Comparative Biochemistry and Physiology Part A 125 (2000) 377 – 388 www.elsevier.com/locate/cbpa Characterization of Na/K-ATPase in Macrobrachium rosenbergii and the effects of changing salinity on enzymatic activity Marcy N Wilder a,*, Do Thi Thanh Huong a, Muharijadi Atmomarsono a,1, Tran Thi Thanh Hien b, Truong Quoc Phu b, Wei-Jun Yang a a Japan International Research Center for Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries, -2 Ohwashi, Tsukuba, Ibaraki Prefecture 305 -8686, Japan b Institute for Marine Aquaculture, College of Agriculture, Cantho Uni6ersity, Cantho, Vietnam Received 10 October 1999; received in revised form 19 January 2000; accepted 31 January 2000 Abstract A ouabain-sensitive Na/K-ATPase kinetic assay system based on the hydrolysis of ATP and the oxidation of NADH was adapted in order to characterize enzymatic activity in gills and examine the effects of changing salinity in Macrobrachium rosenbergii Maximum inhibition by ouabain occurred at a concentration of 1.4 mM, and the Km of the reaction was 0.2 mM In a first experiment, animals were acclimated to freshwater, 1/3 seawater, 2/3 seawater and full seawater for up to week Na/K-ATPase activity in front gills was 1.62 90.19 mmol ADP/mg protein per h in freshwater, and was seen to increase slightly in 1/3 seawater (1.88 0.19 mmol ADP/mg protein per h) and 2/3 seawater (2.099 0.24 mmol ADP/mg protein per h), decreasing slightly in full seawater (1.92 0.43 mmol ADP/mg protein per h); however, differences were not significant Back gills showed slightly higher levels, and a similar pattern of Na/K-ATPase activity In a second experiment, animals were acclimated to 1/3 seawater and 2/3 seawater, and then transferred to freshwater However, no changes in activity were seen, indicating that exposure to dilute media did not effect enzymatic activity Whereas Na/K-ATPase is important in osmoregulatory function in marine euryhaline crustaceans, it may not play a significant role in adaptation in freshwater crustaceans that inhabit a more narrow range of salinities © 2000 Elsevier Science Inc All rights reserved Keywords: Enzymatic activity; Freshwater prawns; Gills; Hemolymph; Ionic regulation; Na/K-ATPase; Osmoregulation; Salinity Introduction The giant freshwater prawn, Macrobrachium rosenbergii, like many other species of the Macrobrachium genus, is primarily a freshwater species * Corresponding author Tel.: +81-298-386630; fax: +81298-386316 E-mail address: marwil@jircas.affrc.go.jp (M.N Wilder) Present address: Research Institute for Coastal Aquaculture, Maros, South Sulawesi, Indonesia that requires brackish water for the survival of its larvae (Ling, 1969; Sandifer et al., 1975) M rosenbergii is a species of considerable economic importance in Southeast Asia (Chavez Justo, 1990), with total global production due to aquaculture being about 27 000 tons per year (de Caluwe et al., 1995) In order to improve existing aquaculture and seed production technology, it is important to understand how this species responds to changing salinity and gain more knowl- 1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc All rights reserved PII: S - 3 ( 0 ) 0 - 378 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 edge of basic underlying mechanisms of osmoregulatory function M rosenbergii shows hyperosmoregulatory ability in fresh water and at low salinities (Wilder et al., 1998), and in this respect is similar to other Macrobrachium species including M acanthurus, M heterochirus, M potiuna (Moreira et al., 1983) and M ohione (Castille and Lawrence, 1981b) In our previous study, we investigated the effects of varying salinity on osmotic and ionic concentrations in the hemolymph, revealing that changes in sodium concentration parallel those in hemolymph osmolality (Wilder et al., 1998) Other studies have demonstrated similarly that osmotic change in prawns is based significantly on changes in hemolymph sodium and chloride concentrations, for example, in M rosenbergii (Castille and Lawrence, 1981b), Penaeus monodon (Ferraris et al., 1987), P setiferus and P stylirostris (Castille and Lawrence, 1981a) As Pequeux (1995) has reviewed, various salttransporting tissues, including the body wall, gastrointestinal tract, and excretory organs play a vital role in osmoregulation, but the gill epithelium is expected to be of foremost significance in maintaining hemolymph NaCl balance in Crustacea Indeed, a number of studies have examined the role of branchial Na/K-ATPase activity in osmoregulation in various species including several crabs (Towle et al., 1976; Savage and Robinson, 1983; Corotto and Holliday, 1996), freshwater euryhaline crayfish (Wheatly and Henry, 1987), and artemia (Holliday et al., 1990) In the purple shore crab, Hemigrapsus nudus, Na/K-ATPase activity was seen to be highest in low to medium salinities, and decreased at higher salinities (Corotto and Holliday, 1996) Other species, including Artemia salina have demonstrated increases in enzymatic activity during the process of adaptation from higher to lower salinities (Holliday et al., 1990) M rosenbergii frequently migrates between freshwater and estuarine or brackish water areas for purposes of spawning, and is therefore considered to make use of osmoregulatory ability in adapting to salinity changes in this range We conducted this investigation in order to examine whether Na/K-ATPase activity in gill tissue is modulated in response to increasing or decreasing salinity in M rosenbergii, similarly to some of the above crustacean species For purposes of determining Na/K-ATPase enzymatic activity, we adapted a ouabain-sensitive kinetic assay system according to McCormick and Bern (1989) This system, based on the hydrolysis of ATP and the oxidation of NADH, has been widely employed in the determination of Na/KATPase activity in salmonid and other fishes Many previous reports in crustaceans and other invertebrates have utilized methodology based on the determination of inorganic phosphorus released after the addition of ATP to tissue homogenates (Ilenchuk and Davey, 1982; Wheatly and Henry, 1987; Ahl and Brown, 1991; Corotto and Holliday, 1996) In order to validate the use of a kinetic-type assay for application to M rosenbergii, we firstly modified existing assay conditions, and characterized optimal parameters for ouabain inhibition, ionic concentrations, pH, and temperature We then applied this system to measure changes in Na/K-ATPase activity in gill tissue in response to changing salinity in prawns initially acclimated to fresh or brackish water Materials and methods 2.1 Animals, rearing and sampling M rosenbergii were generously supplied by Active Rise, Co., Ltd., a commercial source on the island of Kyushu, Southern Japan Prawns were acclimated to 28°C in freshwater (pre-circulated tapwater) in 600-l stock tanks for at least weeks prior to use in experimentation Animals were then transferred to 60-l treatment tanks containing dividers to hold four prawns per tank Treatments consisted of freshwater (8 mOsm), 1/3 seawater (350 mOsm), 2/3 seawater (620 mOsm) and full seawater (920 mOsm); seawater dilutions were made from appropriate quantities of artificial seawater mix (Aqua OceanR, JPDAO-NO 1100) and pre-circulated tapwater Male and female prawns in the intermolt stages ranging from 15 to 40 g body weight (BW) were employed In the first experiment, prawns (four to six individuals for each determination; BW=23.25 93.57) were maintained under these treatments for week (or days only for those individuals exposed to full seawater) Hemolymph was sampled by cardiac puncture at h and at the conclusion of the experiment Gills were dissected out, and the left and right sides were quick-frozen and stored separately at −80°C until use in Na/KATPase determination In a second experiment, M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 after initial acclimation to freshwater, prawns (four individuals for each determination; BW= 29.06 94.50 g) were then acclimated to 1/3 seawater or 2/3 seawater for week They were then transferred back to freshwater and gill tissue was dissected out after day to examine the effects of freshwater exposure on Na/K-ATPase activity Hemolymph was sampled at the start of the experiment, after 1-week acclimation in 1/3 seawater or 2/3 seawater, and day after transfer back to freshwater For the validation of the assay system, male prawns (BW =22.8 93.46) were employed, and four individuals were used for each type of determination, e.g maximal inhibition by ouabain, optimum ionic concentrations in the reaction media, pH, temperature, effects of gill quantity on enzymatic activity, effects of substrate concentration, and time – activity relationship In addition, separate gill filaments in four larger-sized males ranging from 35 to 40 g (BW = 38.11 92.37) were sampled and enzymatic activity in each filament was measured in order to determine whether activity differs according to gill position 2.2 Hemolymph osmolality Osmolality of the hemolymph and treatment tank water was analyzed using 10 ml quantities directly in a Fiske – 10 osmometer (USA) in order to follow changes in response to varying salinitiy 2.3 Basic solutions and protocol A preparation of imidazole buffer (50 mM, pH 7.5) was used to prepare a basic reaction mixture and salt solution (see below) except where the effects of pH were being determined, in which case pH was varied between 6.9 and 8.1 (see assay validation) Sucrose EDTA imidazole buffer (SEI) buffer consisted of 150 mM sucrose, 10 mM EDTA and 50 mM imidazole (pH 7.3), and this was used to prepare 0.1% sodium deoxycholate (SEID) stock solution SEID was diluted 5-fold with SEI to obtain working SEID solution prior to sample homogenization A stock ouabain solution of 20 mM was prepared by dissolving ouabain octahydrate in the appropriate amount of 50 mM imidazole buffer (pH 7.5) with heating A basic reaction mixture consisting of 0.22 mM NADH, 2.8 mM phosphoenolpyruvate (PEP), 0.7 379 mM ATP, 16.8 ml lactic dehydrogenase (LDH)(Sigma: 3.7 mg protein/ml; 490 U/mg protein), and 35.75 ml pyruvate kinase (PK)(Sigma: 12.9 mg protein/ml; 830 U/mg protein), was then prepared in imidazole buffer with a ouabain concentration of or mM (for assay validation, concentrations were tested at 0, 0.5, 1.0, 2.0 and 4.0 mM) A basic salt solution consisting of 180 mM NaCl, 10 mM MgCl2 and 80 mM KCl in imidazole buffer (50 mM, pH 7.5) was also prepared Standards for the assay were prepared as 20, 10, 5, 2.5 and mM ADP in working SEID solution Samples were prepared by homogenizing 30– 40 mg of gill tissue (wet weight) in 300 ml working SEID solution for 30 s on ice using an Omni International hand-held homogenizer (USA) Homogenates were then centrifuged at 500 rpm for The supernatant was diluted from 2- to 8-fold to the appropriate range for activity measurement, but this was usually 4-fold The assay was conducted by adding 10 ml sample or standard in duplicate with 50 ml salt solution and 140 ml assay mixture containing or mM ouabain During this process, the microplate was kept on a cold-pack and pipetting was done as rapidly as possible After initiating the reaction, the plate was read at 2.5-min intervals for 15 at 340 nm in a Bio-Rad Model 3550-UV microplate reader The activity of ouabain-sensitive Na/KATPase was determined as the difference in the rate of NADH oxidation in the presence and absence of ouabain as mOD/10 ml per divided by the standard curve of around 21 mOD/nmole ADP and expressed initially as nmoles ADP/10 ml per Total protein in samples was determined with the Bio-Rad protein assay kit using IgG as the standard and expressed as mg protein/10 ml Final values were then calculated as mmol ADP/ mg protein per h 2.4 Assay 6alidation For validation of this system, standard conditions described below were employed, varying one factor while keeping all other parameters constant Inhibition by ouabain corresponding to the actual measurement of Na/K-ATPase activity, as a function of ouabain concentration in the reaction mixture, was firstly examined Under the standard conditions described above, ouabain concentrations in the basic reaction mixture were 380 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 varied from 0, 0.5, 1.0, 2.0 and 4.0 mM in the reaction mixture, corresponding to a final concentration of 0, 0.35, 0.70, 1.40 and 2.80 mM in wells, and inhibition by ouabain was expressed in terms of percent activity at a specific concentration of total activity observed in the absence of ouabain The outcome is described in detail in Section 3, but maximal inhibition was observed at mM ouabain in the reaction mixture, and thus, this concentration was fixed in the examination of all other parameters in the remainder of the validation The assay system was additionally characterized for the following parameters: final ion concentrations including Na+ (0, 22.5, 45.0, 90.0 mM), K+ (0, 10, 20, 40 mM) and Mg2 + (0, 1.25, 2.50, 5.00 mM) in wells, pH (6.9, 7.2, 7.5, 7.8, 8.1) and temperature (4, 16, 28, 40°C) In addition, the effects of gill quantity on enzymatic activity, relationship of substrate concentration and activity, and time after homogenization on enzymatic activity were examined Optimal conditions for actual analysis of osmoregulatory function in response to changing salinity were set according Fig Determination of standard ouabain conditions Ouabain concentrations in the basic reaction mixture were varied from 0, 0.5, 1.0, 2.0 and 4.0 mM in the reaction mixture, corresponding to a final concentration of 0, 0.35, 0.70, 1.40 and 2.80 mM in wells Inhibition by ouabain was expressed in terms of percent activity of total activity observed in the absence of ouabain Maximal inhibition was observed at mM ouabain in the assay mixture Results are show as the mean S.E to the above results After setting assay conditions, individual gills were measured to determine whether activity varies according to gill position; however, gills and were assayed together due to small size, and gills 3, 4, 5, 6, and were measured individually Differences were not significant, and thereafter, in actual experimentation, front gills (1– 4) and back gills (5– 7) were combined separately for left and right sides, and homogenized in separate batches 2.5 Statistical analysis Duncan’s multiple range test (with PB 0.05 taken as significant) was employed to analyze differences in Na/K-ATPase enzymatic activity among gill samples exposed to differing salinities in two separate experiments Results 3.1 Assay 6alidation Fig shows that percent inhibition of total ATPase activity increased with increasing concentrations of ouabain in the salt solution, reaching a maximum at a concentration of mM (53.0%) in the reaction mixture equivalent to 1.4 mM final concentration in wells At higher concentrations, inhibition was seen to decrease Conditions of mM ouabain in the reaction mixture were thus chosen for subsequent trials in order to fully measure ouabain-sensitive Na/K-ATPase activity Next, concentrations of Na+, K+ and Mg2 + were varied in the test solution A small amount of activity was observed when Na+ concentrations were mM, increasing to maximal activity at 22.5 mM and plateauing from 45 mM When K+ was mM, activity was about half that of maximally observed activity, increasing at 10 mM and then plateauing from 20 mM Activity was nil when Mg2 + concentration was mM, but maximal activity was observed at concentrations of 1.25 mM and higher Based on this, standard assay conditions for these cations were chosen to be 45, 20, and 2.5 mM for Na+, K+ and Mg2 + , respectively (corresponding to 180, 80, and 10 mM in original salt solution) In this set of experiments, maximal activity was approximately 1.5 mmol ADP/mg protein per h These results are shown in Fig M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 381 Fig Determination of standard ionic concentrations The assay system was characterized for optimal ionic concentrations of Na+ (0, 22.5, 45.0, 90.0 mM), K+ (0, 10, 20, 40 mM) and Mg2 + (0, 1.25, 2.50, 5.00 mM) Based on these results, standard assay conditions for these cations were chosen to be 45, 20, and 2.5 mM for Na+, K+ and Mg2 + , respectively (corresponding to 180, 80, and 10 mM in original salt solution) Results are show as the mean 9S.E Fig Effects of temperature on enzymatic activity At 4°C, activity was 0.637 mmol ADP/mg protein per h, increasing to 1.156, 1.554, and 3.318 mmol ADP/mg protein per h at 16, 28 and 40°C For final assay conditions, 28°C was chosen as representative of the physiological temperature at which the experimental animals were maintained Results are show as the mean 9S.E 382 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 Dependence of enzymatic activity on temperature is shown in Fig Activity was 0.637 mmol ADP/mg protein per h, increasing to 1.156, 1.554, and 3.318 mmol ADP/mg protein per h at 16, 28 and 40°C For final assay conditions, 28°C was chosen as representative of the physiological temperature at which the experimental animals were maintained There was no dependence of enzymatic activity on pH in the range tested (data not shown); subsequent determinations were conducted at pH 7.5 In examination of the effects of protein concentration on enzymatic activity, it was seen that in a sample consisting of 1.44 mg gill tissue/10 ml SEID buffer diluted from 2- to 16-fold, activity decreased linearly in proportion to quantity of tissue In the undiluted samples, activity was lower, indicating that the reaction was hampered in the original homogenate (Fig 4) This demonstrated that measurements were valid for samples diluted at least 2-fold, although samples were usually diluted 4-fold in this investigation The relationship between substrate concentration and activity was examined by varying ATP concentration in the reaction mixture from 0- to 4-fold that of standard assay conditions (concentration showed for final concentration in wells) Activity increased up to standard assay conditions, and plateaued upon doubling this con- centration This showed that substrate was used under saturated conditions (Fig 5) Analysis of the data using a Lineweaver– Burke plot (not shown) revealed that the Km for the reaction was 0.2 mM Finally, the effects of time on enzymatic activity were examined Enzymatic activity was assayed at 0, 1, 3, and 24 h after homogenization, keeping extracts at 4°C when not being used Activity did not decline in extracts kept for up to h, declining slightly after 24 h (data not shown) This indicated that enzymatic activity was stable in homogenates and time spent preparing for measurement did not affect results 3.2 Effects of changing salinity on enzymatic acti6ity In the first experiment, prawns were transferred from freshwater to 1/3 seawater, 2/3 seawater and seawater in order to confirm changes in osmolality in response to varying salinity as in our previous investigation (Wilder et al., 1998) In freshwater and 1/3 seawater, hemolymph osmolality was about 480 mOsm, and did not change during the course of the experimentation, while in 2/3 seawater and full seawater, increases began after transfer, becoming equivalent to that of the rearing water These results (data not shown) Fig Effects of protein concentration on enzymatic activity In a sample consisting of 1.44 mg gill tissue/10 ml SEID buffer diluted from 2- to 16-fold, activity decreased linearly in proportion to quantity of tissue The reaction was hampered in the original homogenate Measurements were considered valid for samples diluted at least 2-fold, although samples were usually diluted 4-fold in this investigation M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 383 Fig Relationship between substrate concentration and enzymatic activity ATP concentration in the reaction mixture was varied from to four times that of standard assay conditions (concentration showed for final concentration in wells) Activity increased up to standard assay conditions, and plateaued upon doubling this concentration, showing that substrate was used under saturated conditions Analysis of the data using a Lineweaver – Burke plot revealed that the Km for the reaction was 0.2 mM were the same as those obtained previously (Wilder et al., 1998) Na/K-ATPase activity in gills was measured by pooling front (1– 4) and back gills (5– 7) and homogenizing in separate batches Fig shows results obtained from left-side gills In front gills, activity in freshwater was 1.62 90.19 mmol ADP/ mg protein per h, increasing slightly in 1/3 seawater (1.889 0.19 mmol ADP/mg protein per h) and 2/3 seawater (2.0990.24 mmol ADP/mg protein per h), and decreasing slightly in full seawater (1.92 90.43 mmol ADP/mg protein per h) However, no significant differences were seen among treatments (P \0.05) In back gills, a similar trend was seen with all values being slightly higher than in corresponding treatments in front gills: activity was 1.8190.20, 2.279 0.19, 2.4690.20, 2.20 0.35 mmol ADP/mg protein per h for freshwater, 1/3 seawater, 2/3 seawater and seawater, respectively Differences were also not significant (P \0.05) among back gill treatments In the second experiment, prawns were acclimated to 1/3 seawater and 2/3 seawater for week, and then transferred back to freshwater In prawns sampled at the end of acclimation, activity in right-side front gills was 1.6090.04 mmol ADP/mg protein per h in 1/3 seawater and 2.05 0.16 mmol ADP/mg protein per h in 2/3 seawater, similar to the results of the first experiment One day after transfer to freshwater, activity was 1.47 90.13 mmol ADP/mg protein per h in the 1/3 seawater group and 2.5990.18 mmol ADP/mg protein per h in the 2/3 seawater group; no significant changes (P \0.05) were observed as a result of this exposure These results, as well as those for back gills are shown in Table Discussion Many previous studies on Na/K-ATPase have been conducted in crustaceans and insects utilizing an assay system based on the measurement of inorganic phosphorus (Ilenchuk and Davey, 1982; Wheatly and Henry, 1987; Kosiol et al., 1988; Ahl and Brown, 1991; Corotto and Holliday, 1996) including a study by Stern et al (1984) on M rosenbergii However, Stern et al (1984) did not 384 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 examine the distribution of activity in the branchial tissue and did not investigate the effects of salinity on enzymatic activity, which was one of the objectives of this study In this study, a kinetic assay system measuring directly the oxidation of NADH as is used in many fish species (McCormick and Bern, 1989; Fig Changes in Na/K-ATPase activty with varying salinity In front left gills (a), activity increased slightly in 1/3 seawater and 2/3 seawater, declining slightly in full seawater However, no significant differences were seen among treatments (P\ 0.05) A similar pattern was seen in back gills (b) Results are show as the mean S.E McCormick, 1996), was adapted for use in the giant freshwater prawn, M rosenbergii The assay system was first characterized for conditions of ouabain and ionic concentrations, pH, and temperature Maximum inhibition of non-specific ATPase activity was observed when ouabain concentrations were mM in the reaction mixture (1.4 mM final) The reaction required the presence of Na+, K+ and Mg2 + in the medium Enzymatic activity increased with increasing temperature, but interestingly, variance of pH did not effect activity and activity remained stable for at least h after homogenization of tissues A Lineweaver– Burke plot showed that the Km for the enzyme was 0.2 mM The same data plotted to observe ATP saturation, showed that maximal enzymatic activity was obtained at a concentration of 0.5 mM ATP, as in Stern et al (1984) These results demonstrated this assay was valid for the determination of Na/K-ATPase activity in M rosenbergii gills Several reports have demonstrated that the posterior gills have higher Na/K-ATPase activity, which is more sensitive to changing salinity (Neufeld et al., 1980; Henry and Cameron, 1982; Holliday, 1988; Kamemoto, 1991; Corotto and Holliday, 1996) In H nudus, activity in gills 6, and were approximately double that of activity in gills – 5, and activity in all gills decreased with increasing seawater concentration (Corotto and Holliday, 1996) Considering this possibility, we measured enzyme activity in individual gills in M rosenbergii, but there were no significant differences among individual gills, indicating an even distribution of activity throughout the branchial tissue Similarly, in the euryhaline freshwater crayfish Pacifastacus leniusculus, Na/K-ATPase activity in all gills – was unvaried (Wheatly and Henry, 1987) Concentration of activity on posterior gills may be a characteristic of euryhaline marine species (Kirschner, 1979; Mantel and Farmer, 1983), which hyper-regulate in dilute seawater M rosenbergii may be more similar in this respect to P leniusculus, which is a freshwater species with lower hemolymph ionic concentrations (Wheatly and Henry, 1987) In freshwater, activity was generally 1.5 mmol ADP/mg protein per h, which was lower than that in many crustacean species already reported (Towle et al., 1976; Wheatly and Henry, 1987; Holliday, 1988; Corotto and Holliday, 1996) In other reports, activity for the most part was determined based on differences in inorganic phos- M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 385 Table Gill Na/K-ATPase activity in prawns exposed to freshwater after acclimation to 1/3 seawater and 2/3 seawater for weeka Front gills Back gills a 1/3 Seawater Freshwater 2/3 Seawater Freshwater 1.60 0.04 1.90 0.15 1.47 0.13 2.17 0.31 2.05 0.16 2.48 0.17 2.59 0.18 2.66 0.28 Results are shown for right-side gills as the mean phate in the presence and absence of ouabain, giving units of mmol Pi/mg protein per or h, but final outcome can be considered comparable In H nudus, enzyme activity was approximately 10 and 25 mmol Pi/mg protein per h in anterior and posterior gills respectively, in animals acclimated to 25% salinity, but was decreased to half of these levels in full seawater (Corotto and Holliday, 1996) In the isopod, Idotea wosnesenskii, which has five pairs of biramous gills referred to as pleopods, activity varied above mmol Pi/mg protein per h in front pleopods under all salinities, and was 35 mmol Pi/mg protein per h decreasing to about 15 mmol Pi/mg protein per h in full seawater (Holliday, 1988) Reasons for lower observed enzymatic activity in M rosenbergii compared to other crustacean species may be related to methodology; similar low values have been obtained in fish species such as the coho salmon Oncorhynchus kisutch (McCormick and Bern, 1989), Atlantic salmon Salmo salar (McCormick, 1996) using this kinetic assay system In this study, animals acclimated to freshwater were transferred to 1/3 seawater and 2/3 seawater for week and to full seawater for days In addition, animals acclimated to 1/3 seawater and 2/3 seawater for week were transferred back to freshwater However, none of these treatments produced significant changes in Na/K-ATPase activity in the gills As reviewed by Kamemoto (1991) and Pequeux (1995), it is often the case that Na/K-ATPase increases in animals adapting from higher salinity to lower salinity environments In H nudus, activity was highest in animals adapted to freshwater and 25% seawater, significantly decreasing in those adapted to full seawater (Corotto and Holliday, 1996) A similar pattern is seen for I wosnesenskii (Holliday, 1988) In P leniusculis, however, there were no alterations to Na/K-ATPase activity in most of the gills at 0, 350 or 750 mOsm; only two gills of a total of seven had activity reduced 20% in 750 mOsm (Wheatly and Henry, 1987) In M rosenbergii, activity did not increase significantly in response to higher salinity in contrast to the example of several crab species (Mantel and Landesman, 1977; Spencer et al., 1979) However, Na/K-ATPase activity usually increases upon exposure to dilute mediums (Savage and Robinson, 1983); it is considered that in the natural environment, animals inhabiting saline environments when exposed to more dilute ones must actively engage in ion uptake to maintain hemolymph ionic concentrations Na/K-ATPase is located on the basolateral membrane of the branchial tissue (Towle and Todd Kays, 1986) The distribution of activity appears directly related to the location of salt-transporting cells In marine crabs such as Callinectes sapidus, chloride cells are located in the posterior gills (Copeland and Fitzjarrell, 1968), where Na/K-ATPase activity is most sensitive to salinity fluctuations In crayfish species, the chloride cells are evenly distributed among gills (Dickson and Dillaman, 1985) However, the mechanism of ion transport is more complicated than what can be inferred from the basic stoichiometry of the Na/K-ATPase reaction In this reaction, three Na+ ions are extruded from the cytoplasm with the uptake of two K+ ions with the hydrolysis of ATP More active Na/K-ATPase activity should alter the balance of K+ in the cytoplasm, allowing other transport mechanisms and downhill flow of K+ to occur Pequeux (1995) has reviewed the existence of Cl− channels, a K+ leak pathway, an Na+ –K+ – 2Cl− cotransport system The role of the carbonic anhydrase system that provides for the exchange of H+/ HCO− counterions for the Na+/Cl− uptake is also considered important (Wheatly and Henry, 1987) Besides the functioning of gills, the ability to control urine concentrations, i.e to produce hypoosmotic urine such as in freshwater crayfishes (Bryan, 1960), may be more important in a freshwater species such as M rosenbergii As there is relatively little information on similar prawn species regarding the role of Na/K- 386 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 ATPase in overall osmoregulatory function, it is difficult to compare and fully interpret the results obtained in this investigation However, the fact that Na/K-ATPase activity is generally low in M rosenbergii, and that activity does not change in response to salinity changes is of much significance Whereas in other crustacean species already examined, Na/K-ATPase activity is essential in allowing the animal to exist in dilute environments, in M rosenbergii probably does not have as much of a necessity to engage in active uptake of ions from the ambient environment The observed low levels of enzymatic activity are probably sufficient to enable M rosenbergii to inhabit fresh and brackishwater areas It will be interesting to compare M rosenbergii to other freshwater prawn species exhibiting a similar range of habitats and osmoregulatory patterns; it is possible that such species would constitute a group exhibiting similar characteristics relating to Na/K-ATPase and other ion-transport systems The role of the antennal gland and other salttransporting tissues may be more important in such species However, it is still necessary to consider that Na/K-ATPase activity is important during the early developmental stages, as Charmantier (1998) has reviewed that the development of osmoregulatory ability is often correlated with increased Na/ K-ATPase activity After hatchout, M rosenbergii larvae require brackish water for survival, and following metamorphosis to the juvenile stage after about month, they are able to return freshwater areas where they continue further growth Based on this, it is obvious that is necessary to develop the ability to survive in freshwater in the face of dilution of the hemolymph This may be due to the differentiation of tissues related to osmoregulatory function, including the antennal gland, while the presence of Na/K-ATPase may at the same time be a requirement In the brine shrimp Artemia salina, the ability to osmoreglate is related to de novo synthesis of Na/K-ATPase and membrane activation during development in the nauplius stages (Conte et al., 1977; Peterson et al., 1978) We are currently investigating how Na/K-ATPase activity develops during egg and larval development in M rosenbergii There is also the possibility that Na/K-ATPase has a role in controlling osmoregulation during the molting process in M rosenbergii In A salina (Ahl and Brown, 1991), methyl farnesoate and juvenile hormone stimulated Na/K-ATPase in larval homgenates to the same as pre- and postmolting levels This suggested a role in increasing body osmolarity in order to uptake more water just after ecdysis Towle and Mangum (1985) also showed that Na/K-ATPase activity was highest just prior to molting and in the post-molt stages In subsequent investigations, it will be necessary to examine the relationship between osmoregulatory function and molting, as well as the presence and functioning of other ion transport systems in M rosenbergii in order to obtain a more complete understanding of osmoregulatory mechanisms in freshwater prawns Acknowledgements The authors express gratitude to Active Rise, Ltd., Miyazaki City for providing the experimental animals We thank Prof Toyoji Kaneko and S Hasegawa, the Ocean Research Institute, the University of Tokyo, for assistance in setting up the Na/K-ATPase assay used in this study We also thank M Shigemitsu, the Japan International Research Center for Agricultural Sciences (JIRCAS) for technical assistance and rearing of the experimental animals D.T.T Huong and M Atmomarsono were Visiting Research Fellows at JIRCAS and W.-J Yang was a Science and Technology (STA) Fellow while conducting this study This investigation was conducted as part of the basic research component of an international collaborative program between JIRCAS and Cantho University, Vietnam aiming to improve seed freshwater prawn culture technology We thank Dr M Maeda, Director of the JIRCAS Fisheries Division and Dr N.T Phuong, Vice Director of the Institute for Marine Aquaculture, College of Agriculture, Cantho University, for their guidance and support during this study References Ahl, J.S.B., Brown, J.J., 1991 The effect of juvenile hormone III, methyl farnesoate, and methoprene on Na/K-ATPase activity in larvae of the brine shrimp Artemia Comp Biochem Physiol 100A, 155 – 158 Bryan, G.W., 1960 Sodium regulation in the crayfish Astacus flu6iatilis III Experiments with NaClloaded animals Exp Biol 37, 113 – 128 M.N Wilder et al / Comparati6e Biochemistry and Physiology, Part A 125 (2000) 377–388 Castille, F.L., Lawrence, A.L., 1981a The effect of salinity on 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leniusculus J Exp Biol 113, 73 – 86 Wilder, M.N., Ikuta, K., Atmomarsono, M., Hatta, T., Komuro, K., 1998 Changes in osmotic and ionic concentrations in the hemolymph of Macrobrachium rosenbergii exposed to varying salinities and correlation to ionic and crystalline composition of the cuticle Comp Biochem Physiol 119A, 941 – 950 ... 377–388 examine the distribution of activity in the branchial tissue and did not investigate the effects of salinity on enzymatic activity, which was one of the objectives of this study In this study,... measurement of Na/K -ATPase activity, as a function of ouabain concentration in the reaction mixture, was firstly examined Under the standard conditions described above, ouabain concentrations in the. .. in the determination of Na/KATPase activity in salmonid and other fishes Many previous reports in crustaceans and other invertebrates have utilized methodology based on the determination of inorganic