The ammonium excretion of the shore crab, carcinus maenas, in relation to environmental osmotic conditions

Netherlands journal of Sea Research 15(2): 273-283 (1982)

THE AMMONIUM EXCRETION SHORE CRAB, CARCINUS MAENAS, TO ENVIRONMENTAL OSMOTIC

OF THE IN RELATION CONDITIONS

by D. H. S P A A R G A R E N Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands

CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . II. Material and Methods . . . . . . . . . . . . . . . . . . . . . . III. Results and Interpretation . . . . . . . . . . . . . . . . . . . . a. Ammonia concentrations in the blood of Carcinus maenas . . . . . . . b. Ammonia excretion of Carcinus maenas in relation to osmotic conditions. c. Urea and uric acid in the blood of Carcinus maenas . . . . . . . . . IV. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . V. References . . . . . . . . . . . . . . . . . . . . . . . . . . .

273 274 275 275 278 280 282 283

I. I N T R O D U C T I O N I n a q u a t i c a n i m a l s N H 4 + is the m a i n end p r o d u c t of protein m e t a b o lism. I n the b o d y fluids it plays an i m p o r t a n t p a r t in the stabilization of the p H , essential in g o v e r n i n g the reaction rates of m a n y m e t a b o l i c processes. Its significance for various physiological processes is closely related to its toxicity. T h e N H 4 + tolerance of most animals is low. I n invertebrates internal c o n c e n t r a t i o n s higher t h a n 10 m m o l ' l t c a n rarely be survived; v e r t e b r a t e s are generally even m o r e sensitive (CAMPBELL,1973). Besides the a b o v e m e n t i o n e d effect of N H 4 + on the p H of b o d y fluids, the toxicity of N H 4 ÷ also results from the inhibition o f active t r a n s p o r t systems for N a ÷, C1 - a n d H C O a - (e.g. GUPTA et al., 1977). I n view of the toxicity of a m m o n i a it is generally assumed that in aquatic a n i m a l s the m e t a b o l i c a l l y p r o d u c e d a m m o n i a is released to the e n v i r o n m e n t at the s a m e rate at which it is p r o d u c e d . T h i s release m i g h t be a passive process as N H 4 + c o n c e n t r a t i o n s in n a t u r a l waters are low which in turn relates to the fact t h a t N H 4 + tends to be converted to oxidated n i t r o g e n c o m p o u n d s ( N O 2 -, N O 3 -) in aerobic environments. T h e a m m o n i a concentrations which c a n be found in sea w a t e r are locally v e r y v a r i a b l e (Tijsses, personal c o m m u n i c a t i o n ) but, generally, low e n o u g h to ensure the loss of N H 4 + l?om animals by passive diffusion.

274

D.H. SPAARGAREN

Although the low environmental NH4 + concentrations do not require an active excretion of NH 4 + one may wonder whether the efflux of this metabolically active substance proceeds solely by passive diffusion. The absence of an active regulation, stabilizing the internal NH 4 + concentrations, implies that the fluctuations in the external NH 4 + concentrations are reflected in the internal body fluids. It follows that an active transport system prevents accumulation of produced NH 4 +, and possibly also prevents excessive losses. Such an active transport of NH 4 + has indeed recently been found in annelids and molluscs (MA6NUM et al., 1978). Nitrogen metabolism in general is frequently studied in close connection to water availability in the environment. Within the aquatic environment, however, not much is known about the amount of NH4 + production at various osmotic conditions. In terrestrial animals ammonia plays a part in maintaining the alkali reserve: after the intake of a large quantity of water, the alkaline ions Na + and K + are replaced by NH4 + in the formation of urine to prevent their loss. There are indications that a similar process takes place in a marine prawn, Penaeus japonicus, when water turnover is increased during exposure to hypotonic salinities (SPAARGAREN, RICHARD & CECCALDI, 1982). No data are available concerning the question to which extent internal NH4 + concentrations are affected by the extra loss of NH 4 + under these circumstances. This paper presents data on the NH 4 + concentrations in the blood of shore crabs indicating the regulatory capabilities of this species, as well as data on the NH 4 ÷ excretion at various osmotic circumstances indicating that the NH4 + loss is inversely related to environmental salinity and strongly temperature dependent. II. MATERIAL AND METHODS The experiments were carried out with shore crabs, Carcinus maenas (L.), collected in the Wadden Sea, near the island Texel. In the laboratory the animals were kept in groups of 7 in 35 1 aquaria provided with coal filters for purification and aeration of the water. Using sea water of 6 different salinities from ca 5 to 40~o S, 2 series were made, one kept at 4 ° C and one at 20 ° C. Adult male, t~male and ovigerous female intermoult specimens were used with weights varying between 15 and 40 g. Blood samples were collected after 6 days of acclimatization by piercing the membrane at the base of the 5th walking leg with a glass capillary. After collection of about 1 ml of blood the animals were placed back in their aquaria. Samples of sea water media were collected at 6 and 15 days after incubation. Both blood and medium samples were used immediately tor the determinations.

AMMONIUM

EXCRETION

275

CARCINUS

Ammonia concentrations in the blood and medium samples were determined with a NH 3 selective electrode (Philips, type IS 570-NH3) connected to a digital pH meter (Philips, type PW 9409). Directly preceeding the measurement, 50/~1 of an alkali solution (1 M NaOH, 1 M EDTA) was added to a 500/L1 sample to be analysed. The millivoh readings of the pH meter were converted to log NH 3 values using a calibration line, derived empirically in the concentration range between 10-s and 10 ~* mol NH 3 • 1-1 at the start of each experiment. The sum of ammonia and urea concentration was determined by conversion of urea to NH 4 +. To this end 100/21 of a urease suspension (activity ~> 10 U ' m l - 1 ) , containing also 50 mmol'1-1 phosphate buffer, was added to a 500/~1 sample. After incubation during 10 min at ca 40 ° C the solution was made alkaline by addition of 5/21 10 N NaOH. Subsequently NH 3 was measured as described above. In the calculations the dilutions associated with the additions of alkali solution and enzyme suspension were taken into account. Uric acid concentrations were determined spectrophotometrically at 405 nm. After incubation of 500/xl samples together with 5 ml reagent mixture (ca 700 U ' m 1 - 1 catalase, 20 m m o l ' l -z acetylaceton, 1.7 mol' 1-1 methanol, 0.6 mol' 1-1 ammonium phosphate buffer, pH 7) for 1 hour at 37 ° C, 20/21 uricase (5 U" m1-1, 50o<~ glycerol) was added to one half of the solution, while the other half was used as a sample blank. III. RESULTS AND INTERPRETATION a.

AMMONIA

CONCENTRATIONS

IN

THE

BLOOD

OF

CARCINUS

MAENAS

The ammonia concentrations in the blood of C a r c i n u s m a e n a s appear to be fairly constant, independent of either temperature or salinity (Fig. la). The average values range between 0.25 and 0.55 mmol NH4 ÷ -1-1; for the same species similar values (between 0.22 and 0.61 mmol" 1-1) were mentioned by SourrERBICg. (1935). There is a slight, but not significant, tendency for the blood NH~ + values to become lower at higher salinities, which reflects the lower ammonia concentrations in the surrounding water at these salinities (Fig. lb). In the external media there is also a marked difference between the two series at respectively low (4 ° C) and high (20 ° C) temperature. The differences in external NH4 + concentrations, as related to temperature and salinity, are hardly reflected in the blood NH4 + concentrations (Fig. 2). At low external NH4 + concentrations (below ca 0.4 mmol" 1-1) the blood NH4 + concentrations are hyperionic compared to the environ-

D.HS .PAARGAREN

276

mental values. The occurrence of active NH4 + excretion might be suggested by 2 hypo-ionic values at high external ammonia concentrations. To obtain evidence for the hypothesis that active transport is in[NH4+]i, mmol/I

0.8 ¸

0.6 ¸

iI

0~.'

0.2 ¸

0 IN H4+:];I, m r n o l l l

0.8-

/l 0.6-

A/~ 2

0r

0,2O

b

\.jA... °

~ ° ~ o

Fig. I. A m m o n i a concentration (mmol'l -I) in Carcinus maenas aller 6 days of acclimatization to various salinities at 4 ° C (G) and 20 ° C (A). a. In blood; standard deviations indicated (n = 6). b. In external medium.

AMMONIUM

EXCRETION

277

CARCINUS

[NH4+] mmol/I

0.8-

0.6-

OA-

0.2-

°o

'

d5

,io [NH~]. . . . . ,/,

NH4 ] i , mmol/I

15.-

~

b

1.0-

00

0.5

1.O

1.5

2.0

IN H4*] e, retool/I

2.5

Fig. 2. Blood ammonia concentration (mmol' 1-1) in Carcinus maenas in rclation to the ammonia concentration in the external medium (abscissa). a. Animals acclimated to various salinities at 4 ° C (C)) and 20 ° C ( A ) . b. Animals acclimated to 29.4%0 S at 4 ~' C and various external NH4 + concentrations (obtained by addition of NH~C1 to the water).

278

D. H. SPAARGAREN

volved in the stabilization of the blood N H 4 ÷ concentrations, an experiment was carried out in which the N H 4 + concentration of the external m e d i u m was raised artificially by the addition of various amounts-of NH4C1. It appears then, that even at very high external NH4 + concentrations Carcinus maenas is able to maintain a low and almost stable NH4 + concentration in the blood (Fig. 2b). O n l y at external NH4 + concentrations above 1.5 mmol" 1-1 blood N H 4 ÷ values are found which are significantly above the normal range. The well developed powers of Carcinus maenas to stabilize the blood N H 4 ÷ concentrations seem useful regarding the toxicity of ammonia. I t is questionable whether in natural circumstances the species will routinely meet the high external NH4 ÷ concentrations as experimentally created in the last experiment. I f so, the remarkable capabilities for N H 4÷ regulation will indeed be advantageous to the animals. b. AMMONIA

EXCRETION O F C A R C I N U S M A E N A S IN R E L A T I O N OSMOTIC CONDITIONS

TO

T h a t environmental N H 4 ÷ concentrations m a y be variable was already evident from Fig. lb, showing the external a m m o n i a concentrations 6 days after incubation of 7 animals in 32.8 1 of water. Especially at lower salinities and at the higher temperature, a m m o n i a levels, which at the start of the experiment were below 0.01 mmol" 1 1, are increased considerably. After 15 days the effect is even more pronounced (Fig. 3). Apart from some irregularities almost linear increases in external ammonia concentrations appear. Eventually one expects a steady state to be reached when production of NH4 + equals microbial oxygenation [NH4]e, mmolll A 79%1.0-

~ 31.9%o A 38.0%° A

A

242%°

A 42.5 °X~

32.9*/** 212/~

0

o

.~

~ a

6

~Q~

1~ doys

Fig. 3. C h a n g e s in external NH4 + concentrations (mmol. l -1) o f Carcinus maenas after i n c u b a t i o n (7 animals in 32.8 1) at 4 ° C ( O ) a n d 20 ° C ( A ) , and at indicated salinities

(%0 s).

279

AMMONIUM EXCRETION CARCINUS

and other losses, but under the circumstances described the data obtained here do not show yet such equilibration. From the increases in external ammonia concentration, the water volume and the weight of the animals present, one can derive the NH4 ÷ efllux at various osmotic conditions (Fig. 4). The patterns obtained at the 2 different temperatures are much the same, showing an inverse relation with salinity and a strong influence of temperature. Although the relations look rather complex they may be interpreted by assuming a close connection between NH4 ÷ efllux and extracellular ion regulation: at intermediate salinities (between 15 and 30%0 S) at which the blood ion concentrations are regulated (SHAw, 1961; ENGELSMA,1973; ZANDERS, 1980) the general increase of the ammonia eftlux towards lower salinities is interrupted. At lower salinities ( < 15 ~o S), when blood ion concentrations decrease, NH4 + efllux increases, whereas at higher salinities ( > 30~o S), when blood ion concentrations increase, NH4 + efllux decreases. The positive relation to temperature and the negative relation to salinity of the ammonia efflux works out in such a way that at winter conditions (when the animals are exposed to low temperatures and high salinities) the NH4 + efllux is reduced to almost zero. It is clear that the reduction in protein catabolism in these circumstances will be advantageous to the animals. The close connection between the NH 4 + efl]ux and blood ion concentration favours the hypothesis that the excretion of ammonia plays a part in the conservation of the alkali reserve. At intermediate salinities, where blood Na + and K ÷ concentrations are maintained at constant, (~NH<.+,mg NH 3 .kg-l.doy -~

zoo,

o

~

ib

/

~

~o

Fig. 4. Ammonia excretion (rag. kg-1. d 1) o f Carcinus 4° C (±) and 20° C (•).

.

~o maenas

~:o %.,s

at various salinities at

280

D.H. SPAARGAREN

hyper-ionic levels by active transport of these ions, the ammonia ettlux remains constant. At lower salinities, when blood Na ÷ and K ÷ can no longer be maintained at a constant level, NH4 ÷ is excreted at a higher rate, partly replacing Na ÷ and K + loss in urine. At high salinities alkali levels in the blood rise, concurrent with the external concentrations, and NH4 + etflux drops to low levels. At these high salinities the effect of temperature on NH4 + efllux is high. C. U R E A

AND

URIC

ACID

IN

THE

BLOOD

OF

CARCINUS

MAENAS

The increased N H 4 + ettlux at lower salinities and higher temperature take place at a more or less constant NH4 + level in the blood. It may thus be that the stability of the blood NH4 + level at various osmotic conditions is merely regulated by the variable efllux to the environment. Stabilization of the blood NH4 + level may also be effected by synthesis to urea (or other nitrogenous end products) and subsequent excretion of these substances. To verify whether these other regulatory mechanisms are also active, blood samples were investigated on urea and uric acid content. Despite the high standard deviations the results strongly suggest that at decreasing salinities an increasing amount of urea is present in the blood (Fig. 5). The amount of urea is not related to temperature (Fig. 5b), but only determined by the blood NH4 + level. As soon as blood NH4 ÷ concentration becomes higher than 0.28 mmol-1-1 a rapidly increasing amount of urea is found in the blood whereas below this value no urea is present (Fig. 6). Therefore, the formation of urea may be an effective mechanism to stabilize the blood NH4 + concentration to values between 0.25 and 0.55 mmol" 1-a. The presence of strongly variable blood urea concentrations, ranging between 0 and 1.5 mmol" 1-1, have been shown previously in several crustacean species (e.g. FLORKIN, 1960). The detoxication of NH4 + to urea, commonly described for vertebrates, was not known to occur also in aquatic invertebrates, probably because it only takes place at special osmotic conditions when NH4 + production is extremely high. The concentrations of urea stay below the 1 mmol. 1-2 level. The values found are therefore too low to have a significant osmotic function, comparable to that reported for elasmobranchs. Measurements on the blood uric acid concentrations at various osmotic conditions showed low levels, ranging between 120 and 140 /~mol' 1-1, independent of temperature and salinity. It is therefore not likely that this substance, although indeed formed in this species, plays a part in the regulation of blood NH4 + levels. From the observations as described above it is not unambiguously clear whether urea merely performs a.buffering function in the regu-

AMMONIUM

EXCRETION

281

CARCINUS

lation of blood N H 4 ÷ levels or whether it performs a significant pathway for NH4 ÷ excretion. However, considering the role of NH4 + excretion in alkali conservation the latter does not seem very plausible. [NH4÷ + urea - N H4+I i, mmol/I

1.6-

1.2-

O.B-

0.4-

0 Eureo -NH4~] i , mmol/I

\ 0.8-

\ \

\

\ \

0.6-

\

A

\

\ \\

A

\

\

\

\

\ \\ \

0P-

\

\

\ \

\\ \\

0.2-

li

A

"x

o

,b

~

o

&

~o

Fig. 5. a. Free NH4 + plus urea NH4 + in t h e b l o o d of Carcinus maenas acclimated to v a r i o u s s a l i n i t i e s at 4 ° C ( 0 ) and 20 ° C ( A ) . b. U r e a NH4 +, same experiment, data c o r r e c t e d tbr fi~ee NH4 + (Fig. la).

282

D.H. SPAARGAREN

T h e r e f o r e , it is more likely that the formation o f urea provides additional stability in blood NH4 + concentrations, reducing their toxic toxic effects. [NH4++ureo-NH4~],mmolll 1.5o

/ /

/i

1.0"

urea- NH~ z~

o O5-

/

#////

,, / / ' / / / /

~,

/1

//

7/

~/

%,, ee NH~

// /

//

//

o:5

Fig. 6. Free- and urea-generated NH4 + concentrations (mmol. 1-1) in the blood of Carcinus maenas (Fig. 5a) in relation to the free NH4 + concentration in the blood (Fig. 1a), found at various environmental conditions, showing the conversion of tiee NH4 ÷ into urea above 0.28 mmol' 1-1 NH4+ concentrations in the blood.

IV. SUMMARY A m m o n i a concentrations were measured in blood and external media of shore crabs, Carcinus maenas, acclimated to 6 different salinities at high (20 ° C) and low (4 ° C) temperatures. It is seen that e n v i r o n m e n t a l osmotic conditions ( t e m p e r a t u r e and salinity) have a major influence on NH4 + formation and thus on protein (amino acid) catabolism. Blood a m m o n i a concentrations a p p e a r to be strongly stabilized, ind e p e n d e n t of e n v i r o n m e n t a l osmotic conditions, ranging between 0.25 and 0.55 mmol" 1-1. At normal, low e n v i r o n m e n t a l NH4 + concentrations blood NH4 ÷ is strongly hyper-ionic c o m p a r e d to external concentrations; at high e n v i r o n m e n t a l NH4 + concentrations (even when artificially raised to 2.5 m m o l ' 1 1), blood NH4 ÷ is strongly hypo-ionic. R e g u l a t i o n of the blood NH4 + concentrations takes place by a variable ettlux of N H 4 + ; at high e n v i r o n m e n t a l NH4 + concentrations ( > 0.28 mmol" 1 -x), in addition to a high NH4 + efflux, stabilization of the blood N H 4 ÷ concentrations is effectuated by the formation of urea.

AMMONIUM EXCRETION CARCINUS

283

A m m o n i a efllux to the s u r r o u n d i n g w a t e r is h i g h l y d e p e n d e n t to the o s m o t i c c o n d i t i o n s o f the e n v i r o n m e n t : viz. positively r e l a t e d to t e m p e r a t u r e a n d inversely r e l a t e d to e x t e r n a l salinity, with relatively stable values n e a r the isosmotic salinity. R e l a t e d to the s t r o n g v a r i a t i o n s in a m m o n i a ettlux, e x t e r n a l N H 4 ÷ c o n c e n t r a t i o n s in a closed v o l u m e o f w a t e r are h i g h l y variable. I n the course o f time v e r y h i g h values develop in m e d i a o f low salinity at h i g h t e m p e r a t u r e . A close c o n n e c t i o n b e t w e e n N H 4 + excretion a n d e x t r a c e l l u l a r ion r e g u l a t i o n is i n d i c a t e d .

V. R E F E R E N C E S CAMPBELL,J. W. , 1973. Nitrogen excretion. In: C. L. PROSSER.Comparative animal physiology. Saunders, London: 279-316. ENGELSMA,F.J., 1973. Osmoregulatie van de strandkrab, Carcinus maenas (L.). Interne Verslagen Nederlands Instituut voor Onderzoek der Zee, Texel 1973~3:1 37. FLORKIN,M., 1960. Blood chemistry. In: T. WATERMAN.Physiology of" Crustacea 1. Academic Press, London: 141 160. GUPTA,B. L., R. B. MORSTON,J. L. OSCHMAN• B.J. WALL,1977. Transport of ions and water in animals. Academic Press, London: 1417. MAGNUM, C. P., J. A. DYKENS, R. P. HENRY & G. POLITES, 1978. The excretion of NH4 + and its ouabain sensitivity in aquatic annelids and molluscs. ~ . exp. Zool. 203: 151-157. SHAW, J., 1961. Studies on the ionic regulation in Carcinus maenas L. I. Sodium balance.--J, exp. Biol. 311: 135-153. SOVTERBICQ,J., 1935. Sur le taux de l'ammoniaque dans les liquides du milieu int6rieur de quelques invert6br6s.-42, r. S6anc. Soc. Biol. 120" 453-455. SPAARGAREN, D. H., P. RICHARD& H . J . CEGCALDI, 1982. Excretion of nitrogenous products by Penaeusjaponicus Bate in,relation to environmental osmotic conditions. Comp. Biochem. Physiol. (in press). ZANDERS, I. P.,1980. Regulation of blood ions in Carcinus maenas (L.).~Comp. Biochem. Physiol. 65A: 97-108.