Effect of hydrosoluble polysaccharides of Macrocystis pyrifera on physiological and metabolic responses of Litopenaeus vannamei infected with Vibrio campbellii

Efecto de polisacáridos hidrosolubles de Macrocystis pyrifera sobre las respuestas fisiológicas y metabólicas de Litopenaeus vannamei infectado con Vibrio campbellii

Liliana Noemi Sánchez Campos,¹ Fernando Díaz,¹ Alexei Licea,² Ana Denisse Re,¹ M. Leonardo Lizárraga,³ Maricela Flores,⁴ Ricardo González Sánchez¹ and Caroline Tordoya Romero¹

¹ Laboratorio de Ecofisiología de Organismos Acuáticos, Departamento de Biotecnología Marina. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada–Tijuana #3918. Ensenada Baja California, México.

² Laboratorio de Inmunología Molecular. Departamento de Biotecnología Marina. Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE).

⁴ Nutrición y Ecofisiología de Organismos Acuáticos. Instituto de Investigaciones en Ciencias Veterinarias, Universidad Autónoma de Baja California. Av. Obregón y Julián Carrillo s/n Mexicali Baja California México. E–mail: fdiaz@cicese.mx.

Recibido: 21 de abril de 2010
Aceptado: 30 de noviembre de 2010

ABSTRACT

Adult white shrimp Litopenaeus vannamei between 23 and 32 g of wet weight were injected or submerged in a hydrosoluble polysaccharides extract from Macrocystis pyrifera and infected with Vibrio campbellii. The infection decreased the oxygen consumption rate to 24 mg O2 h^–1 kg^–1 w.w., in shrimps of the control group, which were only infected with V. campbellii. Immunestimulated shrimps did not decrease their oxygen consumption rate at any hour p.i. (46 mg O2 h^–1 kg^–1 w.w.) maintaining it similar to the pre–injection group. Glucose level in the hemolymph of V. campbellii infected shrimps at two hours p.i. was significantly higher (p > 0.05) than the level of the pre–injection group. L. vannamei injected with the extract showed a significant decrease (p > 0.05) in their glucose level at 12 hours p.i., but at 24 hours p.i. it returned to normal level. Shrimps submerged in the extract showed no significant glucose level difference (p < 0.05). Lactate concentration in the hemolymph of the pre–injection group was 11.4 mg dL^–1, but adults injected with the extract had the lowest lactate levels throughout the experiment. Shrimps submerged in the extract decreased lactate levels at 6 and 12 hours p.i. but at 24 hours p.i. the level returned to 9.0 mg dL^–1. The total protein concentration in the hemolymph of pre–injection group was 115.3 mg mL^–1, shrimp injected with saline solution showed no significant differences (p < 0.05) compared to basal control; shrimps injected with V. campbellii had the lowest values of total proteins 6 hours p.i (p > 0.05) immunoestimulated shrimps showed an increase in their total proteins levels. This study concluded that administration of extract of M. pyrifera via injection and immersion in adult white shrimp can be used for immunostimulation purposes.

Key words: Oxygen consumption, metabolites, immunostimulation, Vibrio campbellii.

Adultos de camarón blanco Litopenaeus vannamei de entre 23 y 32 g de peso húmedo fueron inyectados o sumergidos en un extracto de polisacáridos hidrosolubles de Macrocystis pyrifera e infectados con Vibrio campbellii. La infección disminuyó la tasa de consumo de oxígeno a 24 mg O2 h^–1 kg^–1 p.h. en los camarones del grupo control infectados con V. campbellii. Los camarones inmunoestimulados vía inyección e inmersión, mantuvieron una tasa de consumo de oxígeno de 46 mg O2 h^–1 kg^–1 p.h., similar a los camarones control. La concentración de glucosa en la hemolinfa de los adultos infectados con V. campbellii a las dos horas p.i. fue significativamente más alto (p > 0.05) que la del grupo control de pre–inyección. Los inyectados con el extracto e infectados con V. campbellii disminuyeron los niveles de glucosa a las 12 horas p.i., pero a las 24 horas p.i. volvió a los niveles basales. Los camarones inmunoestimulados con el extracto vía inmersión no mostraron ninguna diferencia significativa (p < 0.05) en sus niveles de glucosa a lo largo de las 24 horas p.i. La concentración de lactato en la hemolinfa de los camarones del grupo pre–inyección fue de 11.4 mg dL^–1, pero los adultos inyectados con el extracto tuvieron los niveles más bajos durante todo el experimento. L. vannamei sumergidos en el extracto disminuyeron sus niveles de lactato a las 6 y 12 horas p.i. pero a las 24 horas p.i. el nivel regresó a 9 mg dL^–1. La concentración de proteínas totales en la hemolinfa de los camarones del control basal fue de 115.3 mg mL^–1, los organismos inyectados con V. campbellii tuvieron los valores más bajos (p > 0.05) respecto a los del control basal. En los adultos infectados sumergidos e inyectados con el extracto se obtuvieron los valores más altos de proteínas totales en hemolinfa a las 24 horas p.i. Se concluye que la administración del extracto de M. pyrifera vía inyección e inmersión en adultos de camarón blanco puede ser utilizada para propósitos de inmunoestimulación.

Palabras clave: Consumo de oxígeno, metabolitos, inmunoestimulación, Vibrio campbellii.

INTRODUCTION

The Pacific white shrimp Litopenaeus vannamei (Boone, 1931) is distributed along the Pacific coast, from the Sea of Cortez to Peru. It is the most worldwide cultivated penaeid but in Latin America despite being the most profitable aquatic resource, its culture has moved to second place in recent years due to production losses caused by disease–causing viruses and bacteria (FAO, 2008). The sustainability of shrimp farming depends heavily on disease control (Rodríguez & Le Moullac, 2000), however, the shrimp defense system as in other invertebrates, does not allows the use of vaccines because it does not have specificity or immunological memory and lacks immunoglobulins and lymphoid cells (Muñoz et al., 2000; Rodríguez & Le Moullac, 2000; Montaño–Pérez et al., 2005, Huang et al., 2006).

Immunostimulation has been until now the most viable option to enhance disease resistance in shrimp under culture conditions (Berger, 2000, Vargas–Albores, 2002; Rendón & Balcazar, 2003; Smith et al., 2003). Immunoestimulant are extracted from the cell walls of Gram negative bacteria which contain lipopolysaccharides, Gram positive bacteria with peptidoglycan, as well as fungi, yeasts and algae with β–glucans; their role is to simulate a bacterial infection which activates the cellular and humoral defense system of crustaceans (Rendón et al., 2004).

Approximately 80 to 90% of bacteria isolated from penaeid cultures with mass mortalities belong to the genus Vibrio (Pacini), which behaves as an opportunistic pathogen that takes advantage of environmental conditions that affect the host organism, such as climate changes, population density, poor nutrition and water quality (Leaño et al., 1998). Different species of Vibrio affect penaeid shrimps under culture conditions, such as V. harveyi (Johnson & Shunk), V. campbellii (Baumann), V. parahaemolyticus (Fujino), V. vulnificus (Reichelt) and V. alginolyticus (Miyamoto), which are responsible for the so–called “vibriosis” whose principal symptoms in shrimp are whitening of the thoracic region, lethargy, erratic swimming, loss of appetite, intestine inflammation and necrosis of the appendages (Lavilla et al., 1990).

Crustacean gills are not only involved in gas exchange and ionic regulation, they also contribute to the defense system, it is known that they are the main site of foreign particles and bacterial elimination, which, once removed from the hemolymph, they accumulate and are eventually encapsulated in the gills (Martin et al., 2000).

In aquatic animals, oxygen consumption measurement is a standard method to assess the effect of environmental factors such as temperature, salinity, exposure to pollutants, light intensity or dissolved oxygen, and to determine the energy costs associated with the physiological stress imposed on the organisms (Lemos et al., 2001; Altinok & Grizzle, 2003).

Macrocystis pyrifera (Linnaeus) C. Agardh is a brown algae of the order Laminariales commonly called "kelp" which grows in rocky substrates near the coast, at depths not exceeding 40 meters in temperate or cold waters. It is found mainly along the coasts of North and South America, Australia, New Zealand, Norway, Scotland, Japan, Korea and South Africa (Cruz–Suarez et al., 2000). It is one of the brown seaweed that is exploited commercially as a source of alginates in México; its distribution extends from the border with the United States of America to Punta San Hipolito, Baja California Sur. The estimated harvestable biomass (1 m below the sea surface) varies seasonally from 36,520 tons in the winter to 99,626 tons in the summer (Reyes–Tisnado et al., 2004).

The aim of this study was to assess the effect of hydrosoluble polysaccharides extract of Macrocystis pyrifera on adult Litopenaeus vannamei immunostimulated by immersion or injection via and infected with Vibrio campbellii on some physiological and metabolic responses.

MATERIALS AND METHODS

Macrocystis pyrifera blades were collected at La Mision, B.C., México, during the summer of 2007 and a hydrosoluble extract of polysaccharides was prepared based on the methodology described by Yeh et al. (2006).

A total of 270 adult Litopenaeus vannamei shrimps (previously dried with absorbent paper and weighted on an Ohaus Explore balance) ranging from 23 to 32 g of wet weight (w.w.) from the laboratory La Gloria located in Nayarit, México were kept for two months in 2000 L tanks with continuous replacement of seawater (35 ‰), at a temperature of 26 ± 1 °C, with constant aeration and food satiation (Rangen© with 40% protein). A pathogenic strain of V. campbellii was cultured in Zobell marine medium at 28 °C for 18 hours, then harvested and resuspended in saline solution (9 ‰), at a concentration of 1 × 10⁷ colony–forming units cfu mL^–1. Shrimps were injected with 100 µL of this bacterial suspension, the final concentration was 1 × 10⁶ colony–forming units cfu shrimp^–1.

To measure the shrimp’s oxygen consumption rate, a semiopen respirometer with 21 respirometric chambers of 2800 mL each was used; it was connected to a system similar to that described by Díaz et al. (2007). Twenty shrimp were placed individually in 20 respirometric chambers and leaving one chamber without any organism in order to calculate the oxygen consumption of microorganisms present in the water. Water temperature of the entire system was maintained at 26 ± 1 °C using 1000 watts titanium heater connected to programmable temperature controller. The dissolved oxygen concentration was measured by an oximeter YSI 52 B equipped with a polarographic sensor accuracy ± 0.03 mg L^–1, which was inside an acrylic hermetic chamber of 10 mL with adequate stirring magnet, through tubing system to prevent the water sample had direct contact with air. Before closing the chamber, a seawater sample was taken to measure the initial concentration of oxygen, respirometric chambers were closed for 1 hour because according to Stern et al. (1984) is the adequate time in which dissolved oxygen will not decrease below the 30% of saturation, so it does not cause stress on the organisms.

Oxygen consumption rate (OCR) was calculated according to the following equation (Cerezo Valverde et al., 2006; Zheng et al., 2008)

OCR = (C_t – C₀) V / (W × T)

C₀ is the change of the oxygen content, in the blank (control)

V is the volume of the chamber

W and T are the weight of shrimps in kg of wet biomass, and the time duration in hours, respectively.

Lyophilized hydrosoluble polysaccharides extract (6 mg) of M. pyrifera was dissolved in 6 mL of saline solution (9 ‰), and 60 organisms were injected in the abdominal muscle with 100 µL of the prepared extract, the final concentration was 10 µg g^–1; 24 hours later the same shrimps were injected with 100 µL (1 × 10⁶ cfu shrimp^–1) of the bacterial suspension of V. campbellii into abdominal muscle and 20 organisms were placed individually in each respirometric chamber two hours before starting the oxygen consumption measurements which were conducted at two, four, six, eight, 12 and 24 hours post injection (p.i).

For the immersion test, 3.5 g of the lyophilized hydrosoluble polysaccharides extract of M. pyrifera were dissolved in 10 L of seawater; (final concentration of 350 mg L^–1). Sixty L. vannamei adults were kept for 3 hours in aquariums containing the extract with constant aeration. Subsequently, they were injected with 100 µL of the bacterial suspension of V. campbellii (1 × 10⁶ cfu shrimp¹) and 20 organisms were placed individually in each respirometric chamber two hours before the begin of the oxygen consumption measurements which were conducted for two, four, six, eight, 12 and 24 hours p.i.

As control 150 shrimps were used, 60 were injected with 100 µL of saline solution, 20 were placed individually in each respirometric chamber two hours before starting the measurements at two, four, six, eight, 12 and 24 hours p.i. Other 60 shrimps were injected with 100 µL of the bacterial suspension (1 × 10⁶ cfu shrimp^–1), 20 of which were placed inside the respirometric chambers and the oxygen consumption was measured in the same way as described above.

The remaining 30 organisms were used as basal control (preinjection), these organisms were not subjected to any treatment or infection but the oxygen consumption rate was measured with one repetition of the experiment.

Hemolymph was extracted from 8 shrimps from each experimental condition at two, six, and 12, 24 hours after treatment, a 2:1 ratio anticoagulant solution (Vargas–Albores et al., 1993) was used. All samples were centrifuged at 800 g for 3 min at 4 °C, the supernatant was separated for quantification of each metabolite through commercial kits (Lopez et al., 2003; Pascual et al., 2004; Rosas et al., 2004).

The glucose samples were read on a spectrophotometer (Hach) at 520 nm, the lactate at 546 nm and total proteins at 540 nm, finally, the concentration of each metabolite was calculated from respectively standards (Pointe Scientific Inc.).

RESULTS

The oxygen consumption rate of shrimps from the pre–injection control was 46 mg O2 h^–1 kg^–1 w.w. Saline control organisms increased their oxygen consumption rate at two and 12 hours p.i. (62.1 and 63.8 mg O2 h^–1 kg^–1 w.w., respectively) however, this increase was not significantly different (p < 0.05) compared to preinjection control (Fig. 1A).

Shrimps injected with V. campbellii had an oxygen consumption rate increase at two, four and six hours p.i. of 65.6, 67 and 65.4 mg O2 h^–1 kg^–1 w.w. respectively; while at 8 to 12 hours p.i. the oxygen consumption rate initiate to decline (54.3 and 31 mg O₂ h^–1 kg^–1 w.w., respectively), and at 24 hours p.i. the lowest oxygen consumption rate was registered (24.15 mg O2 h^–1 kg^–1 w.w.) (Fig. 1B).

The oxygen consumption rate of pre–injection shrimps and that of those injected with the hydrosoluble polysaccharides extract of M. pyrifera and infected with V. campbellii was not significantly different (two to 24 hr p.i.), with a range of 35.3 to 44.7 mg O2 h^–1 kg^–1 w.w. (p < 0.05) (Fig. 1C).

There were no significant differences (p < 0.05) between the oxygen consumption rate in the pre–injection shrimps and p.i. measurements in organisms that were submerged in the hydrosoluble polysaccharides extract of M. pyrifera and infected with V. campbellii which had an oxygen consumption rate of 43.3 to 59.8 mg O2 h^–1 kg^–1 w.w. (Fig. 1D).

Glucose concentration on the shrimps controls was 10 mg dL^–1. The adults injected with V. campbellii had the highest glucose concentration at two hours p.i. (32.7 mg dL^–1). Shrimps injected with the hydrosoluble polysaccharides extract of M. pyrifera and infected with V. campbellii showed a decrease in hemolymph glucose concentration at 12 hours p.i. (2.3 mg dL^–1), however, at 24 hours p.i. their glucose concentration returned to normal level (11.7 mg dL^–1). Adults exposed to immersion treatment with the hydrosoluble polysaccharides extract of M. pyrifera and infected with V. campbellii showed no significant differences in their hemolymph glucose concentration, compared to pre–injection control shrimps (p < 0.05) (Fig. 2).

Lactate concentration in the hemolymph of pre–injection L. vannamei was 11.4 mg dL^–1. Adults injected with saline solution and V. campbellii showed no significant differences (p < 0.05) over the pre–injection control, however, the organisms injected with the hydrosoluble polysaccharides extract of M. pyrifera and infected, had the lowest concentrations of lactate in the hemolymph ranging from 0.9 to 3.4 mg dL^–1 at 24 hours p.i. Shrimps submerged in the hydrosoluble polysaccharides extract of M. pyrifera had an hemolymph lactate concentration similar to that of pre–injection shrimps. Lactate concentration decreased to 3.8 and 3.1 mg dL^–1 at six and 12 h p.i., and later, at 24 hours p.i. the level returned to the normal concentration of 9.0 mg dL^–1 (Fig. 3).

Total proteins concentration in the hemolymph of pre–injection adults was 115.3 mg mL^–1, shrimp injected with saline showed no significant differences (p < 0.05) compared to the pre–injection organisms, however, the shrimps infected with V. campbellii had the lowest values of total proteins, compared to pre–injection organisms, with a range of 57 to 86 mg mL^–1 (p > 0.05) (Fig. 4).

DISCUSSION

Shrimps injected only with V. campbellii increased their oxygen consumption rate during the first six hours p.i., and decreased their rate later on, Scholnick et al. (2006) reported a similar effect in L. vannamei also infected with V. campbellii. Burnett et al. (2006) studied the effect of the presence of bacteria and hemocytes in the gills of Callinectes sapidus (Rathbun) and proved that oxygen uptake decreased. Martin et al. (1993, 1998, 2000) reported that hemocytes bind to circulating particles in the hemolymph and form nodules which, due to normal mechanical processes, accumulate in the gills. These nodules may affect the ability of the gills to perform their function gas exchange and ion regulation function, causing hypoxia in the infected shrimp.

The formation of these nodules in the gills of shrimp infected with V. campbellii, was probably the cause of oxygen consumption rate had significant reduction at the time lapses (eight, 12 and 24 hours p.i.). The effect of the M. pyrifera extract in the injection and immersion treatments on shrimp challenged with V. campbellii, prevented the reduction in the oxygen consumption rate as the infection progressed over time, as opposed to the results in the control shrimps only injected with the bacteria.

Several authors mention that immunostimulation via immersion improves the immune response of shrimp (Itami et al., 1998; Alabi et al., 1999; Campa– Córdova et al., 2005; Hou & Chen, 2005; Yeh et al., 2006; Fu et al., 2007) and by injection (Cheng et al., 2004; Hou & Chen, 2005; Yeh et al., 2006; Fu et al., 2007), in the present study, both methods of immunostimulation were effective in fighting infection of V. campbellii, in both methods, the organisms resisted the bacterial infection and the presence of these microorganisms did not reduce the oxygen consumption rate in shrimps exposed to hydrosoluble polysaccharides extract via immersion and injection, as not nodules were formed.

Glucose concentration of pre–injection shrimps was similar to that reported by Racotta and Palacios (1998) in the hemolymph of L. vannamei (15 mg dL^–1), who also proved that hemolymph glucose increased in response to the stress of repeated hemolymph sampling. In this study, the control organisms injected only with V. campbellii showed the highest glucose concentration at two hours p.i. due to the stress caused by infection, the hydrosoluble polysaccharides extract used in this study had a positive effect as none of the organisms of the experimental treatments presented abnormal values of glucose concentration, the only change recorded was at 12 hours p.i. in the injection of the extract treatment, however this effect did not persist, as at the following sampling of hemolymph glucose levels remained normal, indicating that all organisms were no stressed, except for the infected with V. campbellii control group at two hours p.i.

Regarding lactate levels in the hemolymph of shrimp, all organisms from treatments and controls remained below the baseline level, which coincided with the values reported by Racotta and Palacios (1998) of 11 mg dL^–1, these authors conclude that despite lactate is being used as an indicator of stress, their results suggest that the hemolymph glucose level, was more sensitive as an indicator of stress in L. vannamei, because lactate levels of juvenile L. vannamei were less affected by manipulation. The use of the hydrosoluble polysaccharides extract by both methods of immunostimulation in the present study, as evidence by the lactate levels, showed that none of the organisms under treatment were under stress, as to raise their glucose and subsequently lactate levels, upon entering the anaerobic cycle of energy production; Hsieh et al. (2008) reported that juveniles of the same species subjected to infection with V. alginolyticus increased their levels of glucose and lactate, which supports our hypothesis that the extract works to maintain those levels at normal values, even while the organisms were under the effects of the infection with V. campbellii.

The basal concentration of total proteins in the hemolymph of pre–injection shrimps was 117.7 mg mL^–1, similar values of 130, 127 and 103 mg mL^–1 have been reported in the hemolymph of L. vannamei by Racotta and Palacios (1998), Rodriguez et al. (2000) and Pascual et al. (2003), respectively. The levels of protein in the hemolymph are affected by nutritional stress, rather than manipulation stress, suggesting that hemolymph protein levels may reflect changes in the shrimp’s health status, including modification of the immune response (Pascual et al., 2003).

Destomieux et al. (2001), Pascual et al. (2004) and Pérez et al. (2006) mentioned that the use of immunostimulants increased the protein levels because of the production of antimicrobial peptides, using hemocyanin as the precursor of these peptides. Crustacean hemocyanin can be processed by a cysteine–type protease to generate antimicrobial peptides under acidic conditions and its production can be induced by injection of lipopolysaccharides and glucans in the organisms (Lee et al., 2003).

ACKNOWLEDGEMENTS

Special thanks to: Guadalupe Vargas from the Molecular Ecology Laboratory of CICESE for all the support with the V. campbellii culture. Jose M. Dominguez and Francisco Javier Ponce from the Drawing Department of CICESE for preparing the figures.

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