versión On-line ISSN 0718-560X
Lat. Am. J. Aquat. Res. vol.39 no.2 Valparaíso jul. 2011
Lat. Am. J. Aquat. Res., 39(2): 197-204, 2011
Efficacy of marine green alga Ulva fasciata extract on the management of shrimp bacterial diseases
Eficacia del extracto del alga marina verde Ulva fasciata sobre el manejo de las enfermedades bacterianas en camarones
Joseph Selvin1, Aseer Manilal2, Suganthan Sujith3, George Seghal Kiran4 & Aron Premnath Lipton5
1-4Department of Microbiology, Bharathidasan University Tiruchirappalli - 620 024, India
5Centre for Marine Science and Technology, M.S. University Rajakkamankalam Kanyakumari District, India
ABSTRACT. Secondary metabolites of the green algae, Ulva fasciata, were tested to determine the efficacy of controlling shrimp bacterial pathogens. Exploratory experiments indicated that an intermediate dose (1 g kg-1 of shrimp) of Ulva in the diet was highly effective at controlling bacterial pathogens of shrimp, as compared to lower (500 mg kg-1) and higher (1.5 g kg-1) doses. The pilot experiments evaluated the percent of relative protection afforded shrimps treated with Ulva diet and faced with various concentrations of bacterial pathogen. The survival of shrimps treated with Ulva diet was significant (P < 0.01). The present findings indicate that the green U. fasciata may be an excellent source for developing a potent medicated feed for shrimp disease management.
Keywords: Penaeus monodon, bacterial diseases, Vibrio, shell disease, proactive management, Ulva fasciata.
RESUMEN. Metabolites secundario de algas verdes Ulva fasciata fue probado para determinar la eficacia de controlar el camarón pathogens bacterial. Las conclusiones de experimentos exploratorios indicaron que la dosis mediana (1 g kg-1 de camarón) de dieta Ulva era sumamente eficaz en el control de pathogens bacterial de camarón cuando comparado al más abajo (500 mg kg-1) y más alto (1,5 g kg-1) dosis. En los experimentos pilotos, la protección de pariente de por ciento de camarones trató con la dieta Ulva y desafió con varias concentraciones de bacterial patógeno fueron evaluados. La supervivencia de camarones trató con la dieta Ulva era significativo (P < 0,01). Basado en las conclusiones presentes, podría ser deducido que U. verde fasciata puede ser una fuente excelente para desarrollar la comida potente medicinal para la dirección de enfermedad de camarón.
Palabras clave: Penaeus monodon, enfermedades bacteriales, Vibrio, enfermedad de cáscara, dirección proactiva, Ulva fasciata.
Bacterial disease outbreaks particularly vibriosis and black shell disease impose a significant constraint on the sustainable production of shrimp (Bachere et al., 1995; Verschuere et al., 2000; Selvin et al., 2005; Manilal et al., 2010b). In Asia, among the pathogenic Vibrio group, 11 species were reported from the shrimp culture systems (Lavilla-Pitogo, 1995). V. alginolyticus and V. harveyi poses a serious disease problem in cultured black tiger shrimp in India (Karunasagar et al., 1997; Selvin & Lipton 2003a; Manilal et al., 2010b). Vibrio species are considered to be members of the normal bacterial flora of shrimp and the culture environment (Jiravanichpaisal et al., 1994; Otta et al., 1999). Often acting as opportunistic pathogens or secondary invaders, they may cause total mortality of reared shrimps (Lightner, 1988; Nash et al., 1992).
Although few reports evidenced the efficacy of algae-based products in shrimp health management (Furusawa et al., 1991; Yamasaki et al., 1997), the role of marine natural product in shrimp disease management has been realized recently (Selvin & Lipton, 2003, 2004; Selvin et al. 2004b; Huang et al., 2006; Manilal et al., 2009). It was found that marine secondary metabolites are promising resources for the development of eco-friendly management practices for shrimp diseases (Selvin & Lipton, 2003; Selvin et al., 2004, 2004b; Manilal et al., 2009, 2009a). Moreover, secondary metabolites of green algae Ulva fasciata elicited the non-specific defense factors of shrimp against pathogenic invaders (Selvin, 2002; Selvin et al., 2004b). In addition, medicated feed formulated with polysaccharides isolated from the Indian green algae, Acrosiphonia orientalis were found to be effective in control of White Spot Syndrome Virus (Manilal et al., 2009). Recently, Huang et al. (2006) envisages the effect of Sargassum fusiforme polysaccharide extracts on vibriosis resistance and immune activity of the shrimp, Fenneropenaeus chinensis.
Southwest coast of India is a unique marine habitat infested with diverse variety of seaweeds. The biological activity of seaweeds from the southwest coast of India (Kollam coast) is already reported (Manilal et al., 2009, 2009a, 2009b, 2010, 2010a, 2011, 2011a). In this background, the present study aims to establish the effect of algal-based medicated feed on the survival of experimentally infected shrimps.
MATERIALS AND METHODS
Collection of marine algae
The mature plants of Ulva fasciata was handpicked from the intertidal and subtidal habitat of the Kollam (08°54N, 76°38'E) area located on the southwestern coast of India. The collection was performed during the period from June 2007 to August 2007 when algal biomass remains dominant. Live and healthy plants were harvested manually and washed thoroughly in running water. Cleaned plant materials were shade dried under an air jet to prevent photolysis and thermal degradation. The completely dried material was weighed and ground coarsely in a mechanical grinder (Manilal et al., 2010).
Extraction of algae
For extraction, 500 g of finely powdered algal material was refluxed three times in a 5 L capacity round bottom flask in a water bath at 65°C for about 6 h using dichloromethane: methanol (1:1) as a binary azeotropic solvent (Manilal et al., 2009b). The extracts were filtered and concentrated to recover the excess solvents in another distillation system. The concentrated extract (about 100 mL) was again filtered through a Whatman N°1 filter paper fitted with a Buchner funnel using suction pressure.
Finally, it was reduced to thick oily natured crude extract in a rotary vacuum evaporator (Yamato) at 40°C, collected in air-tight plastic vials and stored in the refrigerator for further activity studies.
Determination of median lethal dose (MLD) of algal extract
Primary exploratory (dose selecting) experiments were conducted with broad range of the test compounds. Chosen concentrations such as: 100, 1000 and 2000 mg kg-1 shrimp of U. fasciata extract were prepared in 5% EtOH in normal saline to ensure complete solubility. Ten shrimps (abw = 8.5 g) per group (triplicates) were injected intra-muscularly with 0.1 mL of appropriate dose or 5% EtOH in normal saline (control). The immediate reflexes and mortality were observed every hour for the first 6 h and every 24 h for 7 days. Based on the exploratory experiments, narrow ranges were administered to the experimental shrimp to determine the median lethal dose (LD50).
Preparation of medicated diet
Top-coated medicated feed was prepared with appropriate quantity of Ulva extract on the surface of the feed at a rate of 3.2% of the shrimp body weight daily. To prepare the medicated feed, 30, 60 and 80 g of Ulva extract was incorporated in medicated diet. The shrimp were fed with a commercially formulated shrimp feed with the following proximate composition: 35.6% crude protein, 3.5% lipids, 5.9% fibre, 45.7% nitrogen-free extract and 11.21% ash. Commercial pellet shrimp grower feed No. 1 (C.P. feeds, Cochin) was used for preparing medicated feed for the experiments on leaching of Ulva extract in the water. The extract was dissolved in 50 mL of 4% gelatin water and sprayed on 1 kg of pellet feed using a TLC sprayer. The sprayed medicated feed was dried in a hot air oven at 40°C.
Pathogenicity and LD50 value of shrimp pathogens
Initially, the slant culture of shrimp pathogens (V. fischeri, V. alginolyticus, V. harveyi and Aeromonas sp.) was activated and 1 mL of 18 h culture was inoculated in 25 mL nutrient broth. This was kept overnight at 30 ± 2°C in a shaker at 180 ± 5 rpm. An 18 h shake culture was centrifuged at 4800 x g for 15 min at 40°C (Eppendorf). Cell pellets were washed twice with normal saline (NS), re-suspended and serially diluted in NS and enumerated using a Petroff-Hausser counting chamber by plating on nutrient agar supplemented with 2% NaCl (Himedia). This was also plated on nutrient agar to get the colony forming units (cfu) values. The black tiger shrimp Penaeus monodon post-larvae (PL-20) obtained from the Matsyafed hatchery, Kollam and were reared in a 1000 L fiber reinforced plastic tanks provided with constant aeration and 50% daily water exchange. They were reared at optimum hydrological conditions such as salinity (35 ppt), temperature (30 ± 2°C) and pH (7.8). Healthy juveniles (average body weight = 5.4 ± 2.2 g) were segregated and maintained at 20 shrimps/tank in 200 L high-density plastic (HDP) tanks before starting the experiment. The challenged shrimps were maintained at 10 shrimps/tanks in glass aquaria. Shrimps were intramuscularly injected with 0.1 mL of bacterial inoculums using a 1 mL tuberculin syringe at ventral side between the second and third segment. Preliminary examinations revealed that challenge dose of 103 colony forming unit (cfu) per shrimp could not kill the injected shrimp (Manilal et al., 2010b). Therefore the concentrations of 105 to 108 cfu of the appropriate single (V. fischeri, V. alginolyticus, V. harveyi and Aeromonas sp.) and combined culture (V. alginolyticus + V. harveyi) of bacteria per shrimp were used. Parallel control groups received 0.1 mL of normal saline (NS) only. Ten shrimps were used for each inoculation level. The mortality and reflexes of the shrimps were observed for every 15 min in the first hour of post-inoculation and every 1 h until the 6th h (Manilal et al., 2010b). Subsequent monitoring was done every 12 h for a period of seven days (Tendencia & Dureza, 1997). The appropriate concentration of pathogens (105 to 108 cfu), which causes mortality within 24 h or within 7 days, was considered as LD50 whereas which causes infection or abnormalities was considered as ID50 (Infectivity Dose) (Manilal et al., 2010b). The LD50 dose was calculated by the probit method, after Wardlaw (1985).
Effect of different dose of algal extracts in the survival of experimentally infected shrimps
The shrimps with an average body weight of 5.6 ± 1.8 g range were reared in the circular high density plastic (HDP) tanks at a stocking density of 60 individuals per 600 L of seawater. They were fed with Ulva diet (1 g kg-1 shrimp) in three equal installments at a rate of 3.2% of their body weight for a period of 15 days. On the 16th day, three duplicate groups each of treated shrimps (10 numbers/group) were grouped in to three and intramuscularly injected with LD50 (106 cfu/shrimp) challenge dose and higher challenge dose (107 to 108 cfu/shrimp) of single culture of bacterial pathogens such as V. fischeri, V. harveyi, V. algino-lyticus and Aeromonas sp., and combined culture of pathogen (V. harveyi, 5 x 105 + V. alginolyticus, 5 x 105 = 106 cfu/shrimp) and transferred to the 100 L glass aquaria. They were observed for a period of 15 days for mortality and external clinical symptoms (Austin & Austin, 1989). The Ulva diet was continued after challenge for 15 days. Parallel control shrimps (10 numbers) received 0.1 mL normal saline only. The mortality/infectivity percentages were estimated according to Sung et al. (1994). The Percent Relative Protection (PRP) was determined by the following expression:
To determine if significant differences existed between the treated and control shrimps, all results were analyzed using ANOVA. The significant differences were indicated at P < 0.01.
The Median Lethal Dose (MLD) of Ulva extract in P. monodon was determined to be 1120 mg kg-1 shrimp. During the preliminary experiment, the infection (pathogenicity range) was obtained at a higher dose of 108 cfu/shrimp after 5 days of inoculation with luminescent V. fischeri type culture. The LD50 values of bacterial pathogens were ran6ged as 2.8 x 107 cfu/shrimp for V. fischeri and 106 cfu/shrimp for V. harveyi, V. alginolyticus and Aeromonas sp. The external clinical symptoms were characterized as 'shell necrosis' and 'black spots' on the shell, necrotised chelate legs, anorexia, feeble movement and mortality.
The effect of different algal extracts in the survival of experimentally infected shrimp is presented in Table 1. The shrimp fed with the dose rate of 500-mg kg-1 shrimp had the lowest protection against bacterial infection. At this dose, the shrimp challenged with V. fischeri produced infection in 60% of challenged shrimp, whereas the shrimp challenged with V. harveyi and V. alginolyticus caused infection to the extent of 80%. The experimental group, fed with median (1 g kg-1 shrimp) and higher (1.5 g kg-1 shrimp) doses of medicated feed exhibited more or less similar level of protection. At these treatment levels, no mortality was noticed in the shrimps challenged with V. harveyi and Aeromonas sp. whereas V. fischeri and V. alginolyticus caused 20% infection. The infected shrimps showed external clinical symptoms such as 'shell necrosis' and 'black spots' on the shell. Subsequent symptoms such as necrotised chelate legs, anorexia, feeble movement were culminated in mortality, particularly in control and lower treatments. Based on these findings, it could be envisaged that shrimp fed with 1 g kg-1 will provide high protection compared to other doses.
The survival rate of treated and control shrimp against various bacterial pathogens is depicted in Figures 1 to 4. The shrimps challenged with potentiated pathogen produced more infection or mortality in the treated group than those caused by the LD50 value of individual pathogen. In the case of V. fischeri, the LD50 did not cause mortality in the treated group while all the control shrimp died (Fig. 1). Ulva diet elicited significant survival (60 in terms of PRP) against the lethal dose (108 cfu/shrimp) of V. fischeri. However 20% infection was observed among the surviving shrimp of Ulva fed group (Table 2). The Ulva diet elicited complete protection (100% survival) against the infection of V. harveyi and V. alginolyticus. The inoculation of a higher dose (107 cfu/shrimp) of Aeromonas sp. caused 40% infection in the Ulva treated group (Table 2). It was found that the Ulva diet produced a higher level of protection (80% survival) against the infection caused by the combined pathogens. Invariably, the survival of treated shrimp against the bacterial infection was significant at P < 0.01 level (Table 2).
Based on the findings, shrimp fed with Ulva-based medicated feed at a dose rate of 1 g kg-1 could effectively control the bacterial pathogens challenged in this study. Invariably the shrimp treated and challenged subsequently with the LD50 of appropriate bacteria gave higher survival than the control group. In the control group, early high mortality was observed than the actual LD50. It may be due to the enhanced virulence obtained by the pathogenic isolates passed through the host, prior to the 'in captivity' control experiments. The treated group challenged with higher dose (107 to 108 cfu/shrimp) of bacterial inoculum had low survival. The survival rate of Ulva treated shrimp was 100% against the infection caused by the shrimp bacterial pathogens. Selvin et al. (2004b) reported that inhibition of bacterial propagation was a possible mechanism by which Ulva medication provided protection from infection.
Shrimp disease management using Ulva diet was proven to be an effective eco-friendly management strategy for sustainable shrimp farming (Selvin, 2002; Selvin & Lipton, 2003). Ulva diet was found to be a potent immunomodulator and therefore it was considered as a proactive drug (Selvin et al., 2004b). As per our earlier finding, 88% of viable V. fischeri cells were cleared-off from the haemolymph within 1 h in the Ulva treated group. These findings suggest the quick production of bactericidins in the haemolymph of Ulva treated group. The rapid bacterial clearance rate of shrimp haemocytes was stimulated by Ulva treatment. Therefore it was conjectured that bacteridins found in shrimp plasma might be inducibly released from haemocytes by Ulva medication. Recently Huang et al. (2006) reported the effect of seaweed Sargassum fusiforme polysaccharide extracts on vibriosis resistance and immune activity of the treated shrimp. Literature also evidenced the in vivo antiviral (WSSV) potency of seaweed-based medicated feed in Penaeus monodon (Manilal et al., 2009). Satoh et al. (1987) investigated the effect of Ulva on susceptibility of infectious disease in red sea bream Pagrus major fed with 5% Ulva pertusa meal supplementation diet. The Ulva meal increased the resistance against infection caused by Pasteurella piscicida. A spray-dried preparation of micro-algae Tetraselmis suecica was reported to control a variety of fish pathogens such as A. salmonicida, Staphylococcus liquifaciens, V. anguillarum, V. salmonicida and V. ruckeri (Austin & Day, 1990).
Based on the present study, therapeutic formulation (Ulva diet) can be easily prepared with crude extract. Therefore purification strategies and consequent synthetic analogue development process need not be undertaken. As the extract was prepared from the dried material, such source material can easily be stored for 12 months. To sustain the host-defense system and relative protection against pathogenic invaders, the Ulva medication can be used as a prophylactic agent for the entire culture period at low cost. Based on the present findings, it could be inferred that the secondary metabolites of U. fasciata form an excellent source for developing potent formulations as a package of proactive management practice for sustainable shrimp farming.
The author Aseer Manilal is gratefully acknowledged Council of Scientific and Industrial Research (CSIR), New Delhi for providing Senior Research Fellowship. This work is funded by Department of Biotechnology, New Delhi (BT/PR8064/AAQ/03/290/2006).
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Received: 14 March 2010; Accepted: 13 April 2011
Corresponding author: Aseer Manilal (firstname.lastname@example.org)