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Latin american journal of aquatic research

versión On-line ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.39 no.3 Valparaíso nov. 2011


Lat. Am. J. Aquat. Res., 39(3): 534-543, 2011
DOI: 10.3856/vol39-issue3-fulltext-13

Research Article


Enhancement of superoxide dismutase and catalase activity in juvenile brown shrimp, Farfantepenaeus californiensis (Holmes, 1900), fed β-1.3 glucan vitamin E, and β-carotene and infected with white spot syndrome virus

Incremento de la actividad superóxido dismutasa y catalasa en juveniles de camarón café Farfantepenaeus californiensis (Holmes, 1900) alimentados con β-1,3 glucano vitamina E y β-caroteno e infectados con el virus de la mancha blanca


Rosario Pacheco1, Felipe Ascencio1, Martha Zarain2, Gracia Gómez1 & Ángel Campa1

1Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195 Colonia Playa Palo de Santa Rita, La Paz, Baja California Sur, 23096, México
2Centro de Ciencias de Sinaloa, Avenida de las Américas 2771 Norte Colonia Villa Universidad, Culiacán, Sinaloa 80010, México

Dirección para correspondencia

ABSTRACT. The effect of dietary β-Ο-glucan, vitamin E, and β-carotene supplements in juvenile brown shrimp, Farfantepenaeus californiensis, inoculated with white spot syndrome virus (WSSV) was evaluated. Groups of 30 organisms (weighing 1 ± 0.5 g) were cultured in 60 L fiberglass tanks and fed daily with β-1.3-glucan (0.1%), vitamin E (0.01%), and β-carotene (0.01%) for 23 days; the specimens were then inoculated with WSSV. The antioxidant activity of the enzymes superoxide dismutase (SOD) and catalase (CAT) were determined in the hepatopancreas and muscle at 0, 1, 6, 12, 24, and 48 h after inoculation. Shrimp fed with β-1.3-glucan, vitamin E, and β-carotene significantly increased SOD activity in the hepatopancreas and muscle at 12 and 24 h post-infection, respectively. Shrimp fed with vitamin E and β-1.3-glucan registered an increment in SOD activity from 12 to 48 h post-infection. Shrimp fed with β-carotene increased SOD activity before infection with WSSV, and shrimp fed with β-1.3-glucan and vitamin E increased CAT activity, also before infection. The CAT activity response in shrimp muscle increased with respect to the control group for all treatments tested from 1 to 6 h after inoculation with WSSV. The highest antioxidant response was registered in shrimp fed with vitamin E. Juvenile shrimp fed with vitamin E and later inoculated with WSSV registered 100% mortality at 72 h, but shrimp fed with β-Ο-glucan and β-carotene showed greater resistance to WSSV, with mortality at 144 h post-infection. This study demonstrated the capacity of juvenile Farfantepenaeus californiensis fed β-Ο-glucan, vitamin E, or β-carotene to increase the antioxidant response before and after viral infection.

Keywords: Farfantepenaeus californiensis, shrimp, WSSV, superoxide dismutase, catalase.

RESUMEN. Se evaluó el efecto de β-1,3-glucano, vitamina E y β-caroteno en la dieta de juveniles de camarón café Farfantepenaeus californiensis inoculados con virus del síndrome de la mancha blanca (WSSV). Se colocaron grupos de 30 camarones (peso 1 ± 0,5 g) en contenedores de fibra de vidrio de 60 L y se alimentaron diariamente durante 23 días con β-1,3-glucano (0,1%), vitamina E (0,01%), y β-caroteno (0,01%) y posteriormente se inocularon con WSSV. Se determinó la actividad antioxidante de la enzima superóxido dismutasa (SOD) y catalasa (CAT) en hepatopáncreas y músculo a las 0, 1, 6, 12, 24 y 48 h después de la infección. Los grupos de camarones alimentados con los tratamientos incrementaron la actividad SOD en el hepatopáncreas y músculo a las 12 y 24 h después de la infección, respectivamente. Los juveniles tratados con vitamina E y β-1,3-glucano mantuvieron un incremento en la actividad SOD desde las 12 a 48 h postinfección. Los camarones alimentados con β-caroteno incrementaron la actividad de SOD antes de la infección con WSSV y los que fueron alimentados con β-1,3-glucano y vitamina E incrementaron la actividad CAT también antes de la infección. La actividad CAT en músculo se incrementó respecto al grupo control, con todos los grupos de camarones tratados desde 1 hasta 6 h posteriores a la inoculación con WSSV. La actividad antioxidante más alta se registró en los camarones alimentados con vitamina E. Los juveniles alimentados con vitamina E y posteriormente inoculados con WSSV, registraron 100% de mortalidad a las 72 h, pero los que fueron alimentados con β-1,3-glucano y β-caroteno resistieron la infección hasta las 144 h. Los resultados de Antioxidant response in F. californiensis fed with dietary supplements and infected with WSSV este estudio mostraron la capacidad de juveniles de Farfantepenaeus californiensis alimentados con β-1,3-glucano, vitamina E o β-caroteno, de incrementar la respuesta antioxidante antes y durante una infección viral.

Palabras clave: Farfantepenaeus californiensis, camarón, WSSV, superóxido dismutasa, catalasa.


White spot syndrome virus (WSSV) infection is considered one of the most devastating diseases and is responsible for severe economic losses in shrimp culture industry worldwide (Lightner, 1996). WSSV is highly pathogenic and has a broad host range within decapods crustaceans, it has been found in at least 18 cultured or wild penaeid shrimp species (Durand et al., 1997; Lightner et al., 1998) as well as copepods, marine crabs, prawn and freshwater crabs, rotifer, polychaete worms and some aquatic insect larvae species (Lo et al., 1996b; Flegel, 1997; Ramírez-Douriet et al., 2005; Escobedo-Bonilla et al., 2008). The brown shrimp, F. californiensis, is a commercially important fishery in México, and is being studied as an alternative in tropical shrimp culture or as an alternate culture during the winter season in Mexican northern regions, with extreme climate changes (Martínez-Córdova et al., 1998).

Investigations on the antioxidant system in shrimp have shown that WSSV infection causes oxidative stress by forming excessive amounts of reactive oxygen species (ROS) which are involved in phagocytosis activation (Schwarz, 1996; Mohankumar & Ramasamy, 2006). The phagocytic process is the main cellular defense reaction, and together with humoral components, constitutes the first line of defense of crustaceans against infectious microorganism like bacteria, fungi and viruses (Söderhäll & Cerenius, 1992; Lee & Söderhäll, 2001). This process consists of chemotaxis, adherence, ingestion, pathogen destruction, and exocytosis (Vargas-Albores & Yepiz-Plascencia, 1998). Phagocytic cells destroy the internalized organisms by two routes: (1) an aerobic process which will be described in detail herein subsequently, and (2) other anaerobic process that is attributed to the action of diverse microbicidal enzymes, such as lysozyme and low molecular weight AMP (Nappi & Ottaviani, 2000). The aerobic process uses NADPH or NADH as an electron donor, and reduces an oxygen electron to form a radical superoxide ion. This radical in turn changes to hydrogen peroxide (H2O2) spontaneously or by the action of the superoxide dismutase (SOD), which can easily diffuse through the cell membranes into the cytoplasm where the antioxidant catalase (CAT) reduce the hydrogen peroxide producing a new oxygen molecule and thereby detoxify ROS (Mohankumar & Ramasamy, 2006; Aguirre-Guzmán et al., 2009).

The principal ROS molecules are: hydroxyl radical (OH-), superoxide anion (O2-), transition metals such as iron and copper, nitric oxide (NO) and peroxynitrite (ONOO-) (Dormandy, 1983). Although, the ROS are crucial to normal biological processes, they can cause direct cellular injury by including lipid and protein peroxidation and damage to nucleic acid (Richard et al., 1990). Thus, there is a critical balance in cell between ROS generation and antioxidant defense systems to protect themselves against free radical toxicity and to maintain the cellular homeostasis (Sies, 1991; Winston & Di Giulio, 1991; Livingstone, 2001). These systems also include both enzymatic and nonenzymatic components, where the enzymatic system is comprised of superoxide dismutase (SOD), and CAT acting on O2- and H2O2, respectively (Halliwell & Gutteridge, 1996). The nonenzymatic system includes small water-soluble molecules such as vitamin C, as well as lipid-soluble molecules such as carotenoids and vitamin E (Packer, 1991; Liu et al., 2007). The shrimp antioxidant system can be activated by exposure to different immunos-timulants as sodium alginate (Cheng et al., 2005), zymosan A (Zhang et al., 2008), lipopoly-saccharides (LPS), fucoidan, heat-killed Vibrio penaeicida (Campa-Córdova et al., 2005).

The uses of immunostimulants, such as β-glucan, have been successfully applied to enhance resistance of fishes and crustaceans against bacterial and viral infections (Chang et al., 2000, 2003). Previous studies have been demonstrated that shrimp fed with glucan diets increased activity of SOD than those of a glucan-free group before challenge with pathogenic WSSV and bacteria (Chang et al., 2003; Campa-Córdova et al., 2005). However, these studies only considered SOD and did not consider other antioxidants enzymes such as CAT. The antioxidant activity is also reinforced by several molecules of nutritional interest that are of a chemical structure compatible with the antioxidant properties found in vivo, such as pigments and vitamins, which are also capable of modulating the cellular response to ROS (Muñiz-Rodríguez, 2009). Vitamin E is considered the most important antioxidant in extracellular fluids, since it is capable of protecting polyunsaturated fatty acids from peroxi-dation and it has the ability to scavenge oxygen-derived free radicals (Wang et al., 2006; Liu et al., 2007). Likewise, carotenoids also improve antioxidant defense system (SOD and CAT), protecting the body against free radicals (Flores-Leyva, 2006). Additional benefits of carotenoids include provitamin A activity, as well as enhancing immune response, reproduction, growth, maturation, photoprotection, and defense against hypoxic conditions common in pond cultures of prawns (Lorenz, 1998).

The antioxidant effects of vitamins and carotenoids have poorly been evaluated in shrimp. In addition, antioxidant activity with immunostimulants, vitamins, and carotenoids in brown shrimp (F. californiensis) has not been previously evaluated, although it has been shown to be beneficial in other species of shrimp. Thus, the present work was designed to evaluate the effects of dietary β-Ο-glucan, vitamin E and β-carotene supplementation on the antioxidant activity in muscle and hepatopancreas of F. californiensis infected with WSSV.


Experimental animals

Juvenile brown shrimp (F. californiensis) (1 ± 0.5 g) were captured in Bahía de La Paz, Baja California Sur, México. Before the experiments, the animals were acclimated during 15 days in an aerated 1500 L fiberglass tank containing filtered (0.2 µm ) seawater, salinity at 35 g L-1, pH 7.8, and the temperature was maintained at 25°C. Tank water was changed at the rate of 50% daily. Shrimp were fed ad libitum daily with commercial feed (PIASA, 35% of protein level).

Diet preparation

The composition of the experimental diet is described in Table 1. The diets were prepared at the Aquatic Nutrition Laboratory of the Centro de Investigaciones Biológicas del Noroeste (CIBNOR), the formulation was similar to that described by Villarreal et al. (2004) for F. californiensis. The following were the supplemental compounds used to formulate three experimental diets: β-1.3-glucan from Laminaria digitata (Sigma, Cat. N° L-9634); dl-a-tocopheryl acetate (vitamin E) (Sigma, Cat. N° T-3376) and β-carotene (Sigma, Cat. N° C-9750). The immunos-timulants were supplemented separately to the test diets at doses of 100 mg kg-1 vitamin E (Fernández-Giménez et al., 2004), 100 mg kg1 β-carotene (Flores-Leyva, 2006) and 10 g kg-1 β-1.3-glucan (Chang et al., 2003). All ingredients were thoroughly mixed with cod liver oil and water was added to produce pellet of 2 mm in diameter. Feed was dried at 30°C during 3 h. After drying, the diet was stored at -20°C in dark plastic bags during the experiment.

Proximal analysis of the experimental diets

The different diets were determined in triplicate moisture (AOAC, 1995, N° 930.15), crude protein (AOAC, 1995, N° 976.05), ether extract (AOAC, 1995, N° 920.39), crude fiber (AOAC, 1995, N° 962.09), ash (AOAC, 1995, N° 942.05) and gross energy (cal g-1) in an adiabatic calorimeter (Parr 1261).

Preparation of the WSSV stock solution

Viral extract was prepared following the protocol described by Huang et al. (2001), using shrimp tail muscle that tested positive for WSSV by PCR. Infected tissue was homogenized in TN buffer (Tris-HCl 10 mM, NaCl 400 mM, pH 7.4, 1:5 w/v) and centrifuged at 5,500 x g for 20 min. The supernatant was passed through a 0.45 µm -pore-size filter and then passed through a 0.2 µm -pore size syringe filter (Acrodisc, Pall, Port Washington, NY) to generate aliquots that were stored at -80°C until used. The results of preliminary experiments indicated that the optimal inoculums level of the WSSV stock solution to induce 100% mortality within 72 h in juvenile F. californiensis through intramuscular injection was 20 μL per shrimp.

Experimental protocol

Groups of 30 shrimp were kept in 60 L fiberglass containers. The experiment was designed with four treatments in triplicate: (1) shrimp fed with basal diet without additives (control group); (2) shrimp fed with β-Ο-glucan (10 g kg-1); (3) shrimp fed with vitamin E (100 mg kg-1); (4) shrimp fed with β-carotene (100 mg kg-1). Shrimp were fed twice daily at 8:00 and 17:00 h during 23 days. At the end of the experimental feeding, shrimp from all treatments were injected intramuscularly with 20 μL virus inoculum per shrimp. Four randomly chosen shrimp per treatment were sampled at 0 (before inoculation), 1, 6, 12, 24 and 48 h post-inoculation and stored at -80°C until biochemical analysis. The experimental diets were continued after the inoculation until the end of the experiment, and mortality percentage was determined (López et al., 2003). Pleopods of sampled shrimp were excised and placed in 1.5 mL microcentrifuge tubes and stored at -20°C until WSSV diagnosis by PCR.

WSSV detection by PCR

The PCR reactions were performed according to the technique described by Lo et al. (1996a), and following the recommendations of the manufacturer of commercial kit IQ-2000 (Farming Intelligene Tech Corp, USA).

Antioxidant activity

Frozen hepatopancreas and muscle were thawed, dissected, and 100 mg fragment of each tissue were homogenized with a pestle in 1.5 mL microcentrifuge tubes containing 1mL phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 13,000 x g for 10 min at 4°C. Supernatant was removed and stored at -80°C.

Catalase activity was measured by following the kinetic of reduction of hydrogen peroxide at 240 nm using the extinction coefficient 0.04 mm cm-1 (Downs et al., 2001). The kinetic was determined measuring the absorbance at 240 nm in a BioMate 3 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc. USA). Relative enzyme activity (RCAT) was expressed as the ratio of the specific activity of treated shrimp to that of controls and was used as an index of CAT activity.

Superoxide dismutase activity was determined according the method described by Beauchamp & Fridovich (1971) using nitro blue tetrazolium (NBT) in the presence of riboflavin. The absorbance numbers were input into an in-house generated computer program described elsewhere (Vázquez-Juárez et al., 1993) to calculate specific activity (units per milligram of protein). Relative enzyme activity (RSOD) was expressed as the ratio of the specific activity of treated shrimp to that of controls and was used as an index of SOD activity.

Statistical analysis

One-way analysis of variance (ANOVA) using the F test was applied to analyze the differences among treatments and the control. When ANOVA differences occurred, Tukey post-hoc test was used to identify the nature of these differences. Values were significantly different at P < 0.05.


Proximal analysis of the experimental diets

Table 2 shows the proximal analysis (dry basis) of all treatments. The chemical composition of the six diets was similar, varying only in the inclusion levels of experimental food additives.

Activity of antioxidant enzymes

All shrimp fed with the treatments during 23 days showed a significant (P < 0.05) increase in SOD activity in hepatopancreas at 12 h post-infection (Fig. 1a). RSOD increased significantly (P < 0.05) in muscle at 24 h post-infection with all treatments tested (Fig. 1b). From 12 to 48 h, shrimp exposed to vitamin E and β-Ο-glucan induced significant (P < 0.05) RSOD increase in muscle over the control. Only shrimp fed with β-carotene increased significantly (P < 0.05) SOD activity in muscle than the control group at 0 h (before infection).


Figure 1. a) Relative SOD activity in hepatopancreas, and b) muscle of juvenile F. californiensis infected with WSSV. Error bars: mean ± standard deviation. Significantly different than control (P < 0.05).

Figura 1. a) Actividad relativa de SOD en hepato-pancreas, y b) músculo de juveniles de F. californiensis infectados con WSSV. Barras de error: media ± desviación estándar. *Significativamente diferente al grupo control (P < 0,05).

CAT activity in hepatopancreas increased signifycantly over the control in shrimp fed with vitamin E and β-Ο-glucan before infection (Fig. 2a). Shrimp fed with β-Ο-glucan show high and significant level of RCAT in hepatopancreas at 1 h after infection.


Figure 2. a) Relative CAT activity in hepatopancreas, and b) muscle of juvenile F. californiensis infected with WSSV. Error bars: mean ± standard deviation. Significantly different than control (P < 0.05).

Figura 2. a) Actividad relativa de CAT en hepato-pancreas, y b) músculo de juveniles de F. californiensis infectados con WSSV. Barras de error: media ± desviación estándar. *Significativamente diferente al grupo control (P < 0,05).

Significant increase in RCAT occurred in muscle with all doses tested over the control at 1 and 6 h post-infection (Fig. 2b). Shrimp exposed to β-carotene and vitamin E increased significantly RCAT from 1 to 12 h post-infection. The highest increase of antioxidant response was registered in shrimp exposed to vitamin E (3.5 times than the control) at 12 h post-challenge (Fig. 2b). Table 3 resume SOD and CAT values in hepatopancreas and muscle of juvenile F. californiensis.

Shrimp mortality after WSSV infection

All shrimp fed with the treatments were susceptible to WSSV infection. Clinical signs of infection were presented as reduction in feed intake and lethargy at 6 h post-challenge. PCR results were positive to all infected shrimp. Groups of shrimp fed with vitamin E, β-carotene, and control, showed earliest mortalities at 12 h and β-1.3-glucan at 48 h after the challenge with WSSV (Fig. 3). Feeding juvenile shrimp with β-1.3-glucan and β-carotene showed longest resistance to WSSV infection reaching 90 and 100% of mortality at 144 h, respectively. Organisms fed with vitamin E and the control reached 100% mortality at 72 h after WSSV infection (Fig. 3).


Figure 3. Mortality (%) of juvenile F. californiensis infected with WSSV.

Figura 3. Mortalidad (%) de juveniles de F. califor-niensis infectados con WSSV.


Some authors have reported that the administration of β-glucans enhance the antioxidant and immune response in shrimp (Campa-Córdova et al., 2002; Zhang et al., 2005; Wang et al., 2008). Accumulation of SODs in response to oxidative stress caused by biological agents is one of the main antioxidant defense pathways. Increased levels of SOD have been linked to induce oxidative stress (Fridovich, 1995). Campa-Córdova et al. (2005) exposed juvenile shrimp (Litopenaeus vannamei) to β1.6-glucan by immersion and posterior inoculation with Vibrio penaeicida. They reported increased SOD activity in muscle 2.5 times than the control at 48 h post-infection, similar to our study (Fig. 1b). Glucans are capable of enhancing resistance against various pathogens, including white spot syndrome virus (WSSV) infection in tiger shrimp (Chang et al., 1999), as well as Vibrio damsela infection in post-larvae of black tiger shrimp (Su et al., 1995; Liao et al., 1996). Oral administration of β-glucans has been reported to increase the resistance of Penaeus monodon against to WSSV infection (Chang et al., 2003). However, dose-time interaction effect of glucans has not been investigated so far (Sahoo et al., 2008). Sritunyalucksana et al. (1999) found that P. monodon fed with a diet containing 10 g kg-1 of β1.3-glucan for 20 days increased resistance to WSSV. In this study, shrimp fed with βθ-glucan or β-carotene showed significant (P < 0.05) lowest values of mortality between 24 and 120 h post-infection (Fig. 3). Shrimp fed with vitamin E increased antioxidant response during WSSV infection, but showed low survival (Fig. 3). Fernández-Giménez et al. (2004) recommended 100 mg kg-1 of vitamin E to brown shrimp F. californiensis to improve growth in ponds. However, Lee & Shiau (2004) concluded that 179 mg kg-1 improve growth and immune response in shrimp.

The capacity of shrimp hepatopancreas or muscle to generate antioxidant activity following challenge with WSSV registered indexes from 1.8 to 4.5 times higher than the control. In addition, juvenile shrimp reached maximum antioxidant activity in muscle (Fig. 2b, Table 3). Thus, particular physiological conditions in tissue may cause differences in the capacity of the antioxidant response (Downs et al., 2001). In this study, catalase activity was higher in muscle than in hepatopancreas. In contrast, Tavares-Sánchez et al. (2004) did not find gene expression of catalase in muscle of the shrimp L. vannamei. However, Yang et al. (2010) reported higher catalase activity in muscle than in hepatopancreas of L. vannamei fed with the marine yeast Rhodosporidium paludigenum.

In this study, significant increases in the antioxidant of juvenile F. californiensis fed with β-glucan, vitamin E, or β-carotene were observed, but the antioxidant activities among treatments were different. Shrimp exposed to vitamin E registered a higher response (4.5-fold higher that of control) than shrimp exposed to β-glucan, or β-carotene. The molecular structure, including the molecular weight, and dietary levels of β-glucan, vitamin E, or β- carotene have important role in the biological response (Azad et al, 2007; Sukumaran et al, 2010).

The highest SOD activity in hepatopancreas of juvenile shrimp fed with all treatments was recorded at 12 h post-infection (Fig. 1a), and increased CAT activity with all treatments was recorded in muscle at 1 h post-infection (Fig. 2b). Increased levels of antioxidant activity in cells is related to a rapid detoxifying response and also reflected the important role of SOD and CAT removing excessive reactive oxygen species from cells (Moreno et al., 2005).

Increased SOD and CAT activity was registered before WSSV infection (0 h). Shrimp fed with β-carotene showed significant SOD increase in muscle at day 23 before challenge, and decreased to basal values at 1 h post-challenge. Feeding juvenile shrimp with β-13-glucan and vitamin E during 23 days, showed increased CAT activity in hepatopancreas at 0 h and decreased at 6 h post-infection to basal values. The increase in the antioxidant activity is resulted from upregulated expression of SOD and CAT mRNA (Liu et al., 2007) and protection from oxidative stress and potential pathogens (Lorenzon et al., 2002). However, decreased antioxidant response might indicate an accumulation of superoxide anion radical and consequent oxidative stress in cells and susceptibility to pathogens (Mercier et al., 2006; Mohankumar & Ramasamy, 2006).

This study showed activation of antioxidant defenses generated by feeding β-glucan, vitamin E, and β-carotene in juvenile shrimp F. californiensis during WSSV infection. Additional studies would further the understanding of SOD, CAT, and other antioxidant enzymes in cultivated marine species. Also, assays focusing on appropriate doses and sampling in various tissues help us understand physiological responses by feed additives.


We thank Alejandro Ramos of CIBNOR for providing statistical analyses. Ira Fogel of CIBNOR provided many editorial comments and changes. Funding was provided by the Consejo Nacional de Ciencia y Tecnología (CONACYT Grant 101552 to A.C.). R.P.M. is the recipient of a CONACYT doctoral fellowship.



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Received: 10 June 2011; Accepted: 9 October 2011

Corresponding author: Ángel Campa (

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