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K ratio in seawater on growth and energy budget of juvenile Litopenaeus vannamei
Aquaculture 234 (2004) 485 – 496 www.elsevier.com/locate/aqua-online
Effects of Na/K ratio in seawater on growth and energy budget of juvenile Litopenaeus vannamei Changbo Zhu, Shuanglin Dong *, Fang Wang, Guoqiang Huang Mariculture Research Laboratory, Fisheries College, Ocean University of Qingdao, Qingdao 266003, People’s Republic of China Received 3 July 2003; received in revised form 27 October 2003; accepted 24 November 2003
Abstract The effects of seawater Na/K ratio on growth and energy budget of juvenile Litopenaeus vannamei Boone, 1931 were investigated. Salinity (S = 30 ppt) and the total concentration of sodium and potassium in the experimental water were kept constant. Seven treatments were set: R1, R2, R3c, R4, R5, R6 and R7, the Na/K ratios were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/ mmol), respectively. The shrimp in R7 (Na/K = 187.4) died within 2 weeks after the experiment began. After the 30-day feeding trial, the molting frequency (MF) and feed intake in terms of wet weight (FIw) and energy content (FIe) of the shrimp under the other six treatments were not significantly affected by the different Na/K ratios in the seawater. The final wet weight, weight gain (WG), specific growth rates (SGRw and SGRe) and food conversion efficiencies (FCEd and FCEe) of the shrimp under Treatment R6 were significantly lower than those under the other five treatments. There was a trend that, with all the indices mentioned above, the values of the test shrimp under Treatment R2 was the highest, and the sequence was: R2 (Na/K = 34.1)>R3c (Na/K = 47.3)>R1 (Na/ K = 25.6)>R4 (Na/K = 85.2)>R5 (Na/K = 119.3)>R6 (Na/K = 153.3). In the experiment, the percentages of energy deposited for growth ( G), energy lost for respiration (R), energy lost in excretion (U) and energy lost in exuviae (E) to the energy consumed in food (C) were significantly affected by the different Na/K ratios in seawater, while the percentage of energy lost in feces ( F) to C was not significantly affected by the ratios. The percentage of G to C of the test shrimp under Treatment R6 was significantly lower while the percentage of E to C was significantly higher than those under the other treatments. There were no significant differences among the energy allocations of the test shrimp under Treatment R1, R2, R3c, R4 and R5. According to regression analysis,
* Corresponding author. Tel.: +86-532-2032827; fax: +86-532-2032799. E-mail address: [email protected] (S. Dong). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.11.027
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optimal shrimp growth could be obtained by regulating the Na/K ratio of the seawater to 40 – 43 at a salinity of 30 ppt. D 2004 Elsevier B.V. All rights reserved. Keywords: Na/K ratio; Seawater; Litopenaeus vannamei; Growth; Energy budget
1. Introduction The great tolerance to wide salinity range of some penaeid shrimps, such as Penaeus monodon and Litopenaeus vannamei (Boone, 1931), and the high incidence of epidemic diseases in coastal areas has led to the growth of inland shrimp farming (Boyd, 2001; McIntosh and Fitzsimmons, 2001; Jiang and Gong, 2002). Inland shrimp farming was initiated in Thailand more than a decade ago; it is now undertaken in the United States, Ecuador, Brazil, China and several other countries. Possibly 30– 40% of P. monodon culture in Thailand is in inland ponds (Boyd, 2002). By the end of 2002, this activity was present in nine inland Chinese provinces and the culture area in China has amounted to 14,000 ha (Dong, in press). Saline water for inland shrimp farming can be obtained in two ways. In some areas, aquifers containing naturally saline water exist, and ponds can be filled from wells developed in these aquifers. Where saline water is not available naturally, brine solutions from coastal salt farms or solid salt may be transported to the ponds and mixed with freshwater to provide enough salinity for shrimp culture (Boyd, 2001). In most areas, the latter technique is being much more widely cased by shrimp farmers, which may bring all out significant short-term economic benefits, but at the risk of creating long-term cumulative environmental impacts, including excessive freshwater expenditure, and the salinization of agricultural land and freshwater resources (Flaherty and Vandergeest, 1998; Fegan, 1999; Braaten and Flaherty, 2000, 2001; Jiang and Gong, 2002). There are no such problems in culturing shrimp in the saline ground waters in the inland or coastal low-lying saline-alkaline areas. China has been exploiting and ameliorating the saline-alkaline wetland by using the fish pond-agricultural terrace system in the Yellow River Delta for decades. It is now found that this type of aquaculture could not only reduce the salinization of the soil around the pond area, but also be a good way for the sustainable development of the agricultural economy (Cheng, 1993; Gu, 1994). However, well waters with adequate salinity may be unacceptable for shrimp culture because of low concentrations of potassium, magnesium, or other ions (Boyd, 2002). In the saline-alkaline area of Yellow River Delta in China and some places of New South Wales, Australia, the saline ground waters are both chloride type, and most of their chemical compositions are similar to that of oceanic seawater of the same salinity except potassium in which is 90 – 95% less than similar salinity oceanic seawater (Wang et al., 2001; Allan and Fielder, 2002). Both places have the experience of adding potash to fortify the potassium concentration for shrimp or fish culture (Liu, 2001; Fielder et al., 2001; Allan and Fielder, 2002). It has been reported that the survival of red drum Sciaenops ocellatus was significantly correlated with the Na+/ K+ and K+/Cl ratios of the saline ground water (Forsberg et al., 1996). Yet there are few
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reports on the effects of water Na/K ratio on the growth and other physiological characters of aquatic animals. Therefore, in order to exploit these saline lands, investigating adaptability to water Na/K ratios is a most pressing matter. The aim of the present research was to investigate the relation between the growth and energy budget of L. vannamei and water Na/K ratios, and then consider if Na/K ratio could be used as a water quality index for shrimp ponds in potassium deficient saline areas.
2. Materials and methods 2.1. Source of juvenile L. vannamei and preliminary acclimation Juvenile L. vannamei were obtained from the Jiaozhou Shrimp Farm in Qingdao, People’s Republic of China. When the shrimp were transported to the laboratory, they were stored in three continuously aerated 600-l fiberglass tanks with natural seawater (29 – 31 ppt) to undergo a 10-day preliminary acclimation to the indoor laboratory conditions, during which they were fed ad libitum twice a day (8:00 and 18:00) with commercial shrimp ration pellets (composition: 41.58% crude protein, 8.36% crude lipid, 10.75% ash and 8.74% moisture; energy content: 19.37 kJ/g dry matter). 2.2. Experimental design and artificial seawater preparation In order to eliminate the interference of imbalance from other ions, the experimental water was prepared by adding instant artificial seasalts into fully aerated tap water. The instant artificial seasalts were specially designed and produced by General Sea Salt Factory, Ocean University of Qingdao, in which the sodium and potassium ingredients were precisely compounded so as to keep their total concentration constant while the Na/K ratios varied. The salinity of the newly prepared artificial seawaters was 30 ppt, and pH 8.2 F 0.1. The total concentration of monovalent cations and other ions were kept approximately constant. According to a preliminary acute toxicity test, L. vannamei could just survive in the artificial seawater (S = 30 ppt) within a Na/K ratio range of 25 –204 (mmol/mmol), with a 96-h survival rate above 80%. Therefore, seven treatments were set: R1, R2, R3c, R4, R5, R6 and R7, and the Na/K ratios were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/mmol), respectively. The Na/K ratio of R3c was identical with the value of oceanic seawater, and it was set as control. The concentrations of Na+ and K+ were determined with an inductively coupled plasma-atomic emission spectrophotometer (ICP-OES; VISTA-MPX, VARIAN). The details are given in Table 1. Table 1 Concentrations of Na+, K+(mmol l 1) and Na/K ratios of the experimental artificial seawater (S = 30 ppt) Treatments
R1
R2
R3c
R4
R5
R6
R7
(Na/K ratio) Na+ K+ Na+ + K+
25.6 393.2 15.4 408.6
34.1 398.0 11.7 409.7
47.3 402.0 8.5 410.5
85.2 406.8 4.8 411.5
119.3 408.5 3.4 411.9
153.3 409.5 2.7 412.1
187.4 410.1 2.2 412.3
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2.3. Experimental procedure and acclimation of shrimp At the end of the 10-day preliminary acclimation, 224 juveniles of similar size were selected and transferred into glass aquaria (45 25 30 cm) filled with 30 l artificial seawater (S = 30 ppt) of different Na/K ratio for a 7-day acclimation. Seven treatments, four replicates, in total 28 aquaria were used, each aquarium held eight shrimp. The aquaria were randomly located. In order to prevent the shrimp from jumping out, every aquarium was covered with a mesh cover. The ambient temperature was controlled with an air-conditioner. Aeration was provided continuously and two-thirds of the water volume was exchanged every second day to ensure high water quality. During acclimation and the following experiment, dissolved oxygen was maintained above 6.0 mg/l, pH 8.1 F 0.2, water temperature at 25 F 0.5 jC, and a simulated natural photoperiod (14 light/10 dark) was used. The shrimp were fed ad libitum twice a day (6:00 and 16:00) with the commercial shrimp ration pellets mentioned above. After the 7-day acclimation and 24 h starvation, the shrimp under each treatment were weighted individually. To remove excess moisture, shrimp were blotted dry with paper towel and weighted to the nearest 0.001g using an electronic balance. Then, 22 individuals of similar weight (5.360 F 0.030 g) under each treatment (totally 154) were selected, of which six were sampled and dried in an oven at 65 jC to constant weight, homogenized, and stored at 20 jC to estimate the body composition and energy content of the initial shrimp. The remaining 16 individuals were test shrimp, which were randomly assigned to four aquaria (4 individuals/aquarium) for the following experiment. The rearing conditions were similar to those during the acclimation period. 2.4. Samples collection and analysis During the course of the experiment, the daily food supplied was precisely weighed and recorded. The uneaten feed and feces were collected by siphon within 3 h after each meal. Exuviae (molted exoskeletons) were collected and recorded at all times. The collected uneaten feed, feces and exuviae were dried at 65 jC and kept for further analysis. The experiment lasted for 30 days, from October 11th to November 11th of 2002. At the end of the experiment, all the test shrimp were collected and dried at 65 jC for 48 h. 2.5. The estimation of energy budget The energy content of the shrimp bodies, feed and feces was measured by Parr 1281 Oxygen Bomb Calorimeter. The energy budget was calculated using the following equation for the crustacean energy budget (Petrusewicz and Macfadyen, 1970): C ¼GþF þU þEþR
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where, energy and R, The 1979):
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C is the energy consumed in food; G, the energy deposited for growth; F, the lost in feces, U, the energy lost in excretion; E, the energy lost in exuviae, the energy for respiration. estimation of U was based on the nitrogen budget equation (Levine and Sulkin,
U ¼ ðCN GN FN EN Þ 24 830 where CN is the nitrogen consumed from food; FN , the nitrogen lost in feces; GN, the nitrogen deposited in shrimp body; EN, the nitrogen lost in exuviae; 24 830, the energy content in excreted nitrogen per gram (J/g). The nitrogen contents in shrimp bodies, feed, feces and exuviae were determined by Kjeldahl method. The value of R was calculated as the following energy budget equation: R¼CGF U E 2.6. Calculation and data analysis Weight gain (WG) and molting frequency (MF) were calculated as follows: WGð%Þ ¼ 100ðW2 W1 Þ=W1 MF ¼ ð%day1 Þ ¼ 100Nm =ðNs T Þ specific growth rate (SGRw), feed intake (FIw) in terms of the wet weight were calculated as: SGRw ð%day1 Þ ¼ 100ðlnW2 lnW1 Þ=T FIw ðwt:% body weight day1 Þ ¼ 100C=½T ðW2 þ W1 Þ=2 food conversion efficiency (FCEd) in terms of dry matter was calculated as: FCEd ð%Þ ¼ 100ðW 2V W 1V Þ=C where, W2 and W1 are the final and initial wet body weight of the shrimp; Nm, the number of molts; Ns, the number of shrimp; T, the duration of the experiment; C, the total food consumed; W2V and W1V are the final and initial dried body weights of the shrimp. SGR, FI and FCE in terms of energy content (SGRe, FIe, FCEe) were calculated similarly. Statistics were performed using SPSS 10.0 statistical software (SPSS, 1999). All data were subjected to one-way ANOVA. If significant differences were indicated at the 0.05 level, then Duncan’s multiple range tests were used to test the differences between treatments.
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3. Results 3.1. Survival and molting The survival and molting data of the test shrimp are presented in Table 2. The shrimp under Treatment R7 (Na/K = 187.4) displayed anorexia, low-activity. Death began after 7 days, and all were dead in 16 days, indicating that L. vannamei could only endure the high Na/K ratio seawater for a short time (Treatment R7 was excluded in the following analyses unless specified). The survival of test shrimp under the other six treatments varied from 81.3% to 100%, and mortalities arose from shrimp jumping out when exchanging the water and cannibalism. There were no statistically significant differences in survival among those treatments though survival under Treatment R4 was the highest. According to the result of one-way ANOVA, there were no significant differences among the molting frequencies (MF) of the test shrimp under all of the six treatments ( p>0.05). 3.2. Growth At the beginning of the feeding trial, the body weights of the test shrimp under each treatment were similar (Table 2). At the end of the experiment, the final wet weight of the test shrimp under Treatment R6 was significantly lower than those under treatments with lower Na/K ratios. The test shrimp also showed significant differences in weight gains (WG), in which the test shrimp under Treatment R2 had the highest value and was significantly higher than those under Treatment R5 and R6; and the value under Treatment R6 was significantly lower than those under the other treatments. During the experiment, significant differences also occurred in specific growth rates in terms of wet weight (SGRw) and energy content (SGRe) among treatments. Similar to the results of WG, the highest SGRs occurred under Treatment R2, and the values under Treatment R6 were significantly lower than those under the other treatments. Meanwhile, Table 2 Growth, survival and molting of L. vannamei in the artificial seawater of different Na/K ratios (mean F SE)* Treatmentsy
Body wet weight (g) Initial
Final
R1 R2 R3c R4 R5 R6 R7
5.309 F 0.094 5.237 F 0.048 5.439 F 0.068 5.364 F 0.049 5.400 F 0.100 5.413 F 0.064 5.360 F 0.030
8.212 F 0.113b 8.765 F 0.261b 8.446 F 0.205b 8.262 F 0.214b 7.999 F 0.218b 7.188 F 0.365a –
WG (%)
Survival (%)
MF (% day 1)
54.78 F 2.72bc 67.34 F 4.58c 55.44 F 5.29bc 54.08 F 4.17bc 48.15 F 3.31b 32.67 F 5.74a –
81.3 F 6.3 87.5 F 7.2 93.8 F 6.3 100.0 F 0.0 87.5 F 7.2 87.5 F 12.5 0
7.29 F 0.71 8.75 F 0.54 7.50 F 0.76 8.75 F 0.54 8.81 F 0.83 10.07 F 0.35 –
WG: weight gain; MF: molting frequency. * Values (expressed as mean F SE, n = 4) with different letters in the same column are significantly different from each other ( p < 0.05). y The Na/K ratios of treatments R1, R2, R3c, R4, R5, R6 and R7 were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/mmol), respectively.
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there were no significant differences among the test shrimp under Treatment R1, R2, R3c, R4 and R5 in SGRe, but SGRw under Treatment R5 was significantly lower than that under Treatment R2 (Fig. 1). 3.3. Feed intake Feed intakes in terms of wet weight (FIw) and energy content (FIe) are presented in Fig. 2. It is apparent that FIs under Treatment R2 were the highest, and the values under Treatment R6 were the lowest. However, one-way ANOVA showed that there were no significant differences in FIs among treatments ( p>0.05), which indicated that the Na/K ratios of the artificial seawater had no significant effects on the feed intake of L. vannamei in the experiment. 3.4. Food conversion efficiency Food conversion efficiencies in terms of dry matter (FCEd) and energy content (FCEe) manifested the same tendency: FCE under Treatment R2 was the highest, and the value under Treatment R6 was significantly lower than those under the other treatments, while there were no significant differences in FCEs among the test shrimp under Treatments R1, R2, R3c, R4 and R5 (Fig. 3). 3.5. Energy allocation Table 3 shows the allocation of the energy consumed in food (C) of the test shrimp in artificial seawater of different Na/K ratios. As compiled, except for the energy lost in
Fig. 1. Specific growth rates (SGR) of L. vannamei during the 30-day experiment. R1, R2, R3c, R4, R5 and R6 exhibit the Na/K ratio of 25.6, 34.1, 47.3, 85.2, 119.3 and 153.3, respectively. Means (n = 4) with different letters within the same group are significantly different ( p < 0.05), and bars represent standard errors of the means. The indices SGRw and SGRe indicate specific growth rate in terms of wet weight and energy content, respectively.
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Fig. 2. Feed intakes (FI) of L. vannamei during the 30-day experiment. See legend of Fig. 1 for R1 to R6. Bars represent standard errors of the means (n = 4). The indices FIw and FIe indicate feed intake in terms of wet weight and energy content, respectively.
feces ( F), the other parts of energy allocation of the test shrimp presented significant differences among treatments. The test shrimp under Treatment R6 spent much more energy of the consumed food on respiration and excretion while deposited less energy for
Fig. 3. Food conversion efficiencies (FCE) of L. vannamei during the 30-day experiment. See legend of Fig. 1 for R1 to R6. Means (n = 4) with different letters within the same group are significantly different ( p < 0.05), and bars represent standard errors of the means. The indices FCEd and FCEe indicate food conversion efficiency in terms of dry matter and energy, respectively.
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Table 3 Energy allocation of L. vannamei during the 30-day experiment (mean F SE)* Treatmentsy
G (% C 1)
R (% C 1)
F (% C 1)
U (% C 1)
E z (% C 1)
R1 R2 R3c R4 R5 R6
14.58 F 0.53b 15.57 F 0.45b 14.75 F 0.33b 14.04 F 0.76b 13.48 F 0.94b 7.60 F 2.17a
62.22 F 1.06b 61.73 F 1.53b 64.42 F 0.50ab 64.77 F 0.48ab 62.34 F 1.33b 67.21 F 1.25a
16.00 F 0.90 15.28 F 1.57 13.28 F 0.43 13.35 F 0.57 16.41 F 0.89 16.04 F 0.94
5.83 F 0.10b 5.90 F 0.20b 6.06 F 0.05ab 6.11 F 0.07ab 5.88 F 0.18b 6.53 F 0.22a
1.36 F 0.15b 1.52 F 0.07b 1.49 F 0.19b 1.72 F 0.09b 1.89 F 0.17b 2.62 F 0.26a
* Values (expressed as mean F SE, n = 4) with different letters in the same column are significantly different from each other ( p < 0.05). y The Na/K ratios of treatments R1, R2, R3c, R4, R5 and R6 were 25.6, 34.1, 47.3, 85.2, 119.3 and 153.3 (mmol/mmol), respectively. z C is the energy consumed in food; G, the energy deposited for growth; R, the energy for respiration; F, the energy lost in feces; U, the energy lost in excretion; and E, the energy lost in exuviate.
growth than those shrimp under any other Na/K ratio. In reverse, the shrimp under Treatment R2 deposited the highest percentage of energy for growth and spent less energy on respiration and excretion. Meanwhile, the test shrimp under Treatment R6 spent much more energy in exuviae than the shrimp under any other treatment. However, no significant differences were found in energy allocation among treatments R1, R2, R3c, R4 and R5 ( p>0.05).
4. Discussion Generally, L. vannamei can survive within a salinity range of 2 –78 ppt, and can live normally within a salinity range of 5 –40 ppt (Wang, 2000). According to the present experiment, improper Na/K ratio might be an important limiting factor for the survival and growth of L. vannamei. It could only grow normally in saline water within a limited Na/K ratio range. Fielder et al. (2001) suggested that because salinity of the groundwater could vary and the interactions between ions during animal osmoregulation were complex, it might be more important to consider the ratio of ions rather than the specific concentration of individual ions in the water. In the seven treatments of the present experiment, the absolute concentrations of K+ changed a lot when the Na/K ratio varied. Regardless of the Na/K ratios, the absolute concentrations of K+ of the seven treatments equaled the K+ concentrations of salinities of 54.4, 41.3, 30.0, 16.9, 12.0, 9.5 and 7.8 ppt oceanic seawater. Moreover, it was reported that the growth rates of L. vannamei juveniles (initial mean weight = 1.6 g) at salinity 5 and 15 ppt were significantly greater than at 25 and 35 ppt (Bray et al., 1994), seemingly low K+concentrations (at salinity 5 and 15 ppt) were better for the shrimp. However, the shrimp could hardly survive under Treatment R7, and the growth rates were much better under Treatment R2 and R3c than those under R4, R5 and R6. It is difficult to explain such phenomena merely in terms of K+ concentrations. Similarly, there was no effect of salinity (3 –6 ppt) on survival or growth of red drum S. ocellatus, but its survival in groundwater (S = 15 ppt) was negatively correlated with the ratio of Na+/K+ ( p = 0.02,
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r2 = 0.60) and positively correlated with the ratio of K+/Cl ( p = 0.05, r2 = 0.50) (Forsberg et al., 1996). In the present research, different Na/K ratios in the artificial seawater significantly affected the growth, feed intake and energy allocation of juvenile L. vannamei. The final wet weight and weight gain of the test shrimp under Treatment R2 were both much greater than under any other treatment. Also, R2 had the highest SGRs (even higher than those in the control). Through regression analysis, the correlation of SGRw and Na/K ratio (X) of the artificial seawater was obtained from a non-linear (quadratic) curve: SGRw ¼ 1:4577 þ 0:0043X 5 105 X 2 ðp < 0:001; r2 ¼ 0:567Þ: The maximum value occurred while X = 43. Similarly, the relationship between weight gain (WG) and Na/K ratio (X) of the artificial seawater was WG ¼ 56:1792 þ 0:1611X 0:002X 2 ðp < 0:001; r2 ¼ 0:547Þ; and the maximum value occurred while X = 40.3. Accordingly, juvenile L. vannamei could get the best growth within Na/K ratios of 40– 43 under a salinity of 30 ppt. Such values are lower than the Na/K ratio of oceanic seawater namely 47.3. It might therefore be more favorable for the growth of juvenile L. vannamei if the water K+ concentration was comparatively higher than that of oceanic seawater of the same salinity. Therefore, to some extent, higher K+ concentration or lower Na/K ratio could promote the growth and food conversion efficiency of juvenile L. vannamei. However, when the K+ concentration is too high or Na/K ratio is too low, contrary results occurred. In Treatment R7 of the present experiment where K+ was seriously deficient, the test shrimp displayed anorexia, lowactivity and death. The best growth obtained in Treatment R2 where K+ concentration was higher than that in the oceanic seawater. Similar results were reported in the snapper Pagrus auratus cultured in K+ deficient saline groundwater in New South Wales, Australia (Fielder et al., 2001). No significant differences were demonstrated in feed intake among treatments in the test shrimp, thus the growth of juvenile L. vannamei was affected by Na/K ratios through FCE and the percentage of energy deposited for growth ( G) to the energy consumed in food (C) (see Fig. 3 and Table 3). In the present experiment, the shrimp under Treatment R2 had the highest percentage of G to C, and showed the best growth. On the contrary, the shrimp under Treatment R6 showed the lowest percentage of G to C; as a result the growth was the worst. It was remarkable that the percentage of exuviae (E) to C in the shrimp under Treatment R6 was significantly higher than those under other treatments, which coincided with its high molting frequency (see Table 2). In fact, it was because the Na/K ratio of the artificial seawater in Treatment R6 was so different from the value of the oceanic seawater that stress occurred in the test shrimp. Many factors could enhance molting but have adverse effects on the growth of shrimp (Vijayan and Diwan, 1995), such as low salinity to P. monodon (Allan and Maguire, 1992) and ammonia to Penaeus japonicus (Chen and Kou, 1992). High molting frequency could not only increase the energy expenditure for exuviae, but also affect the entire energy allocation strategy of the animal, for the process of molting involves consumption of oxygen and energy (Water-
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man, 1960; Penkoff and Thurberg, 1982; Cockcroft and Wooldridge, 1985). In conclusion, it was feasible to take Na/K ratio as an index for considering the suitability of potassium deficient saline groundwater for shrimp culture and for the management of water quality. However, because of the complexity and variation of the groundwater chemical compositions, there are still many problems to overcome for exploiting the aquaculture potential of natural saline groundwater.
Acknowledgements This work was funded by the Project under the Major State Basic Research of China (Grant No. G1999012011), the National 10th 5-Year Major Program (Grant No. 2001BA505B) and the State Agriculture Program (Grant No. K2002-16). We thank General Sea Salt Factory, Ocean University of Qingdao, for designing and producing the experimental instant seasalts.
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Levine, D.M., Sulkin, S.D., 1979. Partitioning and utilization of energy during the larval development of the xanthid crab, Rithropanopeus harrisii (Gould). Journal of Experimental Marine Biology and Ecology 40, 247 – 257. Liu, J.M., 2001. Experiment on raising the survival of Chinese shrimp Fenneropenaeus chinensis in saline groundwater. Shandong Fisheries 18 (1), 13 – 14. McIntosh, D., Fitzsimmons, K., 2001. Characterization and evaluation of effluent from an inland shrimp farm as an irrigation source. Book of Abstracts-Aquaculture 2001 (423 pp.). Penkoff, S.J., Thurberg, F.P., 1982. Changes in oxygen consumption of the American lobster Homarus americanus during the molt cycle. Comparative Biochemistry and Physiology 72, 621 – 622. Petrusewicz, K., Macfadyen, A., 1970. Productivity of Terrestrial Animals: Principles and Methods. IBP Handbook, vol. 13. Blackwell, Oxford. 190 pp. SPSS Inc., 1999. SPSS for Windows, release 10.0.1 Standard Version SPSS Inc, Chicago, Illinois, USA. Vijayan, K.K., Diwan, A.D., 1995. Influence of temperature, salinity, pH and light on molting and growth in the Indian white prawn Penaeus indicus (Crustacea: Decapoda: Penaeidae) under laboratory conditions. Asian Fisheries Science 8, 63 – 72. Wang, G.J., 2000. Biology and rearing technology of Litopenaeus vannamei. Chinese Journal of Fishery Science and Technology 85, 17 – 20. Wang, S.S., Wang, R.X., Chen, S.L., 2001. Effects of K+concentration on survival of Chinese shrimp Fenneropenaeus chinensis. China Fisheries 4, 53 – 55. Waterman, T.H. (Ed.), 1960. The Physiology of Crustacea. Metabolism and Growth, vol. 1. Academic Press, London. 670 pp.
Effects of Na/K ratio in seawater on growth and energy budget of juvenile Litopenaeus vannamei Changbo Zhu, Shuanglin Dong *, Fang Wang, Guoqiang Huang Mariculture Research Laboratory, Fisheries College, Ocean University of Qingdao, Qingdao 266003, People’s Republic of China Received 3 July 2003; received in revised form 27 October 2003; accepted 24 November 2003
Abstract The effects of seawater Na/K ratio on growth and energy budget of juvenile Litopenaeus vannamei Boone, 1931 were investigated. Salinity (S = 30 ppt) and the total concentration of sodium and potassium in the experimental water were kept constant. Seven treatments were set: R1, R2, R3c, R4, R5, R6 and R7, the Na/K ratios were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/ mmol), respectively. The shrimp in R7 (Na/K = 187.4) died within 2 weeks after the experiment began. After the 30-day feeding trial, the molting frequency (MF) and feed intake in terms of wet weight (FIw) and energy content (FIe) of the shrimp under the other six treatments were not significantly affected by the different Na/K ratios in the seawater. The final wet weight, weight gain (WG), specific growth rates (SGRw and SGRe) and food conversion efficiencies (FCEd and FCEe) of the shrimp under Treatment R6 were significantly lower than those under the other five treatments. There was a trend that, with all the indices mentioned above, the values of the test shrimp under Treatment R2 was the highest, and the sequence was: R2 (Na/K = 34.1)>R3c (Na/K = 47.3)>R1 (Na/ K = 25.6)>R4 (Na/K = 85.2)>R5 (Na/K = 119.3)>R6 (Na/K = 153.3). In the experiment, the percentages of energy deposited for growth ( G), energy lost for respiration (R), energy lost in excretion (U) and energy lost in exuviae (E) to the energy consumed in food (C) were significantly affected by the different Na/K ratios in seawater, while the percentage of energy lost in feces ( F) to C was not significantly affected by the ratios. The percentage of G to C of the test shrimp under Treatment R6 was significantly lower while the percentage of E to C was significantly higher than those under the other treatments. There were no significant differences among the energy allocations of the test shrimp under Treatment R1, R2, R3c, R4 and R5. According to regression analysis,
* Corresponding author. Tel.: +86-532-2032827; fax: +86-532-2032799. E-mail address: [email protected] (S. Dong). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.11.027
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optimal shrimp growth could be obtained by regulating the Na/K ratio of the seawater to 40 – 43 at a salinity of 30 ppt. D 2004 Elsevier B.V. All rights reserved. Keywords: Na/K ratio; Seawater; Litopenaeus vannamei; Growth; Energy budget
1. Introduction The great tolerance to wide salinity range of some penaeid shrimps, such as Penaeus monodon and Litopenaeus vannamei (Boone, 1931), and the high incidence of epidemic diseases in coastal areas has led to the growth of inland shrimp farming (Boyd, 2001; McIntosh and Fitzsimmons, 2001; Jiang and Gong, 2002). Inland shrimp farming was initiated in Thailand more than a decade ago; it is now undertaken in the United States, Ecuador, Brazil, China and several other countries. Possibly 30– 40% of P. monodon culture in Thailand is in inland ponds (Boyd, 2002). By the end of 2002, this activity was present in nine inland Chinese provinces and the culture area in China has amounted to 14,000 ha (Dong, in press). Saline water for inland shrimp farming can be obtained in two ways. In some areas, aquifers containing naturally saline water exist, and ponds can be filled from wells developed in these aquifers. Where saline water is not available naturally, brine solutions from coastal salt farms or solid salt may be transported to the ponds and mixed with freshwater to provide enough salinity for shrimp culture (Boyd, 2001). In most areas, the latter technique is being much more widely cased by shrimp farmers, which may bring all out significant short-term economic benefits, but at the risk of creating long-term cumulative environmental impacts, including excessive freshwater expenditure, and the salinization of agricultural land and freshwater resources (Flaherty and Vandergeest, 1998; Fegan, 1999; Braaten and Flaherty, 2000, 2001; Jiang and Gong, 2002). There are no such problems in culturing shrimp in the saline ground waters in the inland or coastal low-lying saline-alkaline areas. China has been exploiting and ameliorating the saline-alkaline wetland by using the fish pond-agricultural terrace system in the Yellow River Delta for decades. It is now found that this type of aquaculture could not only reduce the salinization of the soil around the pond area, but also be a good way for the sustainable development of the agricultural economy (Cheng, 1993; Gu, 1994). However, well waters with adequate salinity may be unacceptable for shrimp culture because of low concentrations of potassium, magnesium, or other ions (Boyd, 2002). In the saline-alkaline area of Yellow River Delta in China and some places of New South Wales, Australia, the saline ground waters are both chloride type, and most of their chemical compositions are similar to that of oceanic seawater of the same salinity except potassium in which is 90 – 95% less than similar salinity oceanic seawater (Wang et al., 2001; Allan and Fielder, 2002). Both places have the experience of adding potash to fortify the potassium concentration for shrimp or fish culture (Liu, 2001; Fielder et al., 2001; Allan and Fielder, 2002). It has been reported that the survival of red drum Sciaenops ocellatus was significantly correlated with the Na+/ K+ and K+/Cl ratios of the saline ground water (Forsberg et al., 1996). Yet there are few
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reports on the effects of water Na/K ratio on the growth and other physiological characters of aquatic animals. Therefore, in order to exploit these saline lands, investigating adaptability to water Na/K ratios is a most pressing matter. The aim of the present research was to investigate the relation between the growth and energy budget of L. vannamei and water Na/K ratios, and then consider if Na/K ratio could be used as a water quality index for shrimp ponds in potassium deficient saline areas.
2. Materials and methods 2.1. Source of juvenile L. vannamei and preliminary acclimation Juvenile L. vannamei were obtained from the Jiaozhou Shrimp Farm in Qingdao, People’s Republic of China. When the shrimp were transported to the laboratory, they were stored in three continuously aerated 600-l fiberglass tanks with natural seawater (29 – 31 ppt) to undergo a 10-day preliminary acclimation to the indoor laboratory conditions, during which they were fed ad libitum twice a day (8:00 and 18:00) with commercial shrimp ration pellets (composition: 41.58% crude protein, 8.36% crude lipid, 10.75% ash and 8.74% moisture; energy content: 19.37 kJ/g dry matter). 2.2. Experimental design and artificial seawater preparation In order to eliminate the interference of imbalance from other ions, the experimental water was prepared by adding instant artificial seasalts into fully aerated tap water. The instant artificial seasalts were specially designed and produced by General Sea Salt Factory, Ocean University of Qingdao, in which the sodium and potassium ingredients were precisely compounded so as to keep their total concentration constant while the Na/K ratios varied. The salinity of the newly prepared artificial seawaters was 30 ppt, and pH 8.2 F 0.1. The total concentration of monovalent cations and other ions were kept approximately constant. According to a preliminary acute toxicity test, L. vannamei could just survive in the artificial seawater (S = 30 ppt) within a Na/K ratio range of 25 –204 (mmol/mmol), with a 96-h survival rate above 80%. Therefore, seven treatments were set: R1, R2, R3c, R4, R5, R6 and R7, and the Na/K ratios were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/mmol), respectively. The Na/K ratio of R3c was identical with the value of oceanic seawater, and it was set as control. The concentrations of Na+ and K+ were determined with an inductively coupled plasma-atomic emission spectrophotometer (ICP-OES; VISTA-MPX, VARIAN). The details are given in Table 1. Table 1 Concentrations of Na+, K+(mmol l 1) and Na/K ratios of the experimental artificial seawater (S = 30 ppt) Treatments
R1
R2
R3c
R4
R5
R6
R7
(Na/K ratio) Na+ K+ Na+ + K+
25.6 393.2 15.4 408.6
34.1 398.0 11.7 409.7
47.3 402.0 8.5 410.5
85.2 406.8 4.8 411.5
119.3 408.5 3.4 411.9
153.3 409.5 2.7 412.1
187.4 410.1 2.2 412.3
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2.3. Experimental procedure and acclimation of shrimp At the end of the 10-day preliminary acclimation, 224 juveniles of similar size were selected and transferred into glass aquaria (45 25 30 cm) filled with 30 l artificial seawater (S = 30 ppt) of different Na/K ratio for a 7-day acclimation. Seven treatments, four replicates, in total 28 aquaria were used, each aquarium held eight shrimp. The aquaria were randomly located. In order to prevent the shrimp from jumping out, every aquarium was covered with a mesh cover. The ambient temperature was controlled with an air-conditioner. Aeration was provided continuously and two-thirds of the water volume was exchanged every second day to ensure high water quality. During acclimation and the following experiment, dissolved oxygen was maintained above 6.0 mg/l, pH 8.1 F 0.2, water temperature at 25 F 0.5 jC, and a simulated natural photoperiod (14 light/10 dark) was used. The shrimp were fed ad libitum twice a day (6:00 and 16:00) with the commercial shrimp ration pellets mentioned above. After the 7-day acclimation and 24 h starvation, the shrimp under each treatment were weighted individually. To remove excess moisture, shrimp were blotted dry with paper towel and weighted to the nearest 0.001g using an electronic balance. Then, 22 individuals of similar weight (5.360 F 0.030 g) under each treatment (totally 154) were selected, of which six were sampled and dried in an oven at 65 jC to constant weight, homogenized, and stored at 20 jC to estimate the body composition and energy content of the initial shrimp. The remaining 16 individuals were test shrimp, which were randomly assigned to four aquaria (4 individuals/aquarium) for the following experiment. The rearing conditions were similar to those during the acclimation period. 2.4. Samples collection and analysis During the course of the experiment, the daily food supplied was precisely weighed and recorded. The uneaten feed and feces were collected by siphon within 3 h after each meal. Exuviae (molted exoskeletons) were collected and recorded at all times. The collected uneaten feed, feces and exuviae were dried at 65 jC and kept for further analysis. The experiment lasted for 30 days, from October 11th to November 11th of 2002. At the end of the experiment, all the test shrimp were collected and dried at 65 jC for 48 h. 2.5. The estimation of energy budget The energy content of the shrimp bodies, feed and feces was measured by Parr 1281 Oxygen Bomb Calorimeter. The energy budget was calculated using the following equation for the crustacean energy budget (Petrusewicz and Macfadyen, 1970): C ¼GþF þU þEþR
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where, energy and R, The 1979):
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C is the energy consumed in food; G, the energy deposited for growth; F, the lost in feces, U, the energy lost in excretion; E, the energy lost in exuviae, the energy for respiration. estimation of U was based on the nitrogen budget equation (Levine and Sulkin,
U ¼ ðCN GN FN EN Þ 24 830 where CN is the nitrogen consumed from food; FN , the nitrogen lost in feces; GN, the nitrogen deposited in shrimp body; EN, the nitrogen lost in exuviae; 24 830, the energy content in excreted nitrogen per gram (J/g). The nitrogen contents in shrimp bodies, feed, feces and exuviae were determined by Kjeldahl method. The value of R was calculated as the following energy budget equation: R¼CGF U E 2.6. Calculation and data analysis Weight gain (WG) and molting frequency (MF) were calculated as follows: WGð%Þ ¼ 100ðW2 W1 Þ=W1 MF ¼ ð%day1 Þ ¼ 100Nm =ðNs T Þ specific growth rate (SGRw), feed intake (FIw) in terms of the wet weight were calculated as: SGRw ð%day1 Þ ¼ 100ðlnW2 lnW1 Þ=T FIw ðwt:% body weight day1 Þ ¼ 100C=½T ðW2 þ W1 Þ=2 food conversion efficiency (FCEd) in terms of dry matter was calculated as: FCEd ð%Þ ¼ 100ðW 2V W 1V Þ=C where, W2 and W1 are the final and initial wet body weight of the shrimp; Nm, the number of molts; Ns, the number of shrimp; T, the duration of the experiment; C, the total food consumed; W2V and W1V are the final and initial dried body weights of the shrimp. SGR, FI and FCE in terms of energy content (SGRe, FIe, FCEe) were calculated similarly. Statistics were performed using SPSS 10.0 statistical software (SPSS, 1999). All data were subjected to one-way ANOVA. If significant differences were indicated at the 0.05 level, then Duncan’s multiple range tests were used to test the differences between treatments.
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3. Results 3.1. Survival and molting The survival and molting data of the test shrimp are presented in Table 2. The shrimp under Treatment R7 (Na/K = 187.4) displayed anorexia, low-activity. Death began after 7 days, and all were dead in 16 days, indicating that L. vannamei could only endure the high Na/K ratio seawater for a short time (Treatment R7 was excluded in the following analyses unless specified). The survival of test shrimp under the other six treatments varied from 81.3% to 100%, and mortalities arose from shrimp jumping out when exchanging the water and cannibalism. There were no statistically significant differences in survival among those treatments though survival under Treatment R4 was the highest. According to the result of one-way ANOVA, there were no significant differences among the molting frequencies (MF) of the test shrimp under all of the six treatments ( p>0.05). 3.2. Growth At the beginning of the feeding trial, the body weights of the test shrimp under each treatment were similar (Table 2). At the end of the experiment, the final wet weight of the test shrimp under Treatment R6 was significantly lower than those under treatments with lower Na/K ratios. The test shrimp also showed significant differences in weight gains (WG), in which the test shrimp under Treatment R2 had the highest value and was significantly higher than those under Treatment R5 and R6; and the value under Treatment R6 was significantly lower than those under the other treatments. During the experiment, significant differences also occurred in specific growth rates in terms of wet weight (SGRw) and energy content (SGRe) among treatments. Similar to the results of WG, the highest SGRs occurred under Treatment R2, and the values under Treatment R6 were significantly lower than those under the other treatments. Meanwhile, Table 2 Growth, survival and molting of L. vannamei in the artificial seawater of different Na/K ratios (mean F SE)* Treatmentsy
Body wet weight (g) Initial
Final
R1 R2 R3c R4 R5 R6 R7
5.309 F 0.094 5.237 F 0.048 5.439 F 0.068 5.364 F 0.049 5.400 F 0.100 5.413 F 0.064 5.360 F 0.030
8.212 F 0.113b 8.765 F 0.261b 8.446 F 0.205b 8.262 F 0.214b 7.999 F 0.218b 7.188 F 0.365a –
WG (%)
Survival (%)
MF (% day 1)
54.78 F 2.72bc 67.34 F 4.58c 55.44 F 5.29bc 54.08 F 4.17bc 48.15 F 3.31b 32.67 F 5.74a –
81.3 F 6.3 87.5 F 7.2 93.8 F 6.3 100.0 F 0.0 87.5 F 7.2 87.5 F 12.5 0
7.29 F 0.71 8.75 F 0.54 7.50 F 0.76 8.75 F 0.54 8.81 F 0.83 10.07 F 0.35 –
WG: weight gain; MF: molting frequency. * Values (expressed as mean F SE, n = 4) with different letters in the same column are significantly different from each other ( p < 0.05). y The Na/K ratios of treatments R1, R2, R3c, R4, R5, R6 and R7 were 25.6, 34.1, 47.3, 85.2, 119.3, 153.3 and 187.4 (mmol/mmol), respectively.
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there were no significant differences among the test shrimp under Treatment R1, R2, R3c, R4 and R5 in SGRe, but SGRw under Treatment R5 was significantly lower than that under Treatment R2 (Fig. 1). 3.3. Feed intake Feed intakes in terms of wet weight (FIw) and energy content (FIe) are presented in Fig. 2. It is apparent that FIs under Treatment R2 were the highest, and the values under Treatment R6 were the lowest. However, one-way ANOVA showed that there were no significant differences in FIs among treatments ( p>0.05), which indicated that the Na/K ratios of the artificial seawater had no significant effects on the feed intake of L. vannamei in the experiment. 3.4. Food conversion efficiency Food conversion efficiencies in terms of dry matter (FCEd) and energy content (FCEe) manifested the same tendency: FCE under Treatment R2 was the highest, and the value under Treatment R6 was significantly lower than those under the other treatments, while there were no significant differences in FCEs among the test shrimp under Treatments R1, R2, R3c, R4 and R5 (Fig. 3). 3.5. Energy allocation Table 3 shows the allocation of the energy consumed in food (C) of the test shrimp in artificial seawater of different Na/K ratios. As compiled, except for the energy lost in
Fig. 1. Specific growth rates (SGR) of L. vannamei during the 30-day experiment. R1, R2, R3c, R4, R5 and R6 exhibit the Na/K ratio of 25.6, 34.1, 47.3, 85.2, 119.3 and 153.3, respectively. Means (n = 4) with different letters within the same group are significantly different ( p < 0.05), and bars represent standard errors of the means. The indices SGRw and SGRe indicate specific growth rate in terms of wet weight and energy content, respectively.
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Fig. 2. Feed intakes (FI) of L. vannamei during the 30-day experiment. See legend of Fig. 1 for R1 to R6. Bars represent standard errors of the means (n = 4). The indices FIw and FIe indicate feed intake in terms of wet weight and energy content, respectively.
feces ( F), the other parts of energy allocation of the test shrimp presented significant differences among treatments. The test shrimp under Treatment R6 spent much more energy of the consumed food on respiration and excretion while deposited less energy for
Fig. 3. Food conversion efficiencies (FCE) of L. vannamei during the 30-day experiment. See legend of Fig. 1 for R1 to R6. Means (n = 4) with different letters within the same group are significantly different ( p < 0.05), and bars represent standard errors of the means. The indices FCEd and FCEe indicate food conversion efficiency in terms of dry matter and energy, respectively.
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Table 3 Energy allocation of L. vannamei during the 30-day experiment (mean F SE)* Treatmentsy
G (% C 1)
R (% C 1)
F (% C 1)
U (% C 1)
E z (% C 1)
R1 R2 R3c R4 R5 R6
14.58 F 0.53b 15.57 F 0.45b 14.75 F 0.33b 14.04 F 0.76b 13.48 F 0.94b 7.60 F 2.17a
62.22 F 1.06b 61.73 F 1.53b 64.42 F 0.50ab 64.77 F 0.48ab 62.34 F 1.33b 67.21 F 1.25a
16.00 F 0.90 15.28 F 1.57 13.28 F 0.43 13.35 F 0.57 16.41 F 0.89 16.04 F 0.94
5.83 F 0.10b 5.90 F 0.20b 6.06 F 0.05ab 6.11 F 0.07ab 5.88 F 0.18b 6.53 F 0.22a
1.36 F 0.15b 1.52 F 0.07b 1.49 F 0.19b 1.72 F 0.09b 1.89 F 0.17b 2.62 F 0.26a
* Values (expressed as mean F SE, n = 4) with different letters in the same column are significantly different from each other ( p < 0.05). y The Na/K ratios of treatments R1, R2, R3c, R4, R5 and R6 were 25.6, 34.1, 47.3, 85.2, 119.3 and 153.3 (mmol/mmol), respectively. z C is the energy consumed in food; G, the energy deposited for growth; R, the energy for respiration; F, the energy lost in feces; U, the energy lost in excretion; and E, the energy lost in exuviate.
growth than those shrimp under any other Na/K ratio. In reverse, the shrimp under Treatment R2 deposited the highest percentage of energy for growth and spent less energy on respiration and excretion. Meanwhile, the test shrimp under Treatment R6 spent much more energy in exuviae than the shrimp under any other treatment. However, no significant differences were found in energy allocation among treatments R1, R2, R3c, R4 and R5 ( p>0.05).
4. Discussion Generally, L. vannamei can survive within a salinity range of 2 –78 ppt, and can live normally within a salinity range of 5 –40 ppt (Wang, 2000). According to the present experiment, improper Na/K ratio might be an important limiting factor for the survival and growth of L. vannamei. It could only grow normally in saline water within a limited Na/K ratio range. Fielder et al. (2001) suggested that because salinity of the groundwater could vary and the interactions between ions during animal osmoregulation were complex, it might be more important to consider the ratio of ions rather than the specific concentration of individual ions in the water. In the seven treatments of the present experiment, the absolute concentrations of K+ changed a lot when the Na/K ratio varied. Regardless of the Na/K ratios, the absolute concentrations of K+ of the seven treatments equaled the K+ concentrations of salinities of 54.4, 41.3, 30.0, 16.9, 12.0, 9.5 and 7.8 ppt oceanic seawater. Moreover, it was reported that the growth rates of L. vannamei juveniles (initial mean weight = 1.6 g) at salinity 5 and 15 ppt were significantly greater than at 25 and 35 ppt (Bray et al., 1994), seemingly low K+concentrations (at salinity 5 and 15 ppt) were better for the shrimp. However, the shrimp could hardly survive under Treatment R7, and the growth rates were much better under Treatment R2 and R3c than those under R4, R5 and R6. It is difficult to explain such phenomena merely in terms of K+ concentrations. Similarly, there was no effect of salinity (3 –6 ppt) on survival or growth of red drum S. ocellatus, but its survival in groundwater (S = 15 ppt) was negatively correlated with the ratio of Na+/K+ ( p = 0.02,
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r2 = 0.60) and positively correlated with the ratio of K+/Cl ( p = 0.05, r2 = 0.50) (Forsberg et al., 1996). In the present research, different Na/K ratios in the artificial seawater significantly affected the growth, feed intake and energy allocation of juvenile L. vannamei. The final wet weight and weight gain of the test shrimp under Treatment R2 were both much greater than under any other treatment. Also, R2 had the highest SGRs (even higher than those in the control). Through regression analysis, the correlation of SGRw and Na/K ratio (X) of the artificial seawater was obtained from a non-linear (quadratic) curve: SGRw ¼ 1:4577 þ 0:0043X 5 105 X 2 ðp < 0:001; r2 ¼ 0:567Þ: The maximum value occurred while X = 43. Similarly, the relationship between weight gain (WG) and Na/K ratio (X) of the artificial seawater was WG ¼ 56:1792 þ 0:1611X 0:002X 2 ðp < 0:001; r2 ¼ 0:547Þ; and the maximum value occurred while X = 40.3. Accordingly, juvenile L. vannamei could get the best growth within Na/K ratios of 40– 43 under a salinity of 30 ppt. Such values are lower than the Na/K ratio of oceanic seawater namely 47.3. It might therefore be more favorable for the growth of juvenile L. vannamei if the water K+ concentration was comparatively higher than that of oceanic seawater of the same salinity. Therefore, to some extent, higher K+ concentration or lower Na/K ratio could promote the growth and food conversion efficiency of juvenile L. vannamei. However, when the K+ concentration is too high or Na/K ratio is too low, contrary results occurred. In Treatment R7 of the present experiment where K+ was seriously deficient, the test shrimp displayed anorexia, lowactivity and death. The best growth obtained in Treatment R2 where K+ concentration was higher than that in the oceanic seawater. Similar results were reported in the snapper Pagrus auratus cultured in K+ deficient saline groundwater in New South Wales, Australia (Fielder et al., 2001). No significant differences were demonstrated in feed intake among treatments in the test shrimp, thus the growth of juvenile L. vannamei was affected by Na/K ratios through FCE and the percentage of energy deposited for growth ( G) to the energy consumed in food (C) (see Fig. 3 and Table 3). In the present experiment, the shrimp under Treatment R2 had the highest percentage of G to C, and showed the best growth. On the contrary, the shrimp under Treatment R6 showed the lowest percentage of G to C; as a result the growth was the worst. It was remarkable that the percentage of exuviae (E) to C in the shrimp under Treatment R6 was significantly higher than those under other treatments, which coincided with its high molting frequency (see Table 2). In fact, it was because the Na/K ratio of the artificial seawater in Treatment R6 was so different from the value of the oceanic seawater that stress occurred in the test shrimp. Many factors could enhance molting but have adverse effects on the growth of shrimp (Vijayan and Diwan, 1995), such as low salinity to P. monodon (Allan and Maguire, 1992) and ammonia to Penaeus japonicus (Chen and Kou, 1992). High molting frequency could not only increase the energy expenditure for exuviae, but also affect the entire energy allocation strategy of the animal, for the process of molting involves consumption of oxygen and energy (Water-
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man, 1960; Penkoff and Thurberg, 1982; Cockcroft and Wooldridge, 1985). In conclusion, it was feasible to take Na/K ratio as an index for considering the suitability of potassium deficient saline groundwater for shrimp culture and for the management of water quality. However, because of the complexity and variation of the groundwater chemical compositions, there are still many problems to overcome for exploiting the aquaculture potential of natural saline groundwater.
Acknowledgements This work was funded by the Project under the Major State Basic Research of China (Grant No. G1999012011), the National 10th 5-Year Major Program (Grant No. 2001BA505B) and the State Agriculture Program (Grant No. K2002-16). We thank General Sea Salt Factory, Ocean University of Qingdao, for designing and producing the experimental instant seasalts.
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