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Changes of protein-bound and free amino acids in the muscle of the freshwater prawn Macrobrachium nipponense in different salinities
Aquaculture 233 (2004) 561 – 571 www.elsevier.com/locate/aqua-online
Changes of protein-bound and free amino acids in the muscle of the freshwater prawn Macrobrachium nipponense in different salinities Wei-Na Wang a,b,*, An-Li Wang a, Lai Bao c, Jian–Ping Wang c, Yuan Liu a, Ru-Yong Sun a,b a
College of Life Science, South China Normal University, Guangzhou 510631, PR China b College of Life Science, Beijing Normal University, Beijing 100875, PR China c College of Life Science, Hebei University, Baoding 071002, PR China
Received 16 November 2001; received in revised form 16 September 2003; accepted 24 September 2003
Abstract Growth, hemolymph osmolality, tissue water, ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) ratio, protein-bound amino acids and free amino acids (FAA) of Macrobrachium nipponense were investigated, after they were acclimated to 0x , 7x, 14xand 20xfor 14 days. Growth was significantly ( P < 0.05) influenced by salinity. The highest weight gain of prawns ( P < 0.05) was achieved at a salinity of 14x . The RNA/DNA ratio, total protein-bound amino acid (TAA) and FAA concentrations in the muscle of prawns increased by hyperosmotic stress. The osmolality of prawns haemolymph increased slightly in salinities from 0xto14x . A sharp increase in haemolymph osmotic concentration occurs between 14xand 21x , the isosmotic point being reached at 15x (450 mOsm). It is noted that the relationship of total FAA concentration in muscle and salinity in the range of 0 – 20xrevealed a positive linear correlation. The correlation coefficient (R2) is 0.98. During adaptation, the FAA pool (mainly glycine, alanine and proline) of muscle seems to be directly related to osmoregulation, while the percentage change in alanine in prawns transferred from freshwater to diluted seawater (20x ) was most pronounced (116% approx.). These results are compared with those of M. rosenbergii. D 2004 Elsevier B.V. All rights reserved. Keywords: Macrobrachium nipponense; Salinity; Hemolymph osmolality; Amino acids; Nucleic acids
* Corresponding author. College of Life Science, South China Normal University, Guangzhou 510631, PR China. E-mail address: [email protected] (W.-N. Wang). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.09.042
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1. Introduction Crustacean muscle contains high concentrations of free amino acids, particularly glycine, proline, arginine, glutamate and alanine (Simpson et al., 1959; Schoffeniels and Gilles, 1970; Cobb et al., 1975; D’Aniello, 1980). It has been shown that the rapid and qualitatively important adjustments were necessary for osmoregulation following changes in environmental salinity (Bishop and Burton, 1993). The changes of free amino acid (FAA) pool sizes with environmental salinity have been widely documented in Crustacea. However, comparatively little is known about the regulatory patterns and influence of salinity on the contents of protein-bound and free amino acids in the muscle of the prawn Macrobrachium nipponense (de Haan), a commercially important species, found throughout brackish and freshwaters from North China to Annam, Japan and Taiwan (Holthuis, 1980; Uno, 1971; Wang et al., 1997). Ortmann (1902) pointed out that Macrobrachium was a relatively recent genus apparently in the process of emigrating to freshwater. Comparison of the adult osmoregulatory patterns displayed within the genus Macrobrachium may indicate the evolutionary sequence of the development of osmoregulation in the colonisation of freshwater. The free amino acids have been shown to function as osmoregulators in crustaceans (Fang et al., 1992; Boone and Claybrook, 1977), and they also are a major contributor to the flavor of seafoods (Hashimoto, 1965; Jones, 1969; Thompson et al., 1980). Changes of environmental salinity may possibly be utilized to produce freshwater prawns with different flavor characteristics. In addition, the amino acid composition and concentration in the muscle of prawns may affect the muscle quality of the prawn. We determined the effect of salinity on hemolymph osmolality and the protein-bound amino acid and free amino acid concentrations in the muscle of prawns and studied the role of FAA in intracellular osmoregulation during acclimation from freshwater to diluted seawater. These data are compared with measurements for M. rosenbergii. The relationship between the different salinities and the quality of the prawn muscle was also investigated. The ratio of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) has been known to be very sensitive to changes in feeding levels and short-term growth in adult freshwater fish (Haines, 1973; Bulow, 1970; Buckley, 1979). A reduction in RNA/DNA ratios in response to starvation was observed in postlarvae of the lobster Homarus americanus (Jumio et al., 1992), the crab Callinectes sapidus (Wang and Stickle, 1986), and juvenile Penaeus vannamei (Moss, 1994). Stuck et al. (1996) considered the RNA/DNA ratio to be a useful indicator of prolonged nutritional stress in postlarval P. vannamei. The present study was initiated to determine the changes in the RNA/DNA ratio in muscle of prawns, M. nipponense during the acclimation from freshwater to diluted seawater in order to define the effects of hyperosmotic conditions on growth of M. nipponense.
2. Materials and methods M. nipponense were collected from Bai Yangdian Lake (salinity 0x), Hebei Province, China, on May 1999. Prawns selected for the experiments had a mean weight of
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1.015 F 0.049 g. They were at the intermolt stage as defined by Peebles (1977). Prawns were reared for 14 days at salinities of 0x, 7x, 14xand 20x, adjusted by diluting seawater with dechlorinated tap water. Groups of 35 prawns were kept in 60 30 25-cm tanks supplied with aerated circulating water. Each group was replicated. The culture temperature was maintained at 24 F 1 jC using aquarium heaters. Each day, 25% of the water was replaced by a fresh water supply of the same salinity. The prawns were fed with commercial prawn pellets. Mortality of each aquarium was observed daily and dead animals and uneaten feed were removed daily. Growth is reported as growth percent (weight gain) calculated as follows: Growth percent ð%Þ ¼ 100 ½ðWt W0 Þ=W0 ; where W0 and Wt are the initial and final weights of prawn. After the collection of haemolymph, a small piece of the abdominal musculature was removed from each prawn and homogenized immediately at a temperature between 0 and 4 jC for nucleic acid and free amino acids analysis. The remaining muscle removed from each prawn was freeze-dried for amino acid and tissue water analysis. Osmolality was determined in both haemolymph and the medium. Haemolymph was sampled with a capillary and the equivalent seawater was taken in another capillary. The capillaries were flipped to separate the liquid column into short segments of about 1 mm in length, and then frozen at 35 jC. Haemolymph osmolality (mOsm kg 1) was measured by cryoscopy as described by Huang et al. (1999), using an accurate thermometer ( 30 f 20 jC, with 1/10 jC graduations). Haemolymph osmolality was calculated as follows: Haemolymph osmolality ðmOsm kg1 Þ ¼ Dt jC=1:86: The muscle dry weight of six prawns per treatment was determined by freeze-drying in Freeze Dry Vacuum Instrument (Model LGJ-10 BSSI) until the weight was constant. Tissue water was calculated as the difference between the wet weight and dry weight of the animal muscle and expressed as a percentage of weight. Nucleic acids were extracted and partially purified from 1.4 ml of homogenate using the method of Buckley (1979). RNA and DNA were estimated from the absorbance at 260 nm of the appropriate hydrolysate. The free amino acids in the muscle were determined as described by Boone and Claybrook (1977). The free amino acids in the muscle were extracted with sulfosalicylic acid. Fresh tissue (0.424 g) was homogenized in 25 ml sulfosalicylic acid and centrifuged at 4 jC for 10 min at 4000 rpm. The supernatant obtained was filtered through a 25-Am membrane filter and analyzed for free amino acids. The whole-muscle amino acids in the muscle were determined as described by Shahidi et al. (1990). Whole-muscle was hydrolyzed with 6 mol l 1 HCl for 24 h at 110 jC in sealed tubes after replacing oxygen with nitrogen. The solution obtained was filtered through a 25-Am membrane filter and analyzed for amino acids. Free amino acids and whole-muscle amino acids were analyzed with a HITACHI Model 835-50 high-speed amino acid analyzer.
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Fig. 1. The growth percent (weight gain) of the prawn, M. nipponense at different salinities. Significant differences at P < 0.05 are indicated by different letters above bars.
The protein-bound amino acids in the muscle were calculated as follows: The protein-bound amino acids ðlmol g1 Þ ¼ The whole -muscle amino acids ðlmol g1 dryÞ ð1 tissue waterÞ FAA ðlmol g1 wetÞ: Haemolymph protein was estimated by the biuret method (Gornall et al., 1949) using bovine serum albumin as standard. One-way ANOVA followed by Tukey’s post-hoc test was used to compare means among treatments. A critical level of 0.05 has been used.
3. Results 3.1. Growth and survival Growth was significantly ( P < 0.05) influenced by water salinity. The highest growth percent (weight gain) of prawns ( P < 0.05) was achieved at a salinity of Table 1 Hemolymph osmolality and tissue water of M. nipponense in function of seawater salinity (mean (S.D.), n = 6) Salinity (x ) 0 Hemolymph osmolality (mOsm kg Tissue water (%)
1
)
7 d
330 F 6 79.1 F 0.5a
14 c
374 F 5 78.3 F 0.7a
Significant differences at P < 0.05 are indicated by different letters.
20 b
427 F 9 78.2 F 0.9a
603 F 17a 78.1 F 0.3a
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Fig. 2. The changes in RNA/DNA ratio of the prawn, M. nipponense at different salinities. Significant differences at P < 0.05 are indicated by different letters above bars.
14x(Fig. 1). There was no significant difference in percent survival (83%) between treatments. 3.2. Osmolality and tissue water Changes in hemolymph osmolality and tissue water under different salinities are shown in Table 1. The haemolymph osmolality increased proportionally to salinities from 0x to14x. A sharp increase in haemolymph osmolality occurred between 14xand 21x. The isosmotic point was reached at 15x(450 mOsm). However, tissue water was
Fig. 3. The effect of salinity on the protein concentration of the haemolymph of the prawn, M. nipponense. Significant differences at P < 0.05 are indicated by different letters above bars.
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unaffected by the medium salinities, no significant difference of tissue water was observed ( P>0.05) among four salinity levels. 3.3. RNA/DNA ratio Fig. 2 shows the effect of acclimation to different salinities on RNA/DNA ratio in the muscle of prawns. Compared with the RNA/DNA ratio in muscle of prawns at freshwater, the RNA/DNA ratio in muscle of prawns at diluted seawater in the range 7 – 20x increased 76% to 183% The highest RNA/DNA ratio in muscle ( P < 0.05) was achieved at a salinity of 14x. 3.4. Haemolymph protein concentration Fig. 3 shows the effect of acclimation to different salinities on the haemolymph protein concentration of prawns. Between the salinities of 0xand 7x, the protein concentration stayed fairly constant (around 80 mg ml 1). Beyond this range up to 20 x, protein concentrations increased sharply with the increase of the salinities (around 132 mg ml 1). Table 2 The protein-bound amino acid concentrations in the muscle of M. nipponense (Amol g 1 wet wt.) in function of seawater salinity Amino acid
Salinity (x ) 0
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine Tyrptophane AEAA* Glycine Glutamate Alanine Serine Proline Asp ANEAA** ATAA***
7 a
40.45 F 0.25 53.22 F 0.01a 6.76 F 1.03a 46.12 F 0.02a 86.15 F 1.52a 36.34 F 1.01a 80.58 F 1.23b 17.64 F 0.56c 52.66 F 0.94a 26.86 F 0.58b 446.63 F 8.08b 85.14 F 0.19b 179.47 F 1.92b 97.29 F 1.55a 56.26 F 0.00b 5.60 F 0.00b 126.69 F 0.21c 549.35 F 3.54c 996.11 F 11.58b
14 a
42.58 F 0.13 57.21 F 3.43a 7.94 F 0.62a 51.01 F 0.46a 94.46 F 1.35a 39.63 F 0.62a 87.64 F 2.16a 19.92 F 0.03b 54.17 F 2.33a 31.52 F 0.65a 486.11 F 2.08a 85.52 F 2.02b 196.18 F 3.42a,b 96.69 F 1.54a 59.91 F 0.45a,b 8.45 F 0.97a 140.50 F 2.55a,b 587.23 F 9.63b 1073.28 F 11.71a,b
20 a
41.92 F 0.19 57.93 F 2.09a 8.89 F 0.40a 49.6 F 0.01a 92.36 F 1.51a 39.95 F 0.64a 88.68 F 1.56a 22.79 F 0.24a 57.69 F 1.29a 29.63 F 0.82a,b 489.47 F 4.05a 97.69 F 4.41a 205.03 F 3.35a 102.11 F 5.25a 63.66 F 1.42a 5.49 F 3.09a,b 143.02 F 2.08a 616.99 F 16.96a 1106.46 F 21.02a
38.17 F 1.32a 57.41 F 1.73a 3.02 F 0.05b 48.48 F 0.38a 91.86 F 1.92a 37.59 F 2.14a 85.68 F 2.14a 19.47 F 1.31b,c 55.06 F 3.59a 30.60 F 0.53a,b 467.37 F 11.25b 88.80 F 0.84b 198.35 F 7.00a,b 98.4 F 0.48a 59.09 F 1.04b 5.60 F 0.12b 138.29 F 1.52b 585.82 F 2.23b 1053.19 F 9.03b
Each value represents mean F S.D. for determinations of two pooled muscle samples collected from 25 prawn at each salinity. Significant differences at P < 0.05 are indicated by different letters. * Essential amino acid. ** Non-essential amino acid. *** Total content of protein-bound amino acids.
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3.5. Protein-bound amino acids There was a significant influence of salinity on the amounts of protein-bound amino acids (Table 2). The total protein-bound amino acids (TAA) in muscle of M. nipponense increased with increasing salinities in the range of 0– 14xand reached a maximum level at salinity of 7xand 14x. After the maximum, there was a slight decline. The concentration of essential amino acids (EAA), non-essential amino acids (NEAA) and TAA at salinity 14xwas 9.6%, 12.3% and 11.1% higher than that in freshwater, respectively ( P < 0.05). 3.6. The free amino acid Table 3 shows the variation of FAA content (total, essential and non-essential FAA) in muscle as a function of salinity. It can be seen that the FAA concentration increased with increasing salinity in the range of 0 –20x. The EFAA concentration at 14xis 30.6% higher than that in freshwater, and it did not show significant change with increasing salinity in the range of 14– 20x. It is noted that the correlation between the increase of Table 3 The free amino acid concentrations in the muscle of M. nipponense (Amol g 1 wet wt) in function of seawater salinity Amino acid
Salinity (x ) 0
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine Tyrptophane AEFAA* Glycine Glutamate Alanine Serine Proline Taurine ANEFAA** ATFAA***
7 d
14.36 F 0.87 5.81 F 0.33c 2.89 F 0.13d 2.30 F 0.22a 5.16 F 0.33b 3.38 F 0.09a 4.20 F 0.36c 4.80 F 0.01a 26.80 F 0.84a 1.63 F 0.12b 71.34 F 3.22c 46.10 F 0.02c 2.83 F 0.19c 15.88 F 0.83d 4.90 F 0.23b 29.27 F 3.03c 5.43 F 0.23a 104.41 F 6.53d 175.75 F 9.76d
14 c
15.91 F 0.59 6.89 F 0.33b 3.71 F 0.05c 3.29 F 0.20a 7.36 F 0.46a 4.25 F 0.47a 5.64 F 0.59b 4.58 F 0.28a 30.26 F 2.32a 1.88 F 0.06a,b 83.77 F 1.46b 55.43 F 6.25b 3.82 F 0.03b 19.55 F 0.03c 7.08 F 0.90a 34.54 F 0.72b 4.24 F 0.45a 124.66 F 5.98c 208.43 F 7.44c
20 a
20.44 F 1.53 7.59 F 0.13a 4.82 F 0.18a 4.14 F 0.75a 8.40 F 0.69a 4.77 F 1.22a 6.11 F 0.59b 5.06 F 0.65a 29.75 F 2.04a 2.10 F 0.12a 93.18 F 3.07a 54.79 F 0.14b 4.46 F 1.28a,b 22.54 F 0.58b 8.52 F 0.85a 39.48 F 3.10ab 5.15 F 0.51a 134.49 F 0.86b 228.12 F 5.55b
18.71 F 0.80b 7.99 F 0.16a 4.33 F 0.07b 3.99 F 0.03a 8.50 F 0.37a 5.16 F 1.53a 6.47 F 0.08a 4.48 F 0.64a 30.01 F 0.93a 2.25 F 0.15a 91.89 F 3.21a 87.09 F 2.40a 5.95 F 0.51a 34.27 F 3.37a 7.22 F 0.85a 41.14 F 1.29a 5.97 F 0.52a 181.64 F 4.75a 273.53 F 6.19a
Each value represents mean F S.D. for determinations of two pooled muscle samples collected from 25 prawn at each salinity. Significant differences at P < 0.05 are indicated by different letters. * Essential free amino acid. ** Non-essential free amino acid. *** Total content of free amino acids.
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total FAA concentration and increase of salinity revealed positive linear correlation. The correlation coefficient (R2) is 0.98. The increase of total FAA concentration was about 56% at 20x, as compared to that at 0x. The results showed that muscular tissue was characterized by high concentrations of some specific amino acids such as arginine, which represents about 15.24% of the total FAA pool, glycine (26.2%), proline (16.7%), threonine (8.2%) and alanine (9.0%). These five amino acids were present in high concentrations compared with the other amino acids in the muscular tissue of freshwater prawn, which constitute 75.3% of total. Arginine was comparatively abundant amino acid, but the influence of the salinity on arginine concentration was not significant ( P>0.05) (Table 3). On the other hand, glycine, proline and alanine concentrations increased with increasing salinity in the range of 0 – 20x. There was a sharp increase of proline, glycine, and alanine at salinity of 20x(Table 3). Their concentrations increased 40.6%, 89% and 116%, respectively, compared to that in freshwater ( P < 0.05).
4. Discussion From the results presented, it is evident that the RNA/DNA ratio, TAA and FAA contents in muscle of adult prawn M. nipponense were affected by water salinities. The changes of the RNA/DNA ratio in muscle are similar to those of TAA in muscle of M. nipponense acclimated to different salinities. The RNA/DNA ratio and TAA in muscle of prawn M. nipponense in diluted seawater was higher than those in freshwater, and reached a maximum level at salinity of 14x. The haemolymph protein concentrations also increased with increasing salinity in the range of 7 –20x. These changes are probably associated with the rapid change in protein synthetic activity. Deshimaru and Shigueno (1972) suggested that since muscle is the predominant tissue formed in a growing prawn, its amino acid composition would dictate the dietary amino acid pattern required by the prawn. The higher growth for M. nipponense at 14xmay stem from the reason that salinity 14xwas closest to the isosmotic point (15x) of this species. The ratio of RNA to DNA has been related to both long- and short-term growth in adult freshwater fish. Haines (1973) demonstrated a significant positive correlation between population growth over a 15-month period and the RNA/DNA ratio of muscle tissue from smallmouth bass (Micropterus dolomieui) and carp (Cyprinus carpio). To our knowledge, it is the first time that it has been shown that the RNA/DNA ratio is useful for the diagnosis of the growth response of prawns to different salinities. Observations of the FAA pool in muscle after acclimation suggest that NEFAAs might be major osmolytes for intracellular isosmotic regulation. The EFAA concentration at 14xis 30.6% higher than that in freshwater, but was kept rather constant in the range of 14– 20x. High concentrations of glycine, arginine and proline (detected in the present species) in muscle seem to be a common feature of crustaceans (Schoffeniels and Gilles, 1970), but arginine did not show significant change in the range of 0– 20x. Glycine, glutamic acid, alanine and proline concentration varied significantly in responses to hyperosmotic stress, particularly at salinity of 20x. The glutamic acid has been implicated for its importance in isosmotic regulation in a number of marine invertebrates (Boone and Claybrook, 1977;
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Bishop and Burton, 1993). This amino acid did not appear to play a direct role as osmolyte in M. nipponense due to low contribution below 2% to the free amino acid pool at all salinities. It plays a pivotal role in the other NEFAA synthesis, i.e. as glutamate concentration increases, transamination reaction will also increase to produce alanine, aspartate, glycine, proline, and serine (Tan and Choong, 1981). Glycine, alanine and proline, which constitute 87.4% of total NEFAA in M. nipponense, were also known to be major osmolytes in other marine invertebrates studied (Bishop and Burton, 1993; McCoid et al., 1984). The increase of hemolymph osmolality and muscle FAA of adult M. nipponense with increasing salinity is compared with those of adult M. rosenbergii studied previously by Tan and Choong (1981). These species are similar in several respects. Changes of total FAA concentration in muscle and hemolymph osmolality in response to different salinities were similar. Arginine, proline, glycine, alanine and glutamic acid are quantitatively the most important amino acids. These amino acids (with the exception of arginine) were consistently elevated as the prawns were exposed to increasing salinity. In spite of these similarities, these two species differ in their abilities to regulate osmolality and the percentage of changes in some free amino acids in muscle in diluted seawater compared to freshwater (Table 4). Haemolymph osmolality in freshwater (330 mOsm) and the isosmotic point (450 mOsm) of adult M. nipponense were lower than those (360 mOsm; 485 mOsm) of adult M. rosenbergii (Singh, 1980). Denne (1968) and Moreira et al. (1983) commented on the relationship between the isosmotic point and the prawns distribution; generally freshwater species have lower isosmotic values than species found in both brackish and freshwater environments. Thus, the differences of hemolymph osmolality and isosmotic point of these species may originate from their life cycle; M. nipponense is a true freshwater species, while larvae M. rosenbergii migrate into the sea for growth and development. The percentage of changes in alanine and glutamic acid in muscle of adult M. nipponense was greater than that of adult M. rosenbergii from freshwater to diluted seawater, while the percentage of changes in proline, glycine and total FAA was smaller in M. rosenbergii. Thus, the osmolyte change in M. nipponense resembles more closely the freshwater crayfish in which alanine was the most contributable osmolyte of crayfish muscle under hyperosmotic stress (Okuma and Abe, 1994). Freshwater prawn, M. nipponense contained smaller amounts of total FAA (175 Amol g 1) in muscle than marine prawns, Penaeus japonicus (about 250 f 330 Amol g 1) (Marangos et al., 1989; Dalla Via, 1986). After seawater acclimation, however, total FAA was elevated by 56% (273 Amol g 1) which is almost equivalent to that of marine prawns. Table 4 Comparison of some free amino acid of M. nipponense and M. rosenbergii* in seawater diluted with freshwater
Glycine Glutamic acid Alanine Proline Total FAA * Tan and Choong (1981).
M. nipponense (%) (20xcompared with 0x )
M. rosenbergii* (%) (22.8xcompared with 0x )
89.92 110.25 115.81 40.55 49.08
136.28 70.95 50.27 160.5 68.79
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The good correlation between the FAA and hyperosmotic stress (increased salinities in the range of 0 –20x) suggested that the FAA function as osmoregulators in M. nipponense. At the same time, the flavor of M. nipponense reared in seawater may be improved. This is mainly due to findings that the FAA are also a major contributors to the flavor of seafoods (Fuke and Konosu, 1991; Thompson et al., 1980). In conclusion, the protein concentration and FAA in muscle of M. nipponense were affected by hyperosmotic stress. The FAA pool (mainly glycine, alanine and proline) of muscle seems to be directly related to osmoregulatory status, while alanine was the most significant osmolyte in M. nipponense under hyperosmotic stress. References Bishop, J.S., Burton, R.S., 1993. Amino acid synthesis during hyperosonotic stress in Penaeus aztecus postlarvae. Comp. Biochem. Physiol. 106A, 49 – 56. Boone, W.R., Claybrook, D.L., 1977. The effect of low salinity on amino acid metabolism in the tissues of the common mud crab, Panopeus herbstii (Milne-Edwards). Comp. Biochem. Physiol. 57A, 99 – 106. Buckley, L.J., 1979. Relationships between RNA – DNA ratio, prey density, and growth rate in Atlantic cod (Gadus morhua) larvae. J. Fish. Res. Board Can. 36, 1497 – 1502. Bulow, F.J., 1970. RNA – DNA ratio as indicators of recent growth rates of a fish. J. Fish. Res. Board Can. 27, 2343 – 2349. Cobb, B.F., Conte, F.S., Edwards, M.A., 1975. Free amino acids and osmoregulation in penaeid shrimp. J. Agric. Food Chem. 23, 1172 – 1174. Dalla Via, G.J., 1986. Salinity responses of the juvenile shrimp Penaeus japonicus: II. Free amino acids. Aquaculture 55, 307 – 316. D’Aniello, A., 1980. Free amino acids in some tissues of marine crustacea. Experientia 36, 392 – 393. Denne, L.B., 1968. Some aspects of osmotic and ionic regulation in the prawns Macrobrachium australiensis (Holthuis) and M. equidens (Dana). Comp. Biochem. Physiol. 26A, 17 – 30. Deshimaru, O., Shigueno, K., 1972. Introduction to the artificial diet for prawn, Penaeus japonicus. Aquaculture 1, 115 – 133. Fang, L.S., Tang, C.K., Lee, D.L., Chen, I.M., 1992. Free amino acid composition in muscle and hemolymph of the prawn Penaeus monodon in different salinities. Nippon Suisan Gakkaishi 58, 1095 – 1102. Fuke, S., Konosu, S., 1991. Taste-active components in some foods: a review of Japanese research. Physiol. Behav. 49, 863 – 868. Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum proteins by means of biuret reaction. J. Biol. Chem. 177, 751. Haines, T.A., 1973. An evaluation of RNA – DNA ratio as a measure of long-term growth in fish populations. J. Fish. Res. Board Can. 30, 195 – 199. Hashimoto, Y., 1965. Taste-producing substances in marine products. In: Kreuzer, R. (Ed.), The Technology of Fish Utilization. Fishing News Books, Ltd., London, England, p. 57. Holthuis, L.B., 1980. FAO Species Catalogue, vol. 1. Shrimps and Prawns of the World. FAO Fisheries Synopsis No. 125, vol. 1. FIR/S 125 vol. 1. FAO, Rome. 271 pp. Huang, J., Song, X.L., Yu, J., Zhang, L.J., 1999. The components of an inorganic physiological buffer for Penaeas chinensis. Methods Cell Sci. 21, 225 – 230. Jones, N.R., 1969. Meat and Fish flavors. Significance of ribonucleotides and their metabolites. J. Agric. Food Chem. 17, 712. Jumio, M.A.R., Cobb, J.S., Bengtson, D., Johnson, M., 1992. Changes in nucleic acids over the molt cycle in relation to food availability and temperature in Homarus americanus postlarvae. Mar. Biol. 114, 1 – 10. Marangos, C., Brogren, C.H., Alliot, E., Ceccaldi, H.J., 1989. The influence of water salinity on the free amino acid concentration in muscle and hepatopancreas of adult shrimps Penaeus japonicus. Biochem. Syst. Ecol. 17, 589 – 594.
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McCoid, V., Miget, R. Finne, G., 1984. Effect of environmental salinity on the free amino acid composition and concentration in penacid shrimp. J. Food Sci. 49, 327 – 330, 339. Moreira, G.S., McNamara, J.C., Shumway, S.E., Moreira, P.S., 1983. Osmoregulation and respiratory metabolism in Brazilian Macrobrachium (Decapoda, Palaemonidae). Comp. Biochem. Physiol. 74A, 57 – 62. Moss, S.M., 1994. Use of nucleic acids as indicators of growth in juvenile white shrimp, Penaeus vannamei. Mar. Biol. 120, 359 – 367. Okuma, E., Abe, H., 1994. Total D-amino and other free amino acids increase in muscle of crayfish during seawater acclimation. Comp. Biochem. Physiol. 109A, 191 – 197. Ortmann, A.E., 1902. The geographical distribution of freshwater decapods and its bearing upon ancient geography. Proc. Am. Philos. Soc. 41, 267 – 400. Schoffeniels, E., Gilles, R., 1970. Osmoregulation in aquatic arthropods. In: Florkin, M., Scheer, B.T. (Eds.), Chemical Zoology: Arthropoda, vol. 5, pt. A. Academic Press, New York, pp. 255 – 282. Shahidi, F., Synowiecki, J., Naczk, M., 1990. Seal meat—a potential source of muscle food: chemical composition, essential amino acids and colour characteristics. Can. Inst. Food Sci. Technol. J. 23, 137 – 139. Singh, T., 1980. The isosmotic concept in relation to aquaculture of the giant prawn, Macrobrachium rosenbergii. Aquaculture 20, 251 – 256. Simpson, J.W., Allen, K., Awapara, J., 1959. Free amino acids in some aquatic invertebrates. Biol. Bull. 117, 371 – 381. Stuck, K.C., Watts, S.A., Wang, S.Y., 1996. Biochemical responses during starvation and subsequent recovery in postlarval pacific white shrimp, Penaeus vannamei. Mar. Biol. 120, 359 – 367. Tan, C.H., Choong, K.Y., 1981. Effect of hyperosmotic stress on hemolymph protein, muscle ninydrin-positive substances and free amino acids in Macrobrachium rosenbergii (de man). Comp. Biochem. Physiol. 70A, 485 – 489. Thompson, A.B., McGill, A.S., Murray, J., Hardy, R., Howgate, P.F., 1980. The analysis of a range of nonvolatile constituents of cooked haddock (Gadus aeglefinus) and the influence of these on flavor. In: Connell, J.J. (Ed.), Advances in Fish Science and Technology. Fishing Books, Farnham, Surrey, p. 484. Uno, Y., 1971. Studies on the aquaculture of Macrobrachium nipponense (de Haan) with special reference to breeding cycle, Larval development and feeding ecology. La Mer. 9, 123 – 128. Wang, S.Y., Stickle, W.B., 1986. Changes in nucleic acid concentration with starvation in the blue crab Callinectes sapidus Rathbun. J. Crust. Biol. 6, 49 – 56. Wang, W.N., Wang, A.L., Wei, X.H., Guo, M.S., 1997. Studies on the ultrastructure of hepatopancreas cells of Macrobrachium nipponense. Acta Zool. Sin. 43, 39 – 44 (Suppl.).
Changes of protein-bound and free amino acids in the muscle of the freshwater prawn Macrobrachium nipponense in different salinities Wei-Na Wang a,b,*, An-Li Wang a, Lai Bao c, Jian–Ping Wang c, Yuan Liu a, Ru-Yong Sun a,b a
College of Life Science, South China Normal University, Guangzhou 510631, PR China b College of Life Science, Beijing Normal University, Beijing 100875, PR China c College of Life Science, Hebei University, Baoding 071002, PR China
Received 16 November 2001; received in revised form 16 September 2003; accepted 24 September 2003
Abstract Growth, hemolymph osmolality, tissue water, ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) ratio, protein-bound amino acids and free amino acids (FAA) of Macrobrachium nipponense were investigated, after they were acclimated to 0x , 7x, 14xand 20xfor 14 days. Growth was significantly ( P < 0.05) influenced by salinity. The highest weight gain of prawns ( P < 0.05) was achieved at a salinity of 14x . The RNA/DNA ratio, total protein-bound amino acid (TAA) and FAA concentrations in the muscle of prawns increased by hyperosmotic stress. The osmolality of prawns haemolymph increased slightly in salinities from 0xto14x . A sharp increase in haemolymph osmotic concentration occurs between 14xand 21x , the isosmotic point being reached at 15x (450 mOsm). It is noted that the relationship of total FAA concentration in muscle and salinity in the range of 0 – 20xrevealed a positive linear correlation. The correlation coefficient (R2) is 0.98. During adaptation, the FAA pool (mainly glycine, alanine and proline) of muscle seems to be directly related to osmoregulation, while the percentage change in alanine in prawns transferred from freshwater to diluted seawater (20x ) was most pronounced (116% approx.). These results are compared with those of M. rosenbergii. D 2004 Elsevier B.V. All rights reserved. Keywords: Macrobrachium nipponense; Salinity; Hemolymph osmolality; Amino acids; Nucleic acids
* Corresponding author. College of Life Science, South China Normal University, Guangzhou 510631, PR China. E-mail address: [email protected] (W.-N. Wang). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.09.042
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1. Introduction Crustacean muscle contains high concentrations of free amino acids, particularly glycine, proline, arginine, glutamate and alanine (Simpson et al., 1959; Schoffeniels and Gilles, 1970; Cobb et al., 1975; D’Aniello, 1980). It has been shown that the rapid and qualitatively important adjustments were necessary for osmoregulation following changes in environmental salinity (Bishop and Burton, 1993). The changes of free amino acid (FAA) pool sizes with environmental salinity have been widely documented in Crustacea. However, comparatively little is known about the regulatory patterns and influence of salinity on the contents of protein-bound and free amino acids in the muscle of the prawn Macrobrachium nipponense (de Haan), a commercially important species, found throughout brackish and freshwaters from North China to Annam, Japan and Taiwan (Holthuis, 1980; Uno, 1971; Wang et al., 1997). Ortmann (1902) pointed out that Macrobrachium was a relatively recent genus apparently in the process of emigrating to freshwater. Comparison of the adult osmoregulatory patterns displayed within the genus Macrobrachium may indicate the evolutionary sequence of the development of osmoregulation in the colonisation of freshwater. The free amino acids have been shown to function as osmoregulators in crustaceans (Fang et al., 1992; Boone and Claybrook, 1977), and they also are a major contributor to the flavor of seafoods (Hashimoto, 1965; Jones, 1969; Thompson et al., 1980). Changes of environmental salinity may possibly be utilized to produce freshwater prawns with different flavor characteristics. In addition, the amino acid composition and concentration in the muscle of prawns may affect the muscle quality of the prawn. We determined the effect of salinity on hemolymph osmolality and the protein-bound amino acid and free amino acid concentrations in the muscle of prawns and studied the role of FAA in intracellular osmoregulation during acclimation from freshwater to diluted seawater. These data are compared with measurements for M. rosenbergii. The relationship between the different salinities and the quality of the prawn muscle was also investigated. The ratio of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) has been known to be very sensitive to changes in feeding levels and short-term growth in adult freshwater fish (Haines, 1973; Bulow, 1970; Buckley, 1979). A reduction in RNA/DNA ratios in response to starvation was observed in postlarvae of the lobster Homarus americanus (Jumio et al., 1992), the crab Callinectes sapidus (Wang and Stickle, 1986), and juvenile Penaeus vannamei (Moss, 1994). Stuck et al. (1996) considered the RNA/DNA ratio to be a useful indicator of prolonged nutritional stress in postlarval P. vannamei. The present study was initiated to determine the changes in the RNA/DNA ratio in muscle of prawns, M. nipponense during the acclimation from freshwater to diluted seawater in order to define the effects of hyperosmotic conditions on growth of M. nipponense.
2. Materials and methods M. nipponense were collected from Bai Yangdian Lake (salinity 0x), Hebei Province, China, on May 1999. Prawns selected for the experiments had a mean weight of
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1.015 F 0.049 g. They were at the intermolt stage as defined by Peebles (1977). Prawns were reared for 14 days at salinities of 0x, 7x, 14xand 20x, adjusted by diluting seawater with dechlorinated tap water. Groups of 35 prawns were kept in 60 30 25-cm tanks supplied with aerated circulating water. Each group was replicated. The culture temperature was maintained at 24 F 1 jC using aquarium heaters. Each day, 25% of the water was replaced by a fresh water supply of the same salinity. The prawns were fed with commercial prawn pellets. Mortality of each aquarium was observed daily and dead animals and uneaten feed were removed daily. Growth is reported as growth percent (weight gain) calculated as follows: Growth percent ð%Þ ¼ 100 ½ðWt W0 Þ=W0 ; where W0 and Wt are the initial and final weights of prawn. After the collection of haemolymph, a small piece of the abdominal musculature was removed from each prawn and homogenized immediately at a temperature between 0 and 4 jC for nucleic acid and free amino acids analysis. The remaining muscle removed from each prawn was freeze-dried for amino acid and tissue water analysis. Osmolality was determined in both haemolymph and the medium. Haemolymph was sampled with a capillary and the equivalent seawater was taken in another capillary. The capillaries were flipped to separate the liquid column into short segments of about 1 mm in length, and then frozen at 35 jC. Haemolymph osmolality (mOsm kg 1) was measured by cryoscopy as described by Huang et al. (1999), using an accurate thermometer ( 30 f 20 jC, with 1/10 jC graduations). Haemolymph osmolality was calculated as follows: Haemolymph osmolality ðmOsm kg1 Þ ¼ Dt jC=1:86: The muscle dry weight of six prawns per treatment was determined by freeze-drying in Freeze Dry Vacuum Instrument (Model LGJ-10 BSSI) until the weight was constant. Tissue water was calculated as the difference between the wet weight and dry weight of the animal muscle and expressed as a percentage of weight. Nucleic acids were extracted and partially purified from 1.4 ml of homogenate using the method of Buckley (1979). RNA and DNA were estimated from the absorbance at 260 nm of the appropriate hydrolysate. The free amino acids in the muscle were determined as described by Boone and Claybrook (1977). The free amino acids in the muscle were extracted with sulfosalicylic acid. Fresh tissue (0.424 g) was homogenized in 25 ml sulfosalicylic acid and centrifuged at 4 jC for 10 min at 4000 rpm. The supernatant obtained was filtered through a 25-Am membrane filter and analyzed for free amino acids. The whole-muscle amino acids in the muscle were determined as described by Shahidi et al. (1990). Whole-muscle was hydrolyzed with 6 mol l 1 HCl for 24 h at 110 jC in sealed tubes after replacing oxygen with nitrogen. The solution obtained was filtered through a 25-Am membrane filter and analyzed for amino acids. Free amino acids and whole-muscle amino acids were analyzed with a HITACHI Model 835-50 high-speed amino acid analyzer.
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Fig. 1. The growth percent (weight gain) of the prawn, M. nipponense at different salinities. Significant differences at P < 0.05 are indicated by different letters above bars.
The protein-bound amino acids in the muscle were calculated as follows: The protein-bound amino acids ðlmol g1 Þ ¼ The whole -muscle amino acids ðlmol g1 dryÞ ð1 tissue waterÞ FAA ðlmol g1 wetÞ: Haemolymph protein was estimated by the biuret method (Gornall et al., 1949) using bovine serum albumin as standard. One-way ANOVA followed by Tukey’s post-hoc test was used to compare means among treatments. A critical level of 0.05 has been used.
3. Results 3.1. Growth and survival Growth was significantly ( P < 0.05) influenced by water salinity. The highest growth percent (weight gain) of prawns ( P < 0.05) was achieved at a salinity of Table 1 Hemolymph osmolality and tissue water of M. nipponense in function of seawater salinity (mean (S.D.), n = 6) Salinity (x ) 0 Hemolymph osmolality (mOsm kg Tissue water (%)
1
)
7 d
330 F 6 79.1 F 0.5a
14 c
374 F 5 78.3 F 0.7a
Significant differences at P < 0.05 are indicated by different letters.
20 b
427 F 9 78.2 F 0.9a
603 F 17a 78.1 F 0.3a
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Fig. 2. The changes in RNA/DNA ratio of the prawn, M. nipponense at different salinities. Significant differences at P < 0.05 are indicated by different letters above bars.
14x(Fig. 1). There was no significant difference in percent survival (83%) between treatments. 3.2. Osmolality and tissue water Changes in hemolymph osmolality and tissue water under different salinities are shown in Table 1. The haemolymph osmolality increased proportionally to salinities from 0x to14x. A sharp increase in haemolymph osmolality occurred between 14xand 21x. The isosmotic point was reached at 15x(450 mOsm). However, tissue water was
Fig. 3. The effect of salinity on the protein concentration of the haemolymph of the prawn, M. nipponense. Significant differences at P < 0.05 are indicated by different letters above bars.
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unaffected by the medium salinities, no significant difference of tissue water was observed ( P>0.05) among four salinity levels. 3.3. RNA/DNA ratio Fig. 2 shows the effect of acclimation to different salinities on RNA/DNA ratio in the muscle of prawns. Compared with the RNA/DNA ratio in muscle of prawns at freshwater, the RNA/DNA ratio in muscle of prawns at diluted seawater in the range 7 – 20x increased 76% to 183% The highest RNA/DNA ratio in muscle ( P < 0.05) was achieved at a salinity of 14x. 3.4. Haemolymph protein concentration Fig. 3 shows the effect of acclimation to different salinities on the haemolymph protein concentration of prawns. Between the salinities of 0xand 7x, the protein concentration stayed fairly constant (around 80 mg ml 1). Beyond this range up to 20 x, protein concentrations increased sharply with the increase of the salinities (around 132 mg ml 1). Table 2 The protein-bound amino acid concentrations in the muscle of M. nipponense (Amol g 1 wet wt.) in function of seawater salinity Amino acid
Salinity (x ) 0
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine Tyrptophane AEAA* Glycine Glutamate Alanine Serine Proline Asp ANEAA** ATAA***
7 a
40.45 F 0.25 53.22 F 0.01a 6.76 F 1.03a 46.12 F 0.02a 86.15 F 1.52a 36.34 F 1.01a 80.58 F 1.23b 17.64 F 0.56c 52.66 F 0.94a 26.86 F 0.58b 446.63 F 8.08b 85.14 F 0.19b 179.47 F 1.92b 97.29 F 1.55a 56.26 F 0.00b 5.60 F 0.00b 126.69 F 0.21c 549.35 F 3.54c 996.11 F 11.58b
14 a
42.58 F 0.13 57.21 F 3.43a 7.94 F 0.62a 51.01 F 0.46a 94.46 F 1.35a 39.63 F 0.62a 87.64 F 2.16a 19.92 F 0.03b 54.17 F 2.33a 31.52 F 0.65a 486.11 F 2.08a 85.52 F 2.02b 196.18 F 3.42a,b 96.69 F 1.54a 59.91 F 0.45a,b 8.45 F 0.97a 140.50 F 2.55a,b 587.23 F 9.63b 1073.28 F 11.71a,b
20 a
41.92 F 0.19 57.93 F 2.09a 8.89 F 0.40a 49.6 F 0.01a 92.36 F 1.51a 39.95 F 0.64a 88.68 F 1.56a 22.79 F 0.24a 57.69 F 1.29a 29.63 F 0.82a,b 489.47 F 4.05a 97.69 F 4.41a 205.03 F 3.35a 102.11 F 5.25a 63.66 F 1.42a 5.49 F 3.09a,b 143.02 F 2.08a 616.99 F 16.96a 1106.46 F 21.02a
38.17 F 1.32a 57.41 F 1.73a 3.02 F 0.05b 48.48 F 0.38a 91.86 F 1.92a 37.59 F 2.14a 85.68 F 2.14a 19.47 F 1.31b,c 55.06 F 3.59a 30.60 F 0.53a,b 467.37 F 11.25b 88.80 F 0.84b 198.35 F 7.00a,b 98.4 F 0.48a 59.09 F 1.04b 5.60 F 0.12b 138.29 F 1.52b 585.82 F 2.23b 1053.19 F 9.03b
Each value represents mean F S.D. for determinations of two pooled muscle samples collected from 25 prawn at each salinity. Significant differences at P < 0.05 are indicated by different letters. * Essential amino acid. ** Non-essential amino acid. *** Total content of protein-bound amino acids.
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3.5. Protein-bound amino acids There was a significant influence of salinity on the amounts of protein-bound amino acids (Table 2). The total protein-bound amino acids (TAA) in muscle of M. nipponense increased with increasing salinities in the range of 0– 14xand reached a maximum level at salinity of 7xand 14x. After the maximum, there was a slight decline. The concentration of essential amino acids (EAA), non-essential amino acids (NEAA) and TAA at salinity 14xwas 9.6%, 12.3% and 11.1% higher than that in freshwater, respectively ( P < 0.05). 3.6. The free amino acid Table 3 shows the variation of FAA content (total, essential and non-essential FAA) in muscle as a function of salinity. It can be seen that the FAA concentration increased with increasing salinity in the range of 0 –20x. The EFAA concentration at 14xis 30.6% higher than that in freshwater, and it did not show significant change with increasing salinity in the range of 14– 20x. It is noted that the correlation between the increase of Table 3 The free amino acid concentrations in the muscle of M. nipponense (Amol g 1 wet wt) in function of seawater salinity Amino acid
Salinity (x ) 0
Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Histidine Arginine Tyrptophane AEFAA* Glycine Glutamate Alanine Serine Proline Taurine ANEFAA** ATFAA***
7 d
14.36 F 0.87 5.81 F 0.33c 2.89 F 0.13d 2.30 F 0.22a 5.16 F 0.33b 3.38 F 0.09a 4.20 F 0.36c 4.80 F 0.01a 26.80 F 0.84a 1.63 F 0.12b 71.34 F 3.22c 46.10 F 0.02c 2.83 F 0.19c 15.88 F 0.83d 4.90 F 0.23b 29.27 F 3.03c 5.43 F 0.23a 104.41 F 6.53d 175.75 F 9.76d
14 c
15.91 F 0.59 6.89 F 0.33b 3.71 F 0.05c 3.29 F 0.20a 7.36 F 0.46a 4.25 F 0.47a 5.64 F 0.59b 4.58 F 0.28a 30.26 F 2.32a 1.88 F 0.06a,b 83.77 F 1.46b 55.43 F 6.25b 3.82 F 0.03b 19.55 F 0.03c 7.08 F 0.90a 34.54 F 0.72b 4.24 F 0.45a 124.66 F 5.98c 208.43 F 7.44c
20 a
20.44 F 1.53 7.59 F 0.13a 4.82 F 0.18a 4.14 F 0.75a 8.40 F 0.69a 4.77 F 1.22a 6.11 F 0.59b 5.06 F 0.65a 29.75 F 2.04a 2.10 F 0.12a 93.18 F 3.07a 54.79 F 0.14b 4.46 F 1.28a,b 22.54 F 0.58b 8.52 F 0.85a 39.48 F 3.10ab 5.15 F 0.51a 134.49 F 0.86b 228.12 F 5.55b
18.71 F 0.80b 7.99 F 0.16a 4.33 F 0.07b 3.99 F 0.03a 8.50 F 0.37a 5.16 F 1.53a 6.47 F 0.08a 4.48 F 0.64a 30.01 F 0.93a 2.25 F 0.15a 91.89 F 3.21a 87.09 F 2.40a 5.95 F 0.51a 34.27 F 3.37a 7.22 F 0.85a 41.14 F 1.29a 5.97 F 0.52a 181.64 F 4.75a 273.53 F 6.19a
Each value represents mean F S.D. for determinations of two pooled muscle samples collected from 25 prawn at each salinity. Significant differences at P < 0.05 are indicated by different letters. * Essential free amino acid. ** Non-essential free amino acid. *** Total content of free amino acids.
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total FAA concentration and increase of salinity revealed positive linear correlation. The correlation coefficient (R2) is 0.98. The increase of total FAA concentration was about 56% at 20x, as compared to that at 0x. The results showed that muscular tissue was characterized by high concentrations of some specific amino acids such as arginine, which represents about 15.24% of the total FAA pool, glycine (26.2%), proline (16.7%), threonine (8.2%) and alanine (9.0%). These five amino acids were present in high concentrations compared with the other amino acids in the muscular tissue of freshwater prawn, which constitute 75.3% of total. Arginine was comparatively abundant amino acid, but the influence of the salinity on arginine concentration was not significant ( P>0.05) (Table 3). On the other hand, glycine, proline and alanine concentrations increased with increasing salinity in the range of 0 – 20x. There was a sharp increase of proline, glycine, and alanine at salinity of 20x(Table 3). Their concentrations increased 40.6%, 89% and 116%, respectively, compared to that in freshwater ( P < 0.05).
4. Discussion From the results presented, it is evident that the RNA/DNA ratio, TAA and FAA contents in muscle of adult prawn M. nipponense were affected by water salinities. The changes of the RNA/DNA ratio in muscle are similar to those of TAA in muscle of M. nipponense acclimated to different salinities. The RNA/DNA ratio and TAA in muscle of prawn M. nipponense in diluted seawater was higher than those in freshwater, and reached a maximum level at salinity of 14x. The haemolymph protein concentrations also increased with increasing salinity in the range of 7 –20x. These changes are probably associated with the rapid change in protein synthetic activity. Deshimaru and Shigueno (1972) suggested that since muscle is the predominant tissue formed in a growing prawn, its amino acid composition would dictate the dietary amino acid pattern required by the prawn. The higher growth for M. nipponense at 14xmay stem from the reason that salinity 14xwas closest to the isosmotic point (15x) of this species. The ratio of RNA to DNA has been related to both long- and short-term growth in adult freshwater fish. Haines (1973) demonstrated a significant positive correlation between population growth over a 15-month period and the RNA/DNA ratio of muscle tissue from smallmouth bass (Micropterus dolomieui) and carp (Cyprinus carpio). To our knowledge, it is the first time that it has been shown that the RNA/DNA ratio is useful for the diagnosis of the growth response of prawns to different salinities. Observations of the FAA pool in muscle after acclimation suggest that NEFAAs might be major osmolytes for intracellular isosmotic regulation. The EFAA concentration at 14xis 30.6% higher than that in freshwater, but was kept rather constant in the range of 14– 20x. High concentrations of glycine, arginine and proline (detected in the present species) in muscle seem to be a common feature of crustaceans (Schoffeniels and Gilles, 1970), but arginine did not show significant change in the range of 0– 20x. Glycine, glutamic acid, alanine and proline concentration varied significantly in responses to hyperosmotic stress, particularly at salinity of 20x. The glutamic acid has been implicated for its importance in isosmotic regulation in a number of marine invertebrates (Boone and Claybrook, 1977;
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Bishop and Burton, 1993). This amino acid did not appear to play a direct role as osmolyte in M. nipponense due to low contribution below 2% to the free amino acid pool at all salinities. It plays a pivotal role in the other NEFAA synthesis, i.e. as glutamate concentration increases, transamination reaction will also increase to produce alanine, aspartate, glycine, proline, and serine (Tan and Choong, 1981). Glycine, alanine and proline, which constitute 87.4% of total NEFAA in M. nipponense, were also known to be major osmolytes in other marine invertebrates studied (Bishop and Burton, 1993; McCoid et al., 1984). The increase of hemolymph osmolality and muscle FAA of adult M. nipponense with increasing salinity is compared with those of adult M. rosenbergii studied previously by Tan and Choong (1981). These species are similar in several respects. Changes of total FAA concentration in muscle and hemolymph osmolality in response to different salinities were similar. Arginine, proline, glycine, alanine and glutamic acid are quantitatively the most important amino acids. These amino acids (with the exception of arginine) were consistently elevated as the prawns were exposed to increasing salinity. In spite of these similarities, these two species differ in their abilities to regulate osmolality and the percentage of changes in some free amino acids in muscle in diluted seawater compared to freshwater (Table 4). Haemolymph osmolality in freshwater (330 mOsm) and the isosmotic point (450 mOsm) of adult M. nipponense were lower than those (360 mOsm; 485 mOsm) of adult M. rosenbergii (Singh, 1980). Denne (1968) and Moreira et al. (1983) commented on the relationship between the isosmotic point and the prawns distribution; generally freshwater species have lower isosmotic values than species found in both brackish and freshwater environments. Thus, the differences of hemolymph osmolality and isosmotic point of these species may originate from their life cycle; M. nipponense is a true freshwater species, while larvae M. rosenbergii migrate into the sea for growth and development. The percentage of changes in alanine and glutamic acid in muscle of adult M. nipponense was greater than that of adult M. rosenbergii from freshwater to diluted seawater, while the percentage of changes in proline, glycine and total FAA was smaller in M. rosenbergii. Thus, the osmolyte change in M. nipponense resembles more closely the freshwater crayfish in which alanine was the most contributable osmolyte of crayfish muscle under hyperosmotic stress (Okuma and Abe, 1994). Freshwater prawn, M. nipponense contained smaller amounts of total FAA (175 Amol g 1) in muscle than marine prawns, Penaeus japonicus (about 250 f 330 Amol g 1) (Marangos et al., 1989; Dalla Via, 1986). After seawater acclimation, however, total FAA was elevated by 56% (273 Amol g 1) which is almost equivalent to that of marine prawns. Table 4 Comparison of some free amino acid of M. nipponense and M. rosenbergii* in seawater diluted with freshwater
Glycine Glutamic acid Alanine Proline Total FAA * Tan and Choong (1981).
M. nipponense (%) (20xcompared with 0x )
M. rosenbergii* (%) (22.8xcompared with 0x )
89.92 110.25 115.81 40.55 49.08
136.28 70.95 50.27 160.5 68.79
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