Effect of Dietary Potassium on Growth, Nitrogen Metabolism, Osmoregulation and Immunity of Pacific White Shrimp (Litopenaeus vannamei) Reared in Low Salinity Seawater

LIU Hongyu, ZHANG Xinjie, TAN Beiping,, LIN Yingbo, CHI Shuyan, DONG Xiaohui, and YANG Qihui

1) Laboratory of Aquatic Animal Nutrition and Feed, Fisheries College, Guangdong Ocean University, Zhanjiang 524025, P. R. China

2) Department of Oncology and Pathology, Cancer Center Karolinska, R8:04, Karolinska Hospital, SE-171 76 Stockholm, Sweden

Effect of Dietary Potassium on Growth, Nitrogen Metabolism, Osmoregulation and Immunity of Pacific White Shrimp (Litopenaeus vannamei) Reared in Low Salinity Seawater

LIU Hongyu1), ZHANG Xinjie1), TAN Beiping1),*, LIN Yingbo2), CHI Shuyan1), DONG Xiaohui1), and YANG Qihui1)

1) Laboratory of Aquatic Animal Nutrition and Feed, Fisheries College, Guangdong Ocean University, Zhanjiang 524025, P. R. China

2) Department of Oncology and Pathology, Cancer Center Karolinska, R8:04, Karolinska Hospital, SE-171 76 Stockholm, Sweden

An 8 weeks feeding experiment was conducted to determine the effect of dietary potassium on the growth and physiological acclimation of Pacific white shrimp (Litopenaeus vannamei) reared in diluted seawater (salinity 4). Six semi-purified practical diets containing 0.59, 0.96, 1.26, 1.48, 1.74, and 2.17 g potassium K+per 100 g diet were formulated, respectively. The survival and feed conversion rate did not show significant difference among groups of shrimps given these diets (P>0.05). The shrimps fed the diets containing 0.96-1.48 g K+per 100 g diet gained the highest weight, specific growth rate, and protein efficiency ratio. Their ammonium-N excretion rate as well as hemolymph concentration of Na+and Cl−were significantly lower than those of the control (P<0.05), but a reverse trend was observed for their gill Na+/K+-ATPase. Moreover, the shrimps fed with 1.48 g K+per 100 g diet were the highest in hemolymph urea level, and the phenoloxidase and lysozyme activities were significantly higher than those of the control (P<0.05). The growth and physiological response of the test shrimps suggested that diet containing 1.48 g K+per 100 g diet improved the growth of L. vannamei in low-salinity seawater, and enhanced the physiological acclimation of the organism.

Litopenaeus vannamei; potassium; nitrogen metabolism; osmoregulation; immunity

1 Introduction

The Pacific white leg shrimp, Litopenaeus vannamei, is an important commercial crustacean species with high salinity tolerance, which has been bred worldwide. In recent years, rapid advance in aquaculture has enabled the successful commercialization of L. vannamei culture in low-salinity water, which has been reported not only in coastal and inland regions of China (Cheng et al., 2005) but also in United States (Roy et al., 2012), Thailand (Wudtisin et al., 2011), Ecuador (Boyd et al. 2003; Saoud et al., 2003), Mexico (Castillo-Soriano et al., 2010) and other countries with considerable sizes of inland regions. The strong osmoregulation and ion regulation capacity of L. vannamei enables its tolerance of salinities ranging from 0.5 to 40, and even its survival in freshwater (Bray et al., 1994; Saoud et al., 2003).

Many studies on the culture of L. vannamei in seawater of varying salinity (Ponce-Palafox et al., 1997; Sowers et al., 2005; Palacios et al., 2007) and inland low-salinity well water (Saoud et al., 2003; Roy et al., 2010) have been conducted. However, inconsistent or contradictory results about the effects of low salinity on the growth of L. vannamei have prohibited the revealing of this relationship. Martin et al., (2007) reported that L. vannamei maintained in diluted seawater with a salinity of 2 had a greater final weight and a lower feed conversion rate comparing with shrimps maintained in seawaters with salinities 35 and 50. On the other hand, Laramore et al., (2001) found that L. vannamei did not survive in diluted seawater with a salinity below 2 and that the growth of shrimps in salinity 30 after 18–40 d was statistically better comparing with shrimps in 2 and 3. Atwood et al., (2003) obtained the same results. In addition, high mortality and poor growth of L. vannamei reared in low-salinity seawater are still serious problems, and data on the physiological and nutritional requirements for L. vannamei are still limited. Some articles have reported different nutrition requirements for L. vannamei in low-salinity seawater, such as protein (Martin et al., 2007), highly unsaturated fatty acids (Hurtado et al., 2007), calcium (Ca) and phosphorus (P) (Cheng et al., 2006), vitamins (Liu et al., 2007), and among others. The long-term physiological response of shrimps to acclimatization to a low-salinity environment clearly warrants further studies on their physiological and nutritional requirements.

Potassium (K+) is the principal intracellular cation in animals, and it can contribute to in vivo ion regulation, acid-alkaline balance, and basic metabolism (Pequeux et al., 1995). Shiau and Hseih (2001) reported a beneficial effect of dietary K+supplementation for tiger shrimp Penaeus monodon reared in brackish water. In addition to being important for acid–base balance and the maintenance of membrane potentials, K+also plays a key role in osmoregulatory function (Mantel and Farmer, 1983). The osmoregulation capacity of an organism is the main physiological function for crustaceans to acclimatize to a hyposaline environment (Péqueux, 1995). Many studies have shown that the euryhaline shrimp can adapt to the changes of environmental salinity via osmoregulation and ion transport (McGraw et al., 2002). Although the contribution of K+to the total hemolymph osmolality of the shrimp is small (Sower et al., 2006), its role in maintaining Na+/K+-ATPase activity is crucial (Lucu et al., 2003; Huong et al., 2010). An imbalance between K+and Na+concentrations in the hemolymph can cause mortality in prawns (Prangnell and Fotedar, 2005; Zhu et al., 2004; Sowers et al., 2005). Moreover, shrimps held in different seawaters have shown different energy expenditures associated with osmoregulation (Shiau, 1991). Rosas et al. (2001) also reported a depressed growth of L. vannamei cultured with high salinity (40 vs. 16). They observed significantly higher hemolymph ammonia concentrations among animals held in high salinity (7.2 mg L−1). Despite such knowledge, associations between the different levels of dietary K+, nitrogen metabolism, osmoregulation capacity, and immunity remain largely unknown.

The objective of this work was to elucidate the role of dietary K+supplementation in the growth, physiological status, and osmoregulation process of L. vannamei through investigating the effect of various dietary K+levels on the growth performance, oxygen consumption, nitrogen metabolism, and osmoregulation of L. vannamei reared in low-salinity diluted seawater (salinity 4). This study also explored the optimum dietary K+supplementary level for this shrimp species.

2 Material and Methods

2.1 Diet Preparation

Six isoenergetic and isonitrogenous semi-purified diets (Table 1) were formulated to 0%, 0.3%, 0.6%, 0.9%, 1.2% and 1.5% which contained 0.59, 0.96, 1.26, 1.48, 1.74 and 2.17 g K+per 100 g diet, respectively, supplied as potassium chloride (KCl), a corresponding reduction in the amount of cellulose. Vitamin-free casein, gelatin, fish meal and soybean meal were used as the dietary protein source; corn starch was used as the dietary carbohydrate source; fish oil, soy oil and phospholipid oil were used as the dietary lipid source. The actual content of potassium in diet was mensurated with Hitachi Z5000 flame atomic absorption spectrometry. All dried ingredients were mixed with a food mixer (X4-18, Shanghai Food Co. Ltd, Shanghai, China) for 30 min. The ingredients were mixed to facilitate pelleting by bi-bolt plodder (F-26a, South China University of Science and Technology, Guangzhou, China). After pelleting, the diets were dried and stored at−20℃ until used.

Table 1 Composition of the basal diet (dry weight basis)

2.2 Experimental Shrimp and Feeding Trial

The experiment was carried out in Marine Biology Research Base of Guangdong Ocean University, Donghaidao Island Farm (Zhanjiang, Guangdong Province, China). Healthy postlarvae (Hatchery Factory) were held under quarantine conditions for 2 weeks before feeding trial commenced to domesticate them to the experimental conditions. The post larvae were gradually acclimatized from 28 to 4 by adding exposed tap water to the seawater, with salinity reduced by 1–2 daily. The shrimps were given a commercial feed during this period (crude protein 400 g kg–1, crude fat 40 g kg–1, crude fiber 50 g kg–1, lysine 21 g kg–1, methionine 7.5 g kg–1, Zhanjiang Yuehai Feed Co. Ltd, Zhanjiang, China).

Six triplicate groups of shrimps (initial body weight, 0.276 ± 0.01 g) were fed a test diet containing 0.59, 0.96, 1.26, 1.48, 1.74, or 2.17 g K+(100 g diet)–1in 28 cylindri-cal fiberglass tanks (40 shrimps per tank, 300 L capacity, filled with 200 L of diluted seawater, salinity 4) for 8 weeks. The test diets were administered daily at approximately 8% wet body weight at 06:00, 11:00, 17:00, and 22:00, with a daily ration of 30%, 20%, 30%, and 20%, respectively. The amounts of feed were adjusted daily based on the amount of feed consumed by the shrimps within 1.5 h on the previous day. Feces and feed residues were removed, and 20%–50% of the rearing water each tank was replaced daily. The shrimps were starved for 24 h before being weighed and counted every 4 weeks during the feeding trial. The experimental conditions were as follows: water temperature, 28.5℃ ± 2℃; salinity, 4; dissolved oxygen, 10.30–10.50 mg L−1; pH 7.8 ± 0.5.

2.3 Sampling and Chemical Analysis

Sampling (at morning) was done at the 56th day. For hemolymph sampling, the 25 gauge needle was inserted into the ventrolateral sinus of the cephalothorax and quickly expelled into a 1.5 mL centrifuging tube. Samples of the hemolymph were centrifuged at 5000 g at 0–4℃for 15 min. Osmotic pressure of serum was measured in a Fiske Micro-Osmometer (Model 210, USA). The gills were rapidly dissected and placed in 10 volumes of ice-cold homogenization buffer (pH 7.5) containing 250 mmol L−1sucrose, 6 mmol L−1EDTA-Na2, 10 mmol L−1Tris and 0.1% deoxychoic Na. The diced gills were homogenized at 10000 r min−1for 5 min in homogenization buffer using a high-speed homogenizer. After centrifuging the crude extract at 0–2℃ 10000×g for 30 min, the supernatant was centrifuged again under the same condition for 10 min. The final supernatant was kept in a refrigerator at 0–4℃. The Na+/K+-ATPase activity was mensurated in 6 h.

2.4 Shrimp Nutrient Composition and Growth Performance

Standard method AOAC (1995) was used for analyzing feed and shrimp nutrient composition. Moisture and ash content were determined gravimetrically to constant weight in an oven at 105℃ and 550℃, respectively. Crude lipid was determined gravimetrically after extraction with ethyl ether. Crude protein was determined by Kjeldahl method (total-N 6.25) with a FOSS Kjeltec System (2300, Sweden) using boric acid to trap released ammonia.

Determination of the whole shrimp body potassium: Weigh dry samples at 550℃ after dry ashing, with 1% hydrochloric acid solution with Hitachi-Z5000 flame atomic absorption spectrometry.

Growth performance was calculated for all treatments using the formula:

where Wtis the mean final body weight (g) at the time t (days), Wiis the mean initial body weight (g), Wdis dry diet consumed (g), Wpis dietary crude protein (%).

2.5 Nitrogen Metabolism

Three replicate shrimps were used each treatment and control. At the beginning of each test, specimens were deprived of food for 24 h and transferred to individual sealed respiratory chambers of 1 L, filled up with oxygen-saturated water. The initial levels of dissolved oxygen (Do) were measured just before the chambers were closed and water samples were taken to estimate Ammonia–N nitrogen. Then, DOwas measured at intervals of 1 h during the first 4 h and at 24 h, using YSI 55 oxygen meter (Yellow Springs, USA). Ammonia excretion was determined at the beginning and at 24 h. Water sampled from each treatment and control was tested colorimetrically following the Nessler Method (Conover and Corner, 1968).

Oxygen consumption was calculated for all treatments using the following formula:

where C0is the concentration of dissolved oxygen in water (mg L−1) of the control group at the end of the trial; C1is the concentration of dissolved oxygen in water (mg L−1) of the test group at the end of the trial; V is the volume of water (L); W is the shrimp body weight (g); T is the test duration (h).

Nitrogen excretion was calculated for all treatments using the following formula:

where N0is the concentration of ammonia-N in water (mg L−1) of the control group at the end of the trial; N1is the concentration of ammonia-N in water (mg L−1) of the test group at the end of the trial; V is the volume of water (L); W is the shrimp body weight (g); T is the test duration (h).

The analysis of hemolymph urea, uric acid level and arginase activity was followed the methods of Lee and Chen (2003). Two hundred microliters of hemolymph were added in three times volume of ice cold 10% trichloroacetic acid (TCA) solution and centrifuged at 13000×g for 15 min at 4℃ with a centrifuge (Sanyo, Japan) for the measurements of hemolymph ammonia, urea and uric acid. Hemolymph ammonium was measured based on its reaction with a-ketoglutarate and NADH in the presence of glutamate dehydrogenase to form L-glutamate, using a spectrophotometer, Cary 50 Spectrophotometer (Varian, USA) at 340 nm (Bergmeyer and Beutler, 1983). Hemolymph urea was measured based on the addition of diacetylmonoxime to form a pink complex and hydroxylamine using a spectrophotometer at 525 nm following the method of Rahmatullah and Boyde (1980). Hemolymph uric acid was measured with a spectropho-tometer at 520 nm.

2.6 Osmoregulatory Capacity

Hemolymph and rearing water sample (0.1 mL) were seethed and digested with 1mL HClO4at 90℃ respectively, and then the samples were diluted to a final volume of 25 mL with de-ionized water. The Na+and K+concentration were measured with Hitachi Z5000 flame atomic absorption spectrometry and Cl−was measured with automatic biochemistry analyzer (Model: LX-20; Beckman instrument Company, Ltd, USA).

The method of measuring Na+/K+-ATPase was identical to that of Whealty and Henry (1987). Protein concentration of hemolymph was determined by the modified Coomassie brilliant blue method (Bradford et al., 1976) with bovine serum albumin as the standard. Hemocyanin was determined in accordance with the UV/VIS spectral photometric method based on the Nickerson and Van Hold (1971) by Cary 50 Spectrophotometer (Varian, USA). 0.1 mL of hemolymph was diluted with 9.9 mL of distilled water, and the absorbance was measured at 334 nm (characteristic of oxyhemocyanin)

2.7 Immunity

Preparation of hemolymph samples followed the method of Smith and Söderhäll (1991). 100 μL hemolymph was withdrawn from the ventral sinus cavity of each living shrimp with 1-mL sterile syringe (25 gauge) containing 200 μL of anticoagulant solution (100 mmol L−1EDTA-Na2, 450 mmol L−1NaCl, 10 mmol L−1KCl, 10 mmol L−1HEPES, pH 7.3, osmolarity adjusted with glucose to 850 mosm kg−1) (Gollas-Galván et al., 1997). The diluted hemolymph of four shrimp was pooled and then divided into three parts. A drop of the anticoagulanthemolymph mixture was placed on a hemocytometer (Boaco, Hamburg, Germany) to measure THC using an optical microscope under 400 × magnification. Another part of anticoagulant-hemolymph mixture was used for the measurement of respiratory burst activity of hemocytes. The remaining hemolymph mixture was centrifuged at 700× g at 4℃ for 20 min and the supernatant was separated for subsequent tests.

Respiratory burst activity (O2−) of hemocytes was quantified using the reduction of nitroblue tetrazolium (NBT, Sigma) to formazan as a measure of superoxide anion production following the method of Song and Hsieh (1994). The anticoagulant-hemolymph mixture (100 μL) was added to flat-bottomed 96-well microtiter plates with 50 μL poly-lysine (0.2%, Sigma) and centrifuged at 300×g for 10 min at 4℃. After removing the supernatant, 100 μL of phorbol mysistate acetate (1 μg mL−1Sigma) was added and followed to react for 30 min at 37℃. NBT (100 μL, 0.3%, Sigma) was added to the mixture and incubated for 30 min at 37℃. The staining reaction was terminated by removing the NBT solution and adding 200 μL of methanol. After three washes with 70% methanol, the hemocytes were air-dried and dissolved using 120 μL of 2 mol L−1KOH and 140 μL dimethyl sulfoxide (Sigma). The optical densities of dissolved cytoplasmic formazan were measured at 650 nm with a microplate reader (Model multiskan spectrum, Thermo).

Phenoloxidase (PO) activity was measured spectrophotometrically by recording the formation of dopachrome from L-dihydroxyphenylalanine (L-DOPA, Sigma, St. Louis, MO, USA) as previously described (Gollas-Galván et al., 1997) with some modifications. A 0.01 mol L−1solution of L-DOPA was prepared in 0.1 mol L−1-phosphate buffer (12.3 mL 0.2 mol L−1Na2HPO4, 87.7 mL 0.2 mol L−1NaH2PO4, pH 6.0). 100 μL serum and 100 μL L-DOPA was added to a spectroscopy cell (10.0 mm) containing 3 mL of phosphate buffer. After the mixture was agitated, the optical density was measured at 490 nm every 2 min for 60 min. Phenoloxidase activity was determined by the increase of O.D. per minute under the environmental condition.

Using freeze-dried Micrococus lysoleikticus powder (Sigma) as the substrate, lysozyme (LSZ) activity was determined following the procedure described by Li and Gatlin (2003). One hundred mg M. lysodeikticus was suspended in 500 mL of PBS (0.04 mol L−1phosphate buffer solution, pH 6.4). 42 μL serum sample was added to 5 mL of M. lysodeikticus suspension. The absorbance at 540 nm was measured using a UV-Visible Spectrophotometer (Varian, Model Cary 50 Probe). The absorbance (A0) values at 540 nm were recorded after 30 s and 4 min 30 s respectively. Lysozyme activity was calculated as follows:

Superoxide dismutase (SOD) activity was measured with Assay Kit (Nanjing Jiancheng Bioengineering institute, China) by observing the inhibition of ferricytochrome C reduction at 550 nm (Cooper et al., 2002). Aliquots of serum were added to the solution with 50 mmol L−1potassium phosphate buffer (pH 7.8), 50 μmol L−1ferricytochrome C (sigma), and 15 mmol L−1xanthine (Sigma). The xanthine oxidase (0.2 U mL−1, Sigma) was added to initiate the reaction; while the decrease in absorbance was recorded for 5 min. Specific activity is reported as units per milliliter of serum. Alkaline phosphatase (ALP) activity was measured with LX-20 Automatic Biochemical Analyzer (Beckman Instruments Inc).

2.9 Statistical Analysis

Statistical comparisons of experimental data were performed by one-way analysis of variance (ANOVA) and Duncan’s Multiple Range by the software SPSS 13.0 for Windows. The level of statistical significance was declared at P<0.05.

3 Results

3.1 Growth Performance

According to Table 2, there was no significant differ-ence in survival rate (SR) and feed convert rate (FCR) among all groups (P>0.05). With the increase of dietary K+level, the weight gain (WG), special growth rate (SGR) and protein efficiency rate (PER) increased firstly and then decreased significantly (P<0.05). The shrimps fed with 0.96–1.48 g K+(100 g diet)−1displayed the highest WG (3477.5 ± 165.4), SGR (6.39 ± 0.13) and PER (215.72 ± 3.63). There was no significant difference in body protein, moisture, ash and lipid among treatments (Table 3, P>0.05). The body potassium of shrimps fed with 0.96–1.48 g (100 g K+)−1were higher than the other groups significantly (P<0.05).

Table 2 WG, SR, SGR, FCR and PER of Litopenaeus vannamei fed with different potassium levels

Table 3 Body composition of Litopenaeus vannamei fed with the diets supplemented with different potassium levels (dry weight basis)

3.2 Nitrogen Metabolism

As depicted in Table 4, the ammonium-N excretion rate and oxygen/ammonium-N ratio of L. vannamei reared in low salinity seawater were significantly affected by the experimental diets supplemented with different potassium levels (P<0.05). The ammonium-N excretion rate of shrimp fed with 1.48 g (100 g K+)−1was significantly lower than that of control (P<0.05). In addition, the oxygen/ammonium-N ratio of L. vannamei demonstrated a peak with maximum at the 0.9% supplement group (total 1.48 g (100 g K+)−1in diet, P<0.05).

The different effects of experimental diets supplemented with different potassium levels on hemolymph urea, uric acid level and arginase activity were presented in Table 5. The hemolymph urea increased firstly and then decreased with the highest level (2.4 mmol L−1± 0.02 mmol L−1, P<0.05) in shrimps fed with 1.48 g (100 g K+)−1diet.The hemolymph arginase activity of L. vannamei increased significantly with the increase of dietary K+levels (P<0.05). There was no significant difference in hemolymph ammonia and uric acid levels (P>0.05).

Table 4 Oxygen consumption and ammonia-N excretion rates of L. vannamei fed with the diets supplemented with different potassium levels

Table 5 Hemolymph urea, uric acid levels and arginase activities in L. vannamei fed with the diets supplemented with different potassium levels

3.3 Osmoregulatory Capacity

In the low salinity seawater, the osmoregulatory capacity of L. vannamei was affected by the K+supplementary levels according to Table 6. The hemolymph Na+and Cl−levels in shrimps fed with 1.48 g K+per 100 g diet were the lowest among all groups (P<0.05). Meanwhile, the gill Na+-K+-ATPase in shrimps fed with 1.48 g K+per 100 g diet showed the highest activity in all groups (3.49 ± 0.22 µmol Pi (mg protein)−1h−1, P<0.05). There was no significant difference in hemolymph somorality and K+levels (P>0.05).

Table 6 Hemolymph osmolarity, K+, Na+, Cl−concentrations and gill Na+-K+-ATPase activity of L. vannamei fed with the diets supplemented with different potassium levels

3.4 Immunity

As depicted in Table 7, with the increase of K+in diets, the O2−of all groups showed significant difference with a peak occurring in shrimps fed with 1.26 and 1.48 g K+per 100 g diet (P<0.05). The hemocyanin increased originally and then decreased significantly (P<0.05). Moreover, the PO and LSZ activities in shrimps fed with 1.48 g K+per 100 g diet were significantly higher than those of control (P<0.05). Meanwhile, the SOD activity in shrimps fed with 1.26 and 1.48 g K+per 100 g diet was significantly higher than that in the control shrimps (P<0.05), and tended to increase firstly and then decrease with the increase of dietary K+. However, there was no significant difference in THC and ALP activities among treatments (P>0.05).

Table 7 Effect of dietary potassium on total haemocyte count (THC), respiratory burst activity (O2−), hemocyanin concentration, and the activities of phenoloxidase (PO), lysozyme (LSZ), superoxide dismutase (SOD) and alkaline phosphatase (ALP) in Litopenaeus vannamei

4 Discussion

4.1 Growth and Survival

The culture of penaeid shrimp in low-salinity environments have been established and reported to gradually prevail in low-salinity inland or brackish water (Laramore et al., 2001; McGraw et al., 2002). However, the growth, survival and optimal nutritional requirement of shrimps have been challenged under a rather unusual culture environment (Atwood et al., 2003; Samocha et al., 1998; Gong et al., 2004). In the present study, survival and feed conversion rate did not significantly differ among groups (Table 2). However, the shrimps fed the diet containing 1.48 g K+per 100 g diet displayed the highest weight gain (3477.5 ± 165.4), specific growth rate (6.39 ± 0.13), and protein efficiency ratio (215.72 ± 3.63). Davis et al. (2005) reported that K+supplementation positively affected the survival and growth of the shrimps reared in low-salinity well water (salinity 4); while the survival of the control shrimps were significantly lower. The results may be attributed to the fact that the diluted seawater used in the present study had an appropriate ion ratio that was similar to normal seawater (diluted seawater: Na+, 1688.37 mg L−1; K+, 44.16 mg L−1; Mg2+, 58.26 mg L−1; Ca2+, 20.37 mg L−1; Na+/K+, 38.23; Mg2+/Ca2+, 2.86) (Saoud et al., 2003). Similar findings were reported by Roy et al. (2010) and Zhu et al. (2004), who conducted experiments in low-salinity well water and in seawater with varying salinity, respectively. In addition, Cheng et al. (2005) studied the response of juvenile L. vannamei to dietary Ca and P in low-salinity seawater (2) and found that the low-salinity environment significantly affected tissue mineralization. The effect of K+on shrimps showed the same effect in the present study. The growth performance of the shrimps in this study strongly suggests that supplementation of diets with 0.3%–0.9% K+(total 0.96–1.48 g K+(100 g diet)−1) may help L. vannamei to survive and grow better in low-salinity water.

4.2 Nitrogen Metabolism

The oxygen consumption rate of crustaceans, which is combined with the nitrogen excretion rate, mainly ammonium (Taboadal et al., 1998), appropriately reflects the respiratory capacity and estimated metabolic rate. The oxygen uptake/nitrogen excretion (O/N) ratio is an important indicator of metabolic shifts and the amount of energy available. It characterizes the diverse demands of an organism under several environmental conditions (Rosas et al., 2001) and nutrient status (Wang et al., 2006). As Mayzaud et al. (1988) have pointed out, an O/N ratio of 3:16 indicates that proteins have been used, whereas higher values are related to mixed substrates (50% protein and 50% lipid with an O/N ratio of 50:60). In the present study, the oxygen consumption rate of L. vannamei reared in low-salinity seawater increased depending on the diets supplemented with different K+levels. Rosas et al. (2001) reported that oxygen consumption rates increased according to a reduction in salinity. Zhang et al. (2009) also reported that low salinity significantly influenced oxygen consumption rates, ammonia-N excretion rates, and the O/N ratio of their test shrimps (P<0.05), among which lower survival and food conversion efficiency were observed. In the current study, with dietary K+levels ranging from 0.59 to 2.17 g per 100 g, the ammonia-N excretion rates and O/N ratios displayed reverse changes. The increase in O/N ratio from 6.65 (control group) to 12.39 (group fed the diet containing 1.48 g K+per 100 g diet), and K+supplementation might cause a decline in protein metabolism.

Nitrogen is excreted mainly as ammonia (60%–70% of total nitrogen (total-N)), amino acid (10%), as well as urea and uric acid (in small amounts) in decapod crustaceans. Ammonia is formed by the catabolism of metabolic amino acids, deamination of amides, and deamination of adenylate following the urine nucleotide cycle (Regnault et al., 1987). Urea is formed through the ornithine-urea cycle, hydrolysis of arginine, and degradation of uric acids in the uricolytic pathway. Uric acid is formed via the degradation of nucleic acids (Chen et al., 1996; Lee et al., 2003). In the present study, significant effects of the experimental diets supplemented with different quantities of K+on hemolymph urea and arginase activity were noted. The effect of salinity on the ammonia-N excretion of shrimps has been previously observed in P. monodon (Chen et al., 1994), M. japonicus (Lee et al., 2003), and L. vannamei (Zhang et al., 2009). Lee et al. (2003) found that significantly higher levels of hemolymph urea and uric acid in combination and the increased arginase activity resulted from the activation of ureogenesis and uricogenesis for M. japonicus in hyperosmotic conditions. The present study showed that supplementation with 0.9% K+(total, 1.48 g per 100 g K+) in the diet might help to activate ureogenesis and uricogenesis in hyposaline acclimation of L. vannamei.

4.3 Osmoregulatory Capacity

As a euryhaline decapod crustacean, L. vannamei in a low-salinity environment can maintain the hemolymph osmotic concentrations by absorbing both sodium and chloride from the environment (Pequeux, 1995). The gill epithelium has been reported to be the primary site of the biochemical adaptations involved in ion transport processes upon hyper-regulation (Lucu et al., 2003). The response of the branchial Na+/K+-ATPase demonstrates this activity as a central component of the ion-regulatory process at the biochemical level in euryhaline crustaceans (reviewed by Lucu et al., 2003). In addition, hemocyanin is not only widely known as a dioxygen transporter, but also displays a remarkable range of functions, including phenoloxidase (PO) activity and acting as an osmolyte and storing protein (Fernando et al., 2008). In the present study, the osmoregulatory capacity of L. vannamei in low-salinity seawater was mostly affected by the diets supplemented with varying K+levels. The gill Na+/K+-ATPase in the shrimps fed with the diet containing 1.48 g/100 g K+was significantly higher than those observed in other groups (3.49 ± 0.22 µmol Pi (mg protein)−1h−1; P<0.05). In the primary phase, the iso-osmotic point of L. vannamei was salinity 25.87 (data not shown), which was calculated from the iso-osmotic line between the hemolymph osmotic pressure and the external medium. Similar studies were conducted by Sower et al., (2006) in L. vannamei and by Tantulo et al., (2006) in P. monodon.

Similar trends between the hemolymph Na+and Cl−concentrations of the diets containing different levels of K+were observed in this study (Table 6). Na+is a predominant cation in hemolymph (Sower et al., 2006), whereas K+is mainly distributed intracellularly (Pequeux, 1995). L. vannamei is hyperosmotic in freshwater or diluted seawater, and Na+can be easily discharged through the Na+leak pathway in the cell epithelium of gills. These data clearly indicate that high Na+/K+-ATPase activity is necessary to absorb enough Na+from ambient media to compensate the lost Na+. On the other hand, Na+/K+-ATPase is the most important ion transporter protein of osmoregulation and mainly locates in the basolateral membrane of the gill epithelium (Lucu et al., 2003). Considering the Na+/K+exchange balance controlled by Na+/K+-ATPase, the amount of K+in hemolymph absorbed from the diet must be in an appropriate scale. The current study showed that the optimum dietary K+requirement of L. vannamei in low-salinity seawater is 1.48 g per 100 g. Similar findings have been reported for P. monodon reared in seawater (salinity 21), with an optimum dietary K+requirement of 1.2%–1.5% (Shiau et al., 2001).

4.4 Immunity

L. vannamei reared in low-salinity environments, especially in intensive farming of high density, shows poor immunity as well as low disease-resistance, which can easily leads to disease outbreaks under stress. Previous studies have demonstrated that the growth performanceand alkaline phosphatase activity of L. vannamei reared in low-salinity seawater are significantly lower than those of shrimps in normal seawater (Liang et al., 2008; Li et al., 2007). Similar to other invertebrates, L. vannamei mainly relies on its nonspecific immunity system to form the first line of defense against invading pathogens, including PO, lysozyme, superoxide dismutase (SOD), respiratory burst activity (O2−), and hemocyanin (Söderhäll and Cerenius, 1992; Roch et al., 1999; Campa-Córdova et al., 2002; Destoumieux et al., 2001; Adachi et al., 2003).

The activated hemocytes also produce extra bactericidal substances, such as superoside anion (O2−) that may increase disease resistance (Song et al., 2003). The immunity enzymes and disease resistance of L. vannamei reduced with the decrease of salinity under the low-salt environmental stress have been reported (Pan et al., 2005; Li et al., 2009). In this study, the PO, lysozyme, and SOD activities in the shrimps fed with the diet containing 1.48 g per 100 g K+were the highest and significantly differed from those of the control shrimps (P<0.05), indicating that dietary supplementation of 0.9% K+(total 1.48 g K+per 100 g diet) might considerably enhance the immunity of L. vannamei. With the increase in dietary K+levels, the O2−levels of the shrimps fed with the diets containing 1.26 and 1.48 g K+per 100 g diet were significantly the highest among all groups (P<0.05). These results revealed that dietary supplementation of 0.6%–0.9% K+(total 1.26–1.48 g K+per 100 g diet) might help to activate SOD activity to remove free radicals and protect shrimps from low-salinity stress (Li et al., 2007). Wang and Chen (2005) also demonstrated that the immunity and resistibility of L. vannamei against Vibrio notably decreased under acute hypo-osmotic stress. The findings of the current study showed that dietary supplementation of 0.9% K+(1.48 g K+per 100 g diet) could enhance the immunity of L. vannamei reared in a low-salinity environment.

Acknowledgements

This work was supported by grants No. 30871928 from the National Natural Science Foundation of China, 201003020 from the Special Fund for Agro-scientific Research in the Public Interest, and by GDUPS (2011) from Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme and High Level Talent Project.

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(Edited by Qiu Yantao)

(Received July 28, 2012; revised October 30, 2012; accepted May 10, 2013)

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

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