase, but did not produce chitmase. Partial sequencing of 16S-rRNA gene SKT-b showed SKT-b similarity to Vibrio alginofyticus. Keywords: shrimp larvae / biocontrol bacteria / vibriosis. INTRODUCTION Tiger shrimp (Penaeus monodori) culture in Indonesia has become more intensive and extensive because of high demand and economic value of this export commodity. However, the shrimp culture industry is associated with multiple problems such as diseases and poor environmental quality, which became the main constraint to reach the target of production. Bacterial disease that attacked at hatchery stage is the most serious threat and often caused mass mortality in shrimp larvae which greatly influenced the sustainable supply of healthy fry. This disease is often caused by a luminous bacterium identified as Vibrio harveyi (Lavilla — Pitogo et al. 1990; Pedersen et al. 1998). To prevent such mass mortalities, shrimp hatcheries routinely use antibiotics. However, widespread antibiotics applications could result in antibiotic resistant pathogens (Karunasagar et al. 1994; Tjahjadi et al. 1994; Tendencia and de la Pena * Corresponding author: asuwanto@indo.net. id BIOTROPIA No. 20,2003 2001). Not only can resistant bacteria proliferate after an antibiotic has killed off the other bacteria, but also they can transfer their resistant genes to other bacteria which have never been exposed to the antibiotic (Verschuere et al. 2000). Furthermore, this approach is not predictable since V. harveyi strains that attack shrimp larvae are genetically diverse (Suwanto et al. 1998). The development of vaccine was hampered due to the lack of immune response-memory or the degradation of the absorbed vaccines (Alabi et al. 1999). An alternative method of controlling pathogenic bacterial strains in shrimp cultures could be supplementation of pure cultures of natural bacterial isolates (biocontrol) which might produce chemical substances inhibiting the growth of pathogens. The approach basically employs the activity of microorganism that could suppress or inhibit the growth of V. harveyi without causing bad impact on the equilibrium system in a particular microbial community. This has been an established practice in husbandry of terrestrial animals (Fuller 1992; Ohhira et al. 1996), and has only recently been applied to aquatic systems such as fish culture (Gildberg et al. 1995) '-nd crustacean culture (Riquelme et al. 1997; Rengpipat et al. 1998). Widanarni and Suwanto (2000) reported the presence of several Vibrio isolates associated with the shrimp from the egg stadia to post larva, as well as their rearing environment. Based on their physiological and genetical characters, the isolates were distinguishable from V. harveyi that has been proved to be pathogenic on shrimp larva. Therefore, further studies should be conducted to evaluate its potency as biocontrol in shrimp hatchery. Similar finding was also reported by Riquelme et al. (1997) in which Vibrio isolates associated with scallop larvae were proved to be a potential probiotic in scallop culture in Chile. The purpose of this research was to obtain new bacterial isolates as improved biocontrol agents which can be applied to solve the problem of bacterial disease in shrimp culture, and to study their ability to inhibit the growth of V. harveyi. MATERIALS AND METHODS Isolation of Vibrio candidates for biocontrol Vibrio isolates for biocontrol were isolated from tiger shrimp larvae and hatchery environments, i.e. Labuan, Pangandaran, and Lampung, Indonesia. Samples were taken from eggs, larvae (nauplius, zoea, and mysis) and post-larvae of tiger shrimp, natural shrimp feed (i.e. Artemia and Skeletonemd), seawater, and rearing water of each stage of shrimp larvae. All samples were spread on thiosulphate citrate bile salt agar (TCBS, Oxoid). The culture was incubated at room temperature (28-31)°C, for 24 hours. Subsequently, different morphological types of colonies were randomly selected for further study. Pathogenic V. harveyi MR5339 was obtained from BALITDITA (Research Institute for Coastal Aquaculture) Maros, South-Sulawesi. 12 Potency of Vibrio isolates for biocontrol of vibriosis- Widanarni et al. Sensitivity test of Vibrio to rifampicin Purified isolates of Vibrio candidates for biocontrol and pathogenic V. harveyi MR5339 were grown in seawater complete agar (SWC-agar) (5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water) supplemented with rifampicin (Rf) 50 u.g/ml. After overnight incubation at room temperature (28-31)°C, the bacteria were scored for their antibiotic sensitivity by streaking on the appropriate antibiotic-containing media. Rifampicin-resistant mutant of V. harveyi One milliliter of a 24 hour culture of V. harveyi MR5339 was centrifuged at 5000 rpm for 1 minute. Following the removal of the supernatant, the bacterial pellet was resuspended in 100 (il of sterile seawater. The suspension was then spread onto SWC-agar containing Rf (50 u.g/ml). Colonies of luminescent bacteria grown on SWC-agar + Rf were restreaked onto new media to obtain isolated V. harveyi MR5339 resistant to rifampicin. Determination by in vitro test of biocontrol Vibrio The inhibitory effects of each isolate candidate for biocontrol were tested against V. harveyi MR5339 Rf*, known to be a pathogen of shrimp larvae. Tested isolate candidates for biocontrol was added to 10 ml of SWC-broth in test tubes at 106 cells/ml. Onto the same test tubes were also added overnight liquid culture of V. harveyi MR5339 Rf* at 102cell/ml. All test tubes were incubated overnight at room temperature. Appropriate dilutions will be chosen for seeding the mixed culture on SWC-agar supplemented with Rf (50 |ag/ml). If the number of colony forming unit (CfU) of V. harveyi MR5339 Rf* from the control tubes (i.e. tubes inoculated only with V. harveyi MR5339 Rf1*) is larger than the number of V. harveyi of the mixed cultures (i.e. V. harveyi MR5339 Rf* which is mixed with the candidate biocontrol isolate), this could identify potential candidates of biocontrol isolates that inhibit the growth of the V. harveyi MR5339 Rf* pathogen. Pathogenicity assay of biocontrol bacteria to shrimp larvae Shrimp larvae were incubated with the isolates which showed positive growth inhibitory activity against V. harveyi MR5339 Rf*. Bacterial isolates as candidate of biocontrol agent were resuspended in sterile seawater. Shrimp larvae were cultured in 2-liter shrimp rearing tank (20 individuals per tank). Bacterial suspension was added into the tank at the final concentration 106 CFU/ml. After incubation at room temperature, survival rate of larvae at 5 days was recorded. BIOTROPIA No. 20, 2003 Pathogen challenge test This test was carried out to study the efficacy of Vibrio candidate biocontrol isolates towards pathogenic V. harveyi in shrimp larvae. The best three biocontrol isolates, based on in vitro test, were selected for pathogen-challenged assay. Suspension of each isolate was placed in the shrimp rearing tank for 6 hours before shrimp larvae were introduced into the tank. After cocultivation of isolates with shrimp larvae for 6 hours, V. harveyi MR5339 Rf* was introduced into the culture tank. This experiment was performed in three replications. Tanks treated with only V, harveyi MR5339 Rf* or without bacteria were employed as controls. Total Vibrio population (counted on TCBS media), V. harveyi MR5339 Rf* (counted on TCBS media + Rf) in the rearing media, larval mortality monitored daily for 5 days and the survival rate of shrimp larvae were determined as described previously (Hala 2002). Microbiological and physiological properties Cell shape, motility, and Gram staining were evaluated using an Olympus BH2-RFC microscope. A number of physiological characteristics were analyzed using Microbact kit test (Medved Science Pry. Ltd. Australia) and production of extra-cellular protease, chitinase and amylase monitored on SWC-agar supplemented with skim milk, colloidal chitin and starch were tested, respectively. The presence of extra-cellular hydrolytic enzymes could be used to provide further information on the pathogenicity or virulence of the isolates. Amplification and sequencing of 16S-rRNA gene Since many strains of marine bacteria share similar physiological and morphological characteristics, identification of bacteria strictly based on such parameters is not reliable. Therefore, it is essential to perform DNA sequencing of the 16S-rRNA gene in order to provide reliable additional information for bacterial identification. Modified phenol-chloroform-isoamylalcohol treatment and ethanol precipitation were used to extract the genomic DNA (Sambrook et ul. 1989). The 16S-rRNA genes were amplified by PCR using specific primers of 63f and 1387r from genomic DNA provided by Ready-To-GO PCR Beads (Pharmacia-Biotech, Uppsala, Sweden). These primers were successful to work with a broad range of environmental samples (Marches! et al. 1998). A part of 16S-rRNA gene was sequenced to infer the closest related organism from Ribosomal Database Project (RDP) maintained in the University of Illinois, Urbana-Champaign. The sequencing reactions were done by using the Big Dye Ready Reaction Dye Deoxy Terminator kit, purify with ethanol-sodium acetate precipitation. The reactions were run on an ABI PRISM 377 DNA Sequencer (Perkin-Elmer Cetus, Norwalk, Conn). Potency of Vibrio isolates for biocontrol of vibriosis — Widanami el al. RESULTS AND DISCUSSION Isolation of Vibrio candidates for biocontrol Thirty one Vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i. e. Labuan, Pangandaran, and Lampung (Table 1). Of these isolates, 25 isolates were found to be associated with eggs, larvae, post-larvae, and the rearing water of each stage; 1 isolate was obtained from seawater reservoir: 4 isolates were obtained from natural feed (i.e. 2 isolates from Artemia and 2 isolates from Skeletonema). All of the isolates were non-luminous yellow colonies on TCBS and swarming on SWC-agar media. Table 1. Codes and sources of Vibrio isolates No. Code Source Location 1. SW Seawater Labuan 2. N-a Nauplius Labuan 3. NT Nauplmstank water Labuan 4. Z-a Zoea Labuan 5. PL,,-a Post-larvae 6 Labuan 6. PL,,-b Post-larvae 6 Labuan 7. PU-c Post-larvae 6 Labuan 8. PL,,-d Post-larvae 6 Labuan 9. PLn-a Post-larvae 1 1 Labuan 10. PL,,-b Post-larvae 1 1 Labuan 11. PLnT-a Post-larvae 1 1 tank water Labuan 12. PLnT-b Post-larvae 1 1 tank water Labuan 13. SKT-a Skeletonema Labuan 14. SKT-b Skeletonema Labuan 15. PL2 Post-larvae 2 Pangandaran 16, PL2T-a Post-larvae 2 tank water Pangandaran 17, PL2T-b Post-larvae 2 tank water Pangandaran 18. PL, Post-larvae 3 Pangandaran 19. PL,T Post-larvae 3 tank water Pangandaran 20. PL,, Post-larvae 6 Pangandaran 21. PL,,T-a Post-larvae 6 tank water Pangandaran 22. PL,,T-b Post-larvae 6 tank water Pangandaran 23. PL7 Post-larvae 7 Pangandaran 24. PL7T-a Post-larvae 7 tank water Pangandaran 25. PL7T-b Post-larvae 7 tank water Pangandaran 26. AT-a Artemia Pangandaran 27. AT-b Anemia Pangandaran 28. E Egg Lampung 29. N-b Nauplius Lampung 30. N-c Nauplius Lampung 31. Z-b Zoea Lampung 32. MR5W) (V.harveyi) Infected shrimp larvae Maros-SULSEL BIOTROPIA No. 20,2003 Pathogenic V. harveyi MR5339 was obtained from BALITDITA (Research Institute for Coastal Aquaculture) Maros, South-Sulawesi and was isolated from infected shrimp larvae. This isolate formed green colonies on TCBS and became luminous on TCBS or SWC-agar media. V. harveyi MR5339 was further employed for both in vitro and pathogen challenge assay to screen for potential biocontrol bacteria. Sensitivity test of Vibrio to rifampicin Sensitivity testing of Vibrio isolates including V. harveyi MR5339 showed that all of the isolates were sensitive to rifampicin. Therefore, a rifampicin resistant mutant of V. harveyi MR5339 was employed in the assay for biocontrol in vitro and pathogen challenge to screen for potential biocontrol bacteria. Tjahjadi et al. (1994) reported that almost all of the bacteria that were isolated from seawater and hatchery-rearing water at Kalianget, East Java, including V. harveyi, were resistant to various antibiotics except rifampicin. Rifampicin, a bactericidal antibiotic, is active against gram-positive and some gram-negative bacteria. This drug interferes with transcription processes in bacteria and thus far was never used in shrimp hatchery. In vitro assay for biocontrol activity The inhibitory effects of each Vibrio isolate candidate for biocontrol were tested against V. harveyi MR5339 Rf (Figure 1). Three out of 31 isolates tested i.e. SKT-b, PL2T-a and N-c were isolated from Skeletonema, post-larvae 2 rearing water and nauplii showed the best result. After 24 hours of incubations, only very few colonies (102-103 cells/ml) of V. harveyi MR5339 Rf* were grown on culture media inoculated with V. harveyi MR5339 Rf* at concentration of 102 cells/ml and Vibrio candidates for biocontrol at concentration of 106 cells/ml. The number of colonies in the control experiments (inoculated only with V. harveyi MR5339 Rf*) could reach 5x108 cells/ml. The other tested isolates could not significantly inhibit the growth of MR5339 Rf, Therefore, only SKT- b, PL2T-a, and N-c were further studied in the pathogenicity challenge experiments. Before performing challenge test with V. harveyi MR5339 RFR in shrimp larvae, some potential isolates candidate for biocontrol were analysed for their pathogenicity to shrimp larva. After 5 days of exposure with biocontrol isolates at 1Q6 CFU/ml, it had confirmed that SKT-b, PL2T-a and N-c isolates were not pathogenic to shrimp larvae. These facts were indicated by relatively similar values of survival rate to those of control group (Table 2). Pathogen challenge test Three of the most potential Vibrio isolates i.e. SKT-b, PL2T-a and N-c for biocontrol based on in vitro test, were assayed for their activities in inhibiting colonization of V. harveyi MR5339 Rf* in shrimp larvae. Observations were carried out on larval survival rates and total population of Vibrio and V. harveyi MR5339 Rr* both in the rearing water and in the dead larvae. Results showed that each of the three biocontrol candidates significantly (P<0.05) could increase survival rates of shrimp larvae reared in seawater inoculated with 103 cells/ml of MR5339 Rf". Larval survival rates in each of biocontrol treatment, i.e. SKT-b, PL2T-a and N-c were 93%, 90% and 82%, respectively. On the other hand, treatment with MR5339 Rf* inoculation without biocontrol isolate showed only 68% survival rate of shrimp larvae (Figure 2). The isolates presumably increased survival rates of shrimp larvae as survival rates of control group (without addition of biocontrol or V. harveyi MR5339 RfR isolates) were lower than treatments with biocontrol isolates. However, observation on shrimp larvae fitness after inoculation is required to determine the physiological condition of larvae. Rengpipat et al. (1998) reported the use of Bacillus Strain S l l as a probiotic administered in enriched Artemia to larvae of the black tiger shrimp (P. monodon). At two weeks, P. monodon survival was significantly different between the control groups (85%) and the treated groups (89%). When challenged with a pathogenic V. harveyi strain D331, the shrimp treated with probiotics showed a higher survival (13%) when compared to the control group (4%). Haryanti et el. (2000) also reported that inoculation with BY-9 strain at a dose of 106 CFU/ml to shrimp larvae, resulted to a survival rate of 59.3%, while for the control only 14.7%. The increased survival rate might be due to the growth inhibition of V. harveyi MR5339 Rf* on shrimp larvae by biocontrol bacteria. Although no significant difference was observed in the number of V. harveyi MR5339 Rf* either in culture media (Figure 3) or dead larvae (Figure 4), high mortality in shrimp larvae for treatment without biocontrol isolates indicated the effect of the inhibition. However, the way how the mechanisms occurred need to be studied further. According to Verschuere et al. (2000), action mechanism of probiotic bacteria or biocontrol could be divided into several ways as follows: (1) production of inhibitory compounds, (2) competition for chemicals or available energy, (3) competition for adhesion sites, (4) enhancement of the immune response, (5) improvement of water quality, (6) interaction with phytoplankton. To get an insight about action mechanism of probiotic bacteria, especially on their competition for adhesion sites, biocontrol bacteria should be tagged with molecular marker so that the existence of bacteria in shrimp larvae could be Figure 3. Number of colonies of Vibrio sp. And V. harveyt MR5339 Rľ in larva rearing thanks   Potency of Vibrio isolates for biocontrol of vibriosis- Widanarni et al. detected. One of the molecular markers extensively used for studying bacterial activity in the e nvironment is gf'p (green fluorescent protein) gene isolated from a jellyfish (Aequorea victoria) (Manning 1997). If expressed, the gene will produce green fluorescence GFP protein under UV light, so that the bacteria could be easily observed. Moreover, as molecular gene marker, GFP could provide several advantages such as no requirement for exogenous substrate or energy source for their visualization. GFP assays were reported to be sensitive, stable, non toxic, and did not disturb cell function and growth (Josenhans et al. 1998; Ling et al. 2000). Characterization and identification of biocontrol bacteria SKT-b was Gram negative, short rod-shape, produced yellow colonies on TCBS, and exhibited swarming activity on SWC-agar. This isolate was motile, could utilize glucose and sucrose but not lactose; produced protease and amylase, but did not produce chitinase. Partial sequencing (500 bp of the 5'-end) of 16S-rRNA gene of SKT-b showed that the isolate showed similarity to Vibrio alginolyticus (83% of similarity). Complete sequencing of the 16S- rRNA gene, however, should give more definitive information about the taxonomic position of this isolate. Austin et al. (1995) reported that V. alginolyticus was effective in reducing diseases caused by V. anguillarum and V. ordalii, however, other strains of this bacterium has been associated with vibriosis in shrimp. Therefore, characterization and identification of biocontrol strain were crucial steps to be carried out to assess the pathogenicity of biocontrol bacteria to the shrimp larvae. Moreover, characterization and identification of biocontrol strain were also important for mass production, quality control and patenting to protect commercial interest (Gomez- Gil et al. 2000). According to Riquelme et al. (1997), Vibrio might be more promising to be developed as probiotics when compared to other probiotic species for hatchery applications, since vibrios are commonly associated with larvae in culture, and auto-inhibition could limit the growth of pathogenic Vibrio. In conclusion, Vibrio isolates from tiger shrimp larvae and hatchery environments have the ability in reducing shrimp larvae mortality in pathogen challenge assays. The isolates are potential to be developed as a biocontrol agent and therefore as an alternative to chemical treatment in preventing luminous bacterial diseases in shrimp hatcheries. ACKNOWLEDGMENTS This research was supported by DIP-Funded Research BIOTROP 2001/2002 to AS. BIOTROPIA No. 20, 2003 REFERENCES Alabi, A.O., D.A. Jones and J.W. Latchford. 1999. The efficacy of immersion as opposed to oral vaccination of Penaeus indicus larvae against Vibrio harveyi. Aquaculture, 179:1-11. Austin. II L. F., P. A. Stucken, W. Robertson, I. Effendi and D. R. W. Griffith. 1995. A probiotic strain of Vibrio alginofyticus effective in reducing diseases caused by Aeromonas salmonicida, V. anguillarum and K ordain. J. Fish Diseases, 18:93-96. Fuller, R. 1992. History and Development of Probiotics. In R. Fuller (Ed) Probiotics the Scientific Basis, p.p: 1-8. Chapman and Hall. London. Gildberg, A., A. Johansen, and J. Bogwald. 1995. 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