. Seed were acclimated for 3 days and fed 1.5 × 105 cells · ml–1 of a mixture of Isochrysis galbana Parke, Chaetoceros calcitrans (Paulsen) Takana, and C. gracilis Schütt (1:1:2) before the treatments.
Growth, survival, and superoxide dismutase activity in juvenile Crassostrea corteziensis (Hertlein, 1951) treated with probiotics
Crecimiento, supervivencia y actividad superoxido dismutasa en juveniles de Crassostrea corteziensis (Hertlein, 1951) tratados con probióticos
Angel I. Campa–Córdova1, Héctor González–Ocampo2, Antonio Luna–González2, José M. Mazón–Suástegui1 and Felipe Ascencio1
1 Centro de Investigaciones Biológicas del Noroeste (CIBNOR). Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, México. *E–mail: angcamp04@cibnor.mx
2 Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, Unidad Sinaloa. Boulevard Juan de Dios Batiz Paredes No. 250, Guasave, Sinaloa. 81101. México.
]]>Recibido: 9 de julio de 2008.
Aceptado: 16 de junio de 2009.
ABSTRACT
Juvenile seed of the Cortés oyster Crassostrea corteziensis were exposed to Lactobacillus sp. isolated from Nodipecten subnodosus, a mix of Pseudomonas sp. and Burkholderia cepacia, a marine yeast strain, a commercial probiotic (Epicin®), and oxytetracycline to determine their effect on growth, survival, SOD activity, and protein content. Probiotics at the test dose of 50,000 cells · ml–1, Epicin and oxytetracycline at 7 mg · l–1 were evaluated during 30 days of culture. Results showed that growth of C. corteziensis was significantly improved by Lactobacillus sp. and the bacilli mix significantly enhanced survival and SOD activity at the test dose. Protein content did not significantly increase by the treatments used. This study demonstrated the potential use of marine microbiota to improve cultivation of C. corteziensis.
Key words: Probiotics, Crassostrea corteziensis, SOD, survival, growth.
RESUMEN
Juveniles de Ostión de Cortés Crassostrea corteziensis fueron expuestos a Lactobacillus sp., aislado de Nodipecten subnodosus, una mezcla compuesta de Pseudomonas sp. y Burkholderia cepacia, una levadura marina, un probiótico comercial (Epicin®) y oxitetraciclina, para determinar su efecto en el crecimiento, supervivencia, actividad superóxido dismutasa (SOD) y contenido de proteína. Los probióticos fueron utilizados a una concentración de 50,000 cells · ml–1, el Epicin y la oxitetraciclina a 7 mg · l–1 y sus efectos se evaluaron durante 30 días de cultivo. Los resultados mostraron crecimiento significativo de C. corteziensis con Lactobacillus sp. e incremento significativo en supervivencia y actividad SOD con la mezcla de bacilos. El contenido proteico no registró incremento significativo con los tratamientos utilizados. Este estudio muestra el uso potencial de la microbiota benéfica aislada de invertebrados marinos para mejorar el cultivo de C. corteziensis.
]]> Palabras clave: Probióticos, Crassostrea corteziensis, SOD, supervivencia, crecimiento.
INTRODUCTION
Bivalve mollusk culture is a profitable economic activity worldwide. Cultivation of filter–feeding bivalves is one of the potential and sustainable forms of aquaculture that can be operated on a large scale with no artificial food because bivalves can obtain nutrients from phytoplankton, microphytobenthos, and organic detritus (Hawkins et al., 2001). Cultivation of bivalves is also useful for reducing fishing effort of wild native species (Pipitone et al., 2000). The Cortés oyster Crassostrea corteziensis (Hertlein, 1951) inhabits the Pacific coast from the Gulf of California to Panama (Keen, 1971) and is a suitable candidate for commercial cultivation. Like other bivalves species, cultivation of C. corteziensis has several problems that need to be addressed. One of the main problems is high mortality during larval and juvenile culture, largely caused by bacteria. Vibrio sp., have been recognized as pathogenic for bivalves, including Crassostrea virginica (Gmelin, 1791) (Elston & Leibovitz, 1980), C. gigas (Thunberg, 1793) (Sugumar et al., 1998), Argopecten purpuratus (Lamarck, 1819) (Riquelme et al., 1995), Pecten maximus (Linnaeus, 1758) (Lambert et al., 1999), Ruditapes philippinarum (Adams & Reeve, 1850) (Borrego et al,. 1996), Argopecten ventricosus (Sowerby II, 1842) (Luna–González et al., 2002), Nodipecten subnodosus (Sowerby I, 1835) (Luna–González et al., 2002), and Atrina maura (Sowerby, 1835) (Luna–González et al., 2002).
Apart from good cultivation practices, antibiotic supplements are used to prevent mortality of larvae and juvenile bivalve species (Luna–González et al., 2004). However, there is widespread concern that antibacterial agents in aquaculture lead to the emergence of resistant bacteria (Scholz, 1996).
Probiotic treatment has been successfully carried out in mollusks (Macey & Coney, 2005), fish (Robertson et al., 2000; Brunt et al., 2007), and crustacean species (Harzevili et al., 1998; Rengpipat et al., 2000; Rodríguez et al., 2007). Probiotics used in aquaculture studies include Gram–positive and Gram–negative bacteria, bacteriophages, yeast, and unicellular algae (Irianto & Austin, 2002). Beneficial effects include growth and feed efficiencies (Venkat et al., 2004). Studies demonstrated control of Vibrio tubiashii infections in Crassostrea gigas larvae (Gibson et al., 1998), inhibition of Vibrio sp. that enhanced survival of Pecten maximus larvae (Ruíz–Ponte et al., 1999) and Argopecten purpuratus larvae (Riquelme et al., 2000), and improvement of growth and resistance to disease in Haliotis midae (Linnaeus, 1758) (Macey & Coney, 2005).
The complex antioxidant system of aerobic organisms prevents the effect of reactive oxygen species (ROS), and also protects cells from oxidative stress (Downs et al., 2001). Enzymatic antioxidant defenses include ascorbate peroxidase, glutathione reductase, catalase, peroxidases, and superoxide dismutase (SOD), which scavenges the superoxide anion (Homblad & Söderhall, 1999). SOD plays an important role in modulating oxidative responses leading to increased or decreased SOD activity (Matsuda et al., 2003).
A common way to select probiotics is to perform in vitro antagonism tests, in which pathogens are exposed to candidate probiotics in a liquid or solid medium (Balcázar et al., 2006). It is essential to document the origin, safety, and ability of the strain to survive the transit through the gastrointestinal tract of the host (Gram et al., 2001).
This study reports the in vivo effect of three bacteria species, one marine yeast strain, a commercial probiotic formulation, and a commercial antibiotic on growth, survival, and antioxidant response in C. corteziensis seed. Attention is paid to cellular SOD (Matsuda et al., 2003; Li et al., 2005), which plays an important role in modulating oxidative responses.
]]> MATERIALS AND METHODS
Maintenance and feeding of specimens. Healthy juvenile C. corteziensis (shell length 0.82 ± 0.1 mm) were maintained at the hatchery of Centro de Investigaciones Biológicas del Noroeste, La Paz, Baja California Sur, Mexico, in small polyethylene tanks containing 4–l filtered (1–mm), and aerated seawater at 25 ± 1 °C and salinity of 36. Seed were acclimated for 3 days and fed 1.5 × 105 cells · ml–1 of a mixture of Isochrysis galbana Parke, Chaetoceros calcitrans (Paulsen) Takana, and C. gracilis Schütt (1:1:2) before the treatments.
Bacterial strains and culture conditions. Bacterial strains were previously isolated from the intestine tract of adult lions–paw scallop Nodipecten subnodosus collected from Bahía de La Paz Baja California Sur, Mexico (~24.3°N, ~110.3°W), from the intestinal tract of adult whiteleg shrimp (Litopenaeus vannamei, Boone, 1931) cultured in a shrimp farm near La Paz, B.C.S., and from the intestinal tract of adult C. corteziensis from an oyster farm in the State of Baja California Sur, Mexico. Bacterial strains were selected from in vitro antagonism tests against pathogenic bacteria (Vibrio alginolyticus and V. harveyi), and from hemolytic activity tests (using bovine erythrocytes). Strains were stored in specific medium (MRS medium, or YPD medium) supplemented with 15% glycerol at –80 °C until used. Bacterial strains were identified using the BIOLOG system.
Lactobacillus strain NS6.1, isolated from Nodipecten sub–nodosus, was incubated in MRS agar medium at 30 °C for 24 h. Pseudomonas aeruginosa strain YC58, isolated from Litopenaeus vannamei and Burkholderia cepacia strain Y021, isolated from C. corteziensis, were incubated in YPD agar medium at 30 °C for 24 h, blended in a 1:1 ratio (Mix). The marine yeast Yarrowia lipolytica strain 020 was obtained from the collection at CIBNOR, selected because of its in vitro antagonistic activity (against the pathogen bacteria V. alginolyticus and V. parahaemolyticus) and lack of hemolytic activity (using bovine erythrocytes), and cultured in YPD agar medium at 30 °C for 24 h. A commercial probiotic formulation, Epicin®, (Epicore Bionetworks, Mount Holly, NJ, USA) was tested, as was oxytetracycline (Sigma, #Cat. O4636) as an antibiotic.
Preparation of probiotics for c. corteziensis. Probiotics were thawed and incubated in specific medium at 30 °C for 24 or 48 h. Cells were removed from the culture medium by centrifuga–tion (14,000 × g, 5 min, 4 °C) and resuspended in 3% sterile saline solution at a final concentration of 1 × 109 CFU · ml–1 (stock concentration). The concentration of probiotics in the C. corteziensis culture container was adjusted from the stock concentration. A stock solution was prepared for Epicin and oxytetracycline treatments, adjusted to a concentration of 7 mg · l–1 in seed culture from both stock solutions.
Experimental protocol. Groups of 50 juveniles were cultured in 4 L plastic containers with 1 µm–filtered and aerated seawater at 25 ± 1 °C and salinity of 36. Culture tank water was changed totally every 48 h. Seeds were fed daily with 3 × 105 cells · ml–1 of Isochrysis galbana, Chaetoceros calcitrans, and C. gracilis (1:1:2). Triplicate groups of juveniles were treated with lactobacilli/ bacilli (Mix), or yeast at 5 × 104 CFU · ml–1, and with oxytetracycline or Epicin at 7 mg · l–1 for 30 days. A triplicate control group was cultured in filtered seawater free of any treatment. Temperature and salinity were measured daily. The concentration of treatment ingredients in the containers was restored with every seawater change. Survival and growth were recorded at day 30. Six juveniles from each container were randomly sampled for protein content and SOD activity and stored at –80 °C.
SOD extraction and activity assay. For cell disruption, 0.1 g frozen tissue was removed from seeds and added to a mechanical homogenizer containing 0.5 ml phosphate buffer (50 mM, pH 7.8). The homogenate was centrifuged at 5,724 × g for 5 min at 4 °C (Beckman model GS–15R; Rotor No. F2402). The supernatant was recovered and heated for 5 min at 65 °C. A new supernatant was obtained after a second centrifugation (crude extract) and stored at –20 °C.
SOD activity was determined according to Beauchamp and Fridovich (1971), using nitro blue tetrazolium (NBT) in the presence of riboflavin. Briefly, 2 ml reaction mixture (0.1 mM EDTA, 13 µM methionine, 0.75 mM NBT, and 20 µM riboflavin added to 50 mM phosphate buffer at pH 7.8) and 0 to 100 µl crude extract were placed under fluorescent light for 2 min or until A560 in control tubes reached 0.2 to 0.25 OD. SOD activity (units per milligram protein) was calculated using a computer program (Vázquez–Juárez et al., 1993).
Protein determination. Total soluble protein concentration in juvenile C. corteziensis (from 100 mg tissue) was measured according to Bradford (1976), using bovine serum albumin as a standard. Protein content was expressed in mg · ml–1.
Statistical analysis. One–way ANOVA using Statistica 6.0 software (StatSoft, Tulsa, OK, USA) was used to analyze the difference between treatments and controls. Values of p < 0.05 were considered significantly different. When significant differences were found, Tukey's HSD test, using Statistica software was used to identify the significance of these differences (p < 0.05).
]]> Survival. Juveniles treated with bacilli (Mix) had significantly (p < 0.05) higher survival than the control group (Fig. 1). Survival was significantly (p < 0.05) lower than the control group after exposure to oxytetracycline or Epicin.
Growth. Figure 2 shows growth of juvenile C. corteziensis exposed to various treatments for 30 days. Juveniles exposed to Lactobacillus sp. (NS6.1) showed significantly (p < 0.05) more growth rate than the control group, whereas, juveniles treated with the commercial probiotic (Epicin) had the least growth of all treatments.
Sod activity. Juvenile C. corteziensis exposed to Lactobacillus sp. or yeast cells for 30 days did not differ significantly in SOD activity (Fig. 3) compared with the control group. However, seed treated with the bacteria Mix showed significantly (p < 0.05) greater SOD activity (120.27 U · mg–1) than the control group (105.25 U · mg–1). Juveniles exposed to oxytetracycline, and Epicin had significantly lower SOD activity (Fig. 3).
Protein. Protein concentration of treated C. corteziensis was not significantly (p > 0.05) increased over than the control group (Fig. 4) and seed treated with Epicin had the lowest concentration of protein.
DISCUSSION
In an intensive aquatic production system, disease control plays a key role, where an intimate relationship between bacteria and host is present. Probiotics have proven advantageous in domestic animal production and the evidence supports the same conclusion for microbial management in rearing aquatic animals (Carnevali et al., 2004; Rodríguez et al., 2007). Probiotics can be delivered directly to the water via live carriers, such as Artemia salina (Linnaeus, 1758) nauplii and rotifers, or added to pelleted dry feed (Gomez–Gil et al., 2000). Only a few studies have focused on bacteria that prevent the growth of pathogenic organisms in aquaculture systems (Harzevilli et al., 1998; Kesarcodi–Watson et al., 2008). Vijayan et al. (2006) suggested using probiotic bacteria to inhibit the growth of bacterial mollusk pathogens.
Pathogenic Vibrio cause large die–offs during larval and grow–out phases of mollusks (Vijayan et al., 2006). For at least two decades, prophylactic and therapeutic use of antibiotics has been practiced in commercial hatcheries (Gatesoupe, 1989), but this appears to have let to antibiotic resistance (Sahul Hameed et al., 2003). Antibiotics commonly used in aquaculture are oxyte–tracycline, furazolidone, chloramphenicol, erythromycin, streptomycin, kanamycin, neomycin, and oxolinic acid (Benbrook, 2002). Campa–Córdova et al. (2005) reported higher larval survival in Argopecten ventricosus treated with 6.0 mg · l–1 of chloramphenicol and erythromycin. Our results showed that 7 mg · l–1 of oxytetracycline did not enhance growth, survival, antioxidant activity, or protein content in juvenile C. corteziensis.
]]> Frequently, lactic acid bacteria have been used as probiotics (Carnevali et al., 2004; Rengpipat et al., 2008). In our study, the use of Lactobacillus sp. at 5 x 104 CFU · ml–1 enhanced growth in juvenile C. corteziensis. These bacteria often produce bacteriocins and other chemical compounds that inhibit the growth of pathogen bacteria (Gildberg et al., 1997; Goldschmidt–Clermont et al., 2008), and induce higher growth and feed efficiency (Venkat et al., 2004). Lactobacillus spp. have been reported to provide benefits to human health, such as reducing cholesterol, absorption of nutrients, promoting lactose digestion, ameliorating gastrointestinal microflora, producing some vitamins, preventing some cancer, viral infection, and allergies, and having an immuno–modulatory effect (Kawahara & Otani, 2006). Bacteria used in this study may provide essential nutrients not present in algae or improved feed digestion by contributing enzymes (Verschuere et al., 2000). Moal et al. (1996) reported that bacteria in the gut of bivalve larvae consist of many strains that produce intracellular enzymes, including proteases and lipases.Pseudomonas spp. are common inhabitants of soil, freshwater, and marine environments and are known to produce a wide range of secondary metabolites, such as antibiotics, hydrogen cyanide, or iron–chelating siderophores, and inhibit a wide range of pathogenic bacteria. Pseudomonas spp. and Vibrio spp. are the most common genera associated with aquatic environments (Otta et al., 1999). Chythanya et al. (2002) indicated that the antagonistic action that inhibits vibriosis is pyocyanin, a chloroform–soluble substance. Gram et al. (1999) observed in vitro inhibition of Vibrio anguillarum by Pseudomonas fluorescens and lower mortality in the probiotic–treated fish Oncorhynchus mykiss (Walbaum, 1792). Specific inhibition of V. harveyi by Pseudomonas aeruginosa was reported by Torrento and Torres (1996). Riquelme et al. (2001) observed increased survival in Argopecten purpuratus larvae fed with a Bacillus sp.
Having an open circulation system, bivalve mollusks ingest biotic and abiotic particles, including pathogens from the surrounding water (Allam & Paillard, 1998). If bacteria or other pathogenic microbes enter the body of an invertebrate, a series of immune defense reactions will normally be elicited (Cheng, 1978). One of these defense reactions is the toxic reactive oxygen intermediates formed during a respiratory burst and which play an essential role to clear invading pathogens from the shellfish tissue and hemolymph (Mitta & Vandenbulcke, 2000). If the oxidant/antioxi–dant balance is an important determinant of immune cell function, increased levels of antioxidants will be needed to improve the immune response. SOD eliminates superoxide free radicals and plays an important role in protection against oxidative stress (Leclère, 2004). Gonzalez and Arenas (2002) concluded that SOD activity and production of the superoxide anion in A. purpuratus hemocytes could be used to evaluate the competence of the immune system in mollusks. In our study, seed treated for 30 days with live bacilli mix, significantly increased SOD activity compared to the control group. Decreased antioxidant activity in seed treated with oxytetracycline, and Epicin may induce oxidative stress in C. corteziensis according to Shuhong et al. (2004). They exposed adult Haliotis diversicolor supertexta (Reeve, 1846) to Escherichia coli and Vibrio spp. and found a significant decreased SOD activity in treated groups compared with controls.
In our study, protein content did not show significant (p > 0.05) variation in C. corteziensis exposed to probiotics, but other studies have related protein content to immune response in invertebrates. Downs et al. (2001) related increased protein content after exposure to immunostimulants to the protective effect of the immune system in grass shrimp, Palaemonetes pugio (Holthuis, 1941), against potential pathogens. Campa–Córdova et al. (2002) found a significant increase in protein content in Litopenaeus vannamei hemocytes after exposure to 0.5 mg · ml–1 of ß–glucans.
Yeasts have been reported as promising probiotics (Vine et al., 2006., Macey & Coney, 2006). Shupantharika et al. (2003) reported significantly enhanced phenoloxidase activity of hemo–cytes of the giant tiger prawn Penaeus monodon (Fabricius, 1798) treated with brewer's yeast ß–glucan in vitro and in vivo. In contrast to these studies, the marine yeast (Yarrowia lipolytica strain 020), used in one of our treatments, did not induce a significant increase in growth, survival, or physiological response. This marine yeast species has been reported to incorporate exogenous eicosapentaenoic and docosahexaenoic fatty acids from crude fish oil (Guo et al., 1999).
What these results of treating juvenile C. corteziensis with probiotics show is that some beneficial bacteria increase shellfish well being as indicated by enhanced growth and survival. Further research should provide information to optimize the concentration of probiotics in the diet of juvenile C. corteziensis. We also recommend that specific cells or tissues, especially hemocytes be used to evaluate antioxidant activity and immune response.
ACKNOWLEDGMENTS
We thank María de Jesús Romero and Sergio Hernández for technical support. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT grant 25981), International Foundation for Science (IFS grant AA/14868R) and Centro de Investigaciones Biológicas del Noroeste (CIBNOR grant AC2.2).
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