Characterization of nak ATPase in macrobrachium rosenbergii and the effects of changing salinity on enzymatic activity

Characterization of Na/K-ATPase in Macrobrachiumrosenbergii and the effects of changing salinity on enzymatic activity aJapan International Research Center for Agricultural Sciences, Min

Characterization of Na/K-ATPase in Macrobrachium

rosenbergii and the effects of changing salinity on enzymatic

activity

aJapan International Research Center for Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries,1 - 2Ohwashi,

Tsukuba, Ibaraki Prefecture305 - 8686, Japan

bInstitute 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 Kmof 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 1 week Na/K-ATPase activity in front gills was 1.62 9 0.19 mmol ADP/mg protein per h in freshwater, and was seen to increase slightly in 1/3 seawater (1.88 9 0.19 mmol ADP/mg protein per h) and 2/3 seawater (2.09 9 0.24 mmol ADP/mg protein per h), decreasing slightly in full seawater (1.92 9 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

1 Introduction

The giant freshwater prawn, Macrobrachium

rosenbergii, like many other species of the

Macro-brachium genus, is primarily a freshwater species

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 aqua-culture 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 re-sponds to changing salinity and gain more

knowl-* Corresponding author Tel.: + 298-386630; fax: +

81-298-386316.

E-mail address: marwil@jircas.affrc.go.jp (M.N Wilder)

1 Present address: Research Institute for Coastal

Aquacul-ture, Maros, South Sulawesi, Indonesia.

1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc All rights reserved.

PII: S 1 0 9 5 - 6 4 3 3 ( 0 0 ) 0 0 1 6 2 - 8

edge of basic underlying mechanisms of

osmoreg-ulatory 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

concentra-tions 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

con-centrations, for example, in M rosenbergii

(Castille and Lawrence, 1981b), Penaeus monodon

(Ferraris et al., 1987), P setiferus and P

styliros-tris (Castille and Lawrence, 1981a).

As Pequeux (1995) has reviewed, various

salt-transporting tissues, including the body wall,

gas-trointestinal tract, and excretory organs play a

vital role in osmoregulation, but the gill

epithe-lium is expected to be of foremost significance in

maintaining hemolymph NaCl balance in

Crus-tacea Indeed, a number of studies have examined

the role of branchial Na/K-ATPase activity in

osmoregulation in various species including

sev-eral 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

spe-cies, including Artemia salina have demonstrated

increases in enzymatic activity during the process

of adaptation from higher to lower salinities

(Hol-liday et al., 1990) M rosenbergii frequently

mi-grates between freshwater and estuarine or

brackish water areas for purposes of spawning,

and is therefore considered to make use of

os-moregulatory ability in adapting to salinity

changes in this range We conducted this

investi-gation 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

en-zymatic 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/K-ATPase activity in salmonid and other fishes Many previous reports in crustaceans and other invertebrates have utilized methodology based on the determination of inorganic phosphorus re-leased after the addition of ATP to tissue ho-mogenates (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

con-ditions, and characterized optimal parameters for ouabain inhibition, ionic concentrations, pH, and temperature We then applied this system to mea-sure changes in Na/K-ATPase activity in gill tis-sue in response to changing salinity in prawns initially acclimated to fresh or brackish water

2 Materials and methods

2.1 Animals, rearing and sampling

M rosenbergii were generously supplied by

Ac-tive 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 2 weeks prior to use in experimentation Animals were then transferred to 60-l treatment tanks contain-ing dividers to hold four prawns per tank Treat-ments 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 artifi-cial seawater mix (Aqua OceanR

, JPDAO-NO 1100) and pre-circulated tapwater Male and fe-male prawns in the intermolt stages ranging from

15 to 40 g body weight (BW) were employed In the first experiment, prawns (four to six individu-als for each determination; BW = 23.25 9 3.57) were maintained under these treatments for 1 week (or 3 days only for those individuals ex-posed to full seawater) Hemolymph was sampled

by cardiac puncture at 0 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/K-ATPase determination In a second experiment,

after initial acclimation to freshwater, prawns

(four individuals for each determination; BW =

29.06 9 4.50 g) were then acclimated to 1/3

seawa-ter or 2/3 seawaseawa-ter for 1 week They were then

transferred back to freshwater and gill tissue was

dissected out after 1 day to examine the effects of

freshwater exposure on Na/K-ATPase activity

Hemolymph was sampled at the start of the

ex-periment, after 1-week acclimation in 1/3 seawater

or 2/3 seawater, and 1 day after transfer back to

freshwater

For the validation of the assay system, male

prawns (BW = 22.8 9 3.46) were employed, and

four individuals were used for each type of

deter-mination, e.g maximal inhibition by ouabain,

optimum ionic concentrations in the reaction

me-dia, pH, temperature, effects of gill quantity on

enzymatic activity, effects of substrate

concentra-tion, and time – activity relationship In addiconcentra-tion,

separate gill filaments in four larger-sized males

ranging from 35 to 40 g (BW = 38.11 9 2.37) were

sampled and enzymatic activity in each filament

was measured in order to determine whether

ac-tivity 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 1 – 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

solu-tion of 20 mM was prepared by dissolving

oua-bain 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

(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 0 or 2 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 0 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) Ho-mogenates were then centrifuged at 6 500 rpm for

2 min The supernatant was diluted from 2- to 8-fold to the appropriate range for activity mea-surement, but this was usually 4-fold The assay was conducted by adding 10 ml sample or stan-dard in duplicate with 50 ml salt solution and 140

ml assay mixture containing 0 or 2 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 min at 340

nm in a Bio-Rad Model 3550-UV microplate reader The activity of ouabain-sensitive Na/K-ATPase was determined as the difference in the rate of NADH oxidation in the presence and absence of ouabain as mOD/10 ml per min divided

by the standard curve of around 21 mOD/nmole ADP and expressed initially as nmoles ADP/10 ml per min 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 condi-tions described below were employed, varying one factor while keeping all other parameters con-stant Inhibition by ouabain corresponding to the actual measurement of Na/K-ATPase activity, as

a function of ouabain concentration in the reac-tion mixture, was firstly examined Under the standard conditions described above, 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

concen-tration 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

concentra-tion of total activity observed in the absence of

ouabain The outcome is described in detail in

Section 3, but maximal inhibition was observed at

2 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

concentra-tions 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,

rela-tionship of substrate concentration and activity,

and time after homogenization on enzymatic

tivity were examined Optimal conditions for

ac-tual analysis of osmoregulatory function in

response to changing salinity were set according

to the above results After setting assay condi-tions, individual gills were measured to determine whether activity varies according to gill position; however, gills 1 and 2 were assayed together due

to small size, and gills 3, 4, 5, 6, and 7 were measured individually Differences were not sig-nificant, and thereafter, in actual experimentation, front gills (1 – 4) and back gills (5 – 7) were com-bined separately for left and right sides, and ho-mogenized in separate batches

2.5 Statistical analysis Duncan’s multiple range test (with P B 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

3 Results

3.1 Assay 6alidation

Fig 1 shows that percent inhibition of total ATPase activity increased with increasing concen-trations of ouabain in the salt solution, reaching a maximum at a concentration of 2 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 2

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 0 mM, increasing to maximal activity at 22.5

mM and plateauing from 45 mM When K+ was

0 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 0 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 +, respec-tively (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 2

Fig 1 Determination of standard ouabain conditions

Oua-bain 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 2

mM ouabain in the assay mixture Results are show as the

mean 9 S.E.

Fig 2 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 Mg 2 + (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 Mg 2 + , respectively (corresponding to 180, 80, and 10 mM

in original salt solution) Results are show as the mean 9 S.E.

Fig 3 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 9 S.E.

Dependence of enzymatic activity on

tempera-ture is shown in Fig 3 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

tem-perature at which the experimental animals were

maintained There was no dependence of

enzy-matic activity on pH in the range tested (data not

shown); subsequent determinations were

con-ducted 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

investi-gation The relationship between substrate

con-centration 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 Kmfor the reaction was 0.2 mM Finally, the effects of time on enzymatic activity were examined Enzymatic activity was assayed at 0, 1, 3, 6 and 24 h after homogeniza-tion, keeping extracts at 4°C when not being used Activity did not decline in extracts kept for up to

6 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 osmolal-ity in response to varying salinosmolal-ity as in our previ-ous investigation (Wilder et al., 1998) In freshwater and 1/3 seawater, hemolymph osmolal-ity 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 4 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.

Fig 5 Relationship between substrate concentration and enzymatic activity ATP concentration in the reaction mixture was varied from

0 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 Kmfor 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 6 shows

results obtained from left-side gills In front gills,

activity in freshwater was 1.62 9 0.19 mmol ADP/

mg protein per h, increasing slightly in 1/3

seawa-ter (1.88 9 0.19 mmol ADP/mg protein per h) and

2/3 seawater (2.09 9 0.24 mmol ADP/mg protein

per h), and decreasing slightly in full seawater

(1.92 9 0.43 mmol ADP/mg protein per h)

How-ever, 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 1.81 9 0.20, 2.27 9 0.19, 2.46 9 0.20,

2.20 9 0.35 mmol ADP/mg protein per h for

fresh-water, 1/3 seafresh-water, 2/3 seawater and seafresh-water,

respectively Differences were also not significant

(P \ 0.05) among back gill treatments.

In the second experiment, prawns were

accli-mated to 1/3 seawater and 2/3 seawater for 1

week, and then transferred back to freshwater In

prawns sampled at the end of acclimation, activity

in right-side front gills was 1.60 9 0.04 mmol ADP/mg protein per h in 1/3 seawater and 2.05 9 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 9 0.13 mmol ADP/mg protein per h in the 1/3 seawater group and 2.59 9 0.18 mmol ADP/mg protein per h in the 2/3 seawater group; no

signifi-cant 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 1

4 Discussion

Many previous studies on Na/K-ATPase have been conducted in crustaceans and insects utiliz-ing 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

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;

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 tem-perature Maximum inhibition of non-specific ATPase activity was observed when ouabain con-centrations were 2 mM in the reaction mixture (1.4 mM final) The reaction required the presence

of Na+, K+ and Mg2 + in the medium Enzy-matic activity increased with increasing tempera-ture, but interestingly, variance of pH did not effect activity and activity remained stable for at least 6 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 concentra-tion 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 pos-terior gills have higher Na/K-ATPase activity, which is more sensitive to changing salinity (Neu-feld et al., 1980; Henry and Cameron, 1982; Holl-iday, 1988; Kamemoto, 1991; Corotto and

Holliday, 1996) In H nudus, activity in gills 6, 7

and 8 were approximately double that of activity

in gills 1 – 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

differ-ences 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 1 – 7 was unvaried (Wheatly and Henry, 1987) Concentration of activity on poste-rior gills may be a characteristic of euryhaline marine species (Kirschner, 1979; Mantel and Farmer, 1983), which hyper-regulate in dilute

sea-water M rosenbergii may be more similar in this respect to P leniusculus, which is a freshwater

species with lower hemolymph ionic concentra-tions (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 deter-mined based on differences in inorganic

phos-Fig 6 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 9 S.E.

Table 1

Gill Na/K-ATPase activity in prawns exposed to freshwater after acclimation to 1/3 seawater and 2/3 seawater for 1 week a

1.60 9 0.04

Back gills

a 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 min 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

accli-mated to 25% salinity, but was decreased to half

of these levels in full seawater (Corotto and

Holl-iday, 1996) In the isopod, Idotea wosnesenskii,

which has five pairs of biramous gills referred to

as pleopods, activity varied above 5 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

ob-served enzymatic activity in M rosenbergii

com-pared 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 1 week and to full seawater for 3 days In

addition, animals acclimated to 1/3 seawater and

2/3 seawater for 1 week were transferred back to

freshwater However, none of these treatments

produced significant changes in Na/K-ATPase

ac-tivity 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

environ-ments In H nudus, activity was highest in

ani-mals 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

sig-nificantly 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 ex-posure to dilute mediums (Savage and Robinson, 1983); it is considered that in the natural environ-ment, 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 sensi-tive 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 stoi-chiometry 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− cotrans-port system The role of the carbonic anhydrase system that provides for the exchange of H+

/ HCO3− 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 simi-lar prawn species regarding the role of

Na/K-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

signifi-cance Whereas in other crustacean species

al-ready 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

environ-ment The observed low levels of enzymatic

activ-ity 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

salt-transporting 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

osmoregula-tory 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

af-ter about 1 month, they are able to return

fresh-water 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 lar-val homgenates to the same as pre- and post-molting 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 osmoregula-tory 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 experimen-tal 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 (JIR-CAS) for technical assistance and rearing of the experimental animals D.T.T Huong and M At-momarsono were Visiting Research Fellows at JIRCAS and W.-J Yang was a Science and Tech-nology (STA) Fellow while conducting this study This investigation was conducted as part of the basic research component of an international col-laborative 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

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Artemia Comp Biochem Physiol 100A, 155 – 158.

Bryan, G.W., 1960 Sodium regulation in the crayfish

Astacus flu6iatilis III Experiments with

NaCl-loaded animals Exp Biol 37, 113 – 128