Active uptake of sodium by the freshwater notostracan riops longicaudatus

Comp. Biochem. Physiol., 1967, Vol. 21, pp. 525 to 531. Pergamon Press Ltd. Printed in Great Britain

ACTIVE U P T A K E OF S O D I U M BY T H E FRESHWATER N O T O S T R A C A N TRIOPS LONGICAUDATUS FRANCIS R. HORNE* Department of Zoology, University of Wyoming, Laramie, Wyoming, U.S.A.

(Received 12 December 1966) Abstract--1. The American tadpole shrimp, Triops longicaudatus, has been shown to possess an active sodium uptake system. The sodium uptake system becomes more active in low environmental concentrations of sodium when animals have been acclimated to similar conditions. 2. Although uptake rates are greatest in sodium chloride, animals take up sodium from both the chloride and sulfate salts. 3. Rapid depletion of hemolymph sodium and chloride does not elicit an increase in ion influx. 4. Since excised gill tissue takes up sodium more rapidly than the whole animal, it is suggested that gill tissue is one of the primary sites of active sodium influx. INTRODUCTION HYPEROSMOTIC fresh-water animals are confronted with continuous salt depletion. To compensate for such losses, efficient physiological mechanisms are essential. Most hyperosmofic crustaceans rely primarily on the active uptake of salts from the environment, while others seem to depend more on food for their salts. In starved animals active uptake mechanisms are the only mode of salt replenishment (Potts & Parry, 1964). Both the gills (Koch et al., 1954; Green et al., 1959; Bielawski, 1964) and the gut (Burger, 1957; Croghan, 1958a) of Crustacea have been shown to be involved in salt acquisition. Recently Quinn & Lane (1966) showed that gill tissue of the semi-terrestrial crab, Cardisoma guanhumi, possesses a Na-K-stimulated ATPase system. This enzyme system has been suggested as the enzymatic basis for sodium and potassium transport (Skou, 1965). Of special interest are the ionic regulatory mechanisms of inhabitants of osmotically unstable environments (e.g. estuaries and temporary ponds). Although considerable work has been conducted on estuarine forms, little is known of the ionic regulatory mechanisms of the crustacean inhabitants of ephemeral ponds, For instance, even though a wide range of salinities are tolerated by phyllopods (Home, 1967a), direct evidence has not yet been presented showing active ion transport to occur in this ancient taxon. Both Chirocephalus diaphanus and Triops * Present address: Department of Zoology, Southwest Texas State College, San Marcos, Texas, U.S.A. 525

526

FRANCISR. HORNE

longicaudatus have been cited as probably having such mechanisms (Panikkar, 1941; Home, 1966), and in the brine shrimp, Artemia salina, Croghan (1958b) and Copeland (1966) have presented indirect evidence suggesting that the first ten branchiae are involved in active sodium transport. Quite to the contrary Krogh (1939) and Parry (1961) proposed that Lepidurus apus and Triops cancriformis procure their salts from food. It therefore seems likely that different ionic regulatory mechanisms have evolved in this primitive group. The present study was initiated to clarify the role of active transport mechanisms in sodium balance of the American notostracan, Triops longicaudatus, and to show the effect of acclimation to differing external sodium concentrations on the influx of sodium. MATERIALS AND METHODS Laboratory-reared Triops weighing between 100 and 300 mg were used in all experiments. Culture techniques are presented elsewhere (Horne, 1967b). The radioactive isotope of sodium (**Na) was obtained from the Atomic Corporation of America as both the chloride and sulfate salts and was used to measure sodium influx. Groups of Triops, which had previously been acclimated to 1.0 mmoles Nail, were subjected to six different sodium chloride concentrations (0-04--2.0 mmoles/l) containing ~Na to attain a typical uptake rate curve. In addition, Triops was acclimated to two sodium chloride solutions (0.25 and 40.0 mmoles/1), and then exposed to four different concentrations of labelled sodium chloride to determine the influence of acclimation on the comparative sodium uptake rates. To show that sodium was actually being actively transported, and not just moving down an electrochemical gradient, five Triops were subjected to a 22Na-labelled sodium sulfate solution (0.7 mmoles/l). These animals were previously acclimated to 0.5 mmoles Na/1. In all of the 22Na experiments in which whole animals were used, animals were subjected to the radioactive solution for 1 0 4 0 rain. The short exposure was sufficient to give good radioactive counts. Hemolymph samples were taken as previously described (Horne, 1966); once withdrawn, the sample was transferred to a planchet containing 0.2 ml of water and dried. Standard counting equipment (Model 151A Nuclear-Chicago Scaler with a Geiger-Muller tube) was used and both a sample of the medium and of the hemolymph were counted for 15 rain. Corrections for background and counting efficiency were made. In an attempt to ascertain whether one of the sites of active transport of sodium might be located in the highly vascular epipodites (branchia) of the thoracic and abdominal phyllopodia, all phyllopodia from the second thoracic to the last abdomenal appendage were excised. Inasmuch as each animal provided two groups of tissue, one from each side of the animal, one group of tissue was denatured in a "boiling" (93°C) water-bath for 10 rain and the other group was untreated. Both groups of tissue then were subjected to the desired solutions of labelled sodium chloride (0-4mmoles/1 and 1.0 mmoles/1). Following 10-rain exposure periods to the labelled sodium, both groups of tissue were washed three

A C T I V E U P T A K E O F S O D I U M B Y TRIOPS L O N G I C A U D A T U S

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times with distilled water, placed in planchets and ashed. The counting procedure was the same as in the previous experiments. All experiments were conducted at 24 + 3°C. RESULTS AND DISCUSSION The effects of acclimation on the active transport of sodium are shown in Fig. 1 and Table 1. An inverse relationship exists between the sodium acclimation concentration and the rate of active sodium transport. The sodium uptake system becomes more active at low environmental concentrations of sodium when the 16-0"25 /

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FIG. 1. The effect of acclimation to differing sodium concentrations on the influx/ external concentration curves of Triops longicaudatus. (acclimation salinities in mmole Na/l -- 0"25, 1"0 and 40.0). animals have been acclimated to similar conditions. Triops can take up sodium from both the chloride and sulfate salts, althought the uptake is much slower from sodium sulfate. In five animals acclimated to 0.5 mmoles/l, sodium influx was 3"45 _+S.E. 0.72 mmoles. 1-1 hr -1 in a sodium sulfate solution (0.35 mmoles/1). Even though Triops is capable of actively transporting sodium from both salts, the differences in uptake rates suggest that an active chloride system may also be operating. In numerous aquatic crustaceans, sodium ions are transported by a saturable, rate-limited system, which has a high affinity for sodium ions (Shaw, 1959a, b; Shaw & Sutcliffe, 1961; Parry & Potts, 1965). The equation that Shaw used to

528

FRANCIS R. HOraCE

express this phenomenon is analogous to the Michaelis-Menten equation V = [VmC/(Km+C)]. At high external concentrations the active transport system becomes saturated, while at low external concentrations ionic availability restricts ionic influx. T h e sodium influx rate curve of T. longicaudatus quite closely approximates the Michaelis-Menten expression. As Triops is acclimated to decreasing external sodium concentrations active sodium influx increases and the uptake rate curve shifts to the left. This shift suggests that there is an increase in quantity or activity of the system; a significant change in the system's affinity does not seem to occur. T h e system's affinity is estimated to be about 0.2 mmoles Na/1. Apparently only an increase in uptake rate, not a change in the system's ionic affinity, is involved in the adaptation of Triops to low salinities. T h e active uptake T A B L E 1 - - I N F L U X E S OF SODIUM IN

Triops longicaudatus IN

Medium in which the influxes were measured (mmole Na/1)

0"25

0"04 0"10 0"30 0"40 0"60 1"00 2"00

-5"75 + 1"43 (7) -10"24 _+1"02 (7) -12-65+1"21 (7) 13-80+1"15 (7)

DIFFERENT MEDIA

Acclimation salinity* (mmole Na/1) 1"0 2-78 +_0"35 (6) -5"84 _+0"94 (7) 7"71 _+0'97 (6) 8.63 + 0.37 (6) 9"38_+0"74(6) 9"91 _+0'48 ( 1 4 )

40"0 -1"87 _+0"23 (6) -2"97 + 0"62 (12) -6"10+0"85 (6) 5"29+1"15 (6)

* The acclimation period was 2 weeks. Readings expressed as mmole. 1-1 hr-1 + S.E. (no. of observations). systems of the fresh-water crustaceans, Austropotamobius pallipes, Gammarus pulex and Potamon niloticus are half-saturated (estimate of ionic affinity) at external concentrations of 0.2-0.3, 0.15 and 0.1 mmoles Na/l, respectively (Shaw, 1959a, b; Shaw & Sutcliffe, 1961). These values are similar to the value reported here for

Triops. Unlike some other crustaceans (Shaw, 19593; Lockwood, 1964), activation of the Triops active sodium transport system does not occur immediately after alteration of the internal sodium and chloride concentrations. By subjecting animals that had been previously acclimated to 40 mmoles Na/1 to deionized water for 6 hr and depleting the internal sodium and chloride content at the rates of approximately 3.2 and 3.4 mmoles.1-1 hr -1 (Home, 1966), it was shown that eight animals took up 2-25 + S.E. 0.63 mmoles Na. 1-1 hr -1 from a 0.4 mmole Na/1 solution. This rate is slightly less than that for similarly acclimated animals that had not been exposed to deionized water. Response to changes in external and internal sodium and chloride concentrations is apparently slow in Triops. Austropotamobius pallipes and Gammarus duebeni respond to exposure to deionized water

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with an enhanced sodium uptake rate (Shaw, 1959a; Lockwood, 1964), and salt-loaded Palaemonetes antennarius have lower sodium uptake rates than saltdepleted animals (Parry & Potts, 1965). In Crustacea the precise factors responsible for stimulating the monitoring systems that regulate active ion transport are not well known. However, these data suggest that they may differ among crustaceans. When compared to other fresh-water crustaceans, Triops has. a higher ionic efflux in deionized water than similarly treated Austropotamobius, Gammarus and Potomon (Shaw, 1959a, b; Shaw & Sutcliffe, 1961), but slower rates than Palaemonetes (Parry & Ports, 1965). Sodium influx, however, is highest in Triops when compared with five fresh-water crustaceans that have been acclimated and exposed to similar solutions (Table 2). Even though the efflux and influx rates cannot be compared without reservation, they do provide a basis for comparing differences in ionic regulatory mechanisms. TABLE 2--COMPARATIVE SODIUM UPTAKE RATES OF SOME FRESH-WATERCRUSTACEANS

Animal

Austropotamobius pallipes Gammarus pulex Palaemonetes antennarius Potamon niloticus Triops longicaudatus

Acclimation cone. (mmole/l)

Experimental cone. (mmole/1)

0-150

0"3

0"3

Shaw, 1959a

7"5

0' 1

0"5

9"7*

0"5

0"5

1.5 10"2"

0"5 0"2

0.5 0"4

Shaw & Sutcliffe, 1961 Parry & Ports, 1965 Shaw, 1959b T h i s paper

U p t a k e rate (mmole.kg -1 hr -1)

Author

* Value based on m m o l e N a (1. of blood)-X hr-1.

Gill tissue appears to be the primary site of sodium uptake since excised tissue actively transports sodium much faster than the whole animal. Excised phyllopodia (partly gill tissue) of animals acclimated to 1 mmole Na/1 took up 19.8 + S.E. 9.9 mmole Na (kg tissue) -t hr -x and 35"4 + S.E. 9.4 mmole Na (kg tissue) -x hr -1 when subjected to 0.4 mmole Na/l and 1.0 mmole Na/l, respectively. Each value is the mean uptake rate of the gill tissue from five animals. The barely detectable quantities of ~2Na taken up by the control tissue (denatured) could be accounted for by diffusion. Since the epipodites (branchia) of the phyllopodia are highly vascular and are most permeable to methylene blue, it seems likely that they are responsible for active ion transport in the phyllopodia. Direct evidence has been presented that shows gill tissue of Austropotamobius, Eriocheir and Uca to be a site of active ion transport (Koch et al., 1954; Green et al., 1959; Bielawski, 1964), and in phyllopods the first ten branchiae of Artemia and Chirocephalus are probably involved in active ion transport (Croghan, 1958b).

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FRANCIS R. HORNE

According to Shaw (1961) the adaptation of Crustacea to fresh water involves a reduction in ionic permeability and the acquisition of a high affinity uptake system. Production of hyposmotic urine and the reduction of the hemolymph osmotic concentration are considered of lesser importance. Adaptation of Triops to fresh water does not seem to have progressed according to Shaw's criteria since they have not solved the problem of being somewhat permeable to ions (Horne, 1966). Triops has, however, evolved an active transport system with high ionic affinity. T h e low hemolymph osmotic content in notostracans undoubtedly aids in reducing the rate of ionic efflux. And since a lower ionic concentration gradient between the h e m o l y m p h and the medium would result in fewer ions being lost to the environment, less energy would be required to maintain the hemolymph osmotic concentration. Nothing is known of the role of the large renal organ in ionic regulation. T h a t Triops has an active sodium uptake system is of phylogenetic interest since this genus has evolved only slightly since Carboniferous times (Tasch, 1963). Also of interest is that the notostracans Lepidurus apus and Triops cancriformis appear to depend on ionic regulatory mechanisms different from that of Triops longicaudatus. Only further studies will clarify these problems.

REFERENCES BIELAWSKI J. (1964) Chloride transport and water intake into isolated gills of crayfish. Comp. Biochem. Physiol. 13, 423-432. BURGERJ. W. (1957) The general form of excretion in the lobster, Homarus. Biol. Bull., Woods Hole 113, 207-223. COPELAND E. (1966) Salt transport organeUe in Artemia salina (brine shrimp). Science, N.Y. 151, 470-471. CROGHAN P. C. (1958a) The mechanism of osmotic regulation of Artemia salina (L.): the physiology of the gut. J. exp. Biol. 35, 243-249. C R o c ~ P. C. (1958b) The mechanism of osmotic regulation of Artemia salina (L.): the physiology of the branchiae. J. exp. Biol. 35, 234-242. GREENJ. W., HARSCHU., BARRL. & PROSSERC. L. (1959) The regulation of water and salt by the fiddler crabs Uca pugnax and Uca pugilator. Biol. Bull., Woods Hole 116, 76-87. HORNE F. R. (1966) Some aspects of ionic regulation in the tadpole shrimp Triops longicaudatus. Comp. Biochem. Physiol. 19, 313-316. HORNE F. R. (1967a) Effects of physical-chemical factors on the distribution and occurrence of some southeastern Wyoming phyllopods. Ecology. (In press.) HORNE F. R. (1967b) Survival and ionic regulation by Triops longicaudatus in various salinities. Physiol. Zool. (In press.) KOCH H. J., EVANSJ. & SCHICKSE. (1954) The active absorption of ions by the isolated gills of the crab, Eriocheir sinensis (M. Edw.). Meded. K. vlaam. Acad. 16, 1-16. KROOH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge. LOCKWOODA. M. P. (1964) Activation of the sodium uptake system at high blood concentrations in the amphipod Gamrnarus duebeni. J. exp. Biol. 41, 447-458. PANIKKAR N. K. (1941) Osmotic behavior of the fairy shrimp Chirocephalus diaphanus Prevost. J. exp. Biol. 18, 110-114. PARRY G. (1961) Chloride regulation in Triops. Nature, Lond. 192, 468-469.

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PARRY G. & POTTS W. T . W. (1965) Sodium balance in the freshwater prawn, Palaemonetes antennarius, ft. exp. Biol. 42, 415-421. POTTS W. T. W. & PARRY G. (1964) Osmotic and Ionic Regulation in Animals. Cambridge University Press, Cambridge. QUINN D. J. & LANE C. E. (1966) Ionic regulation and N a + - K + stimulated ATPase activity in the land crab, Cardisoma guanhumi. Comp. Biochem. Physiol. 19, 533-543. SHAW J. (1959a) T h e absorption of sodium ions by the crayfish, Astacus pallipes Lereboullet - - I . T h e effect of external and internal concentrations, ft. exp. Biol. 36, 126-144. SHAW J. (1959b) Salt and water balance in the East African freshwater crab, Potomon niloticus (M. Edw.). ft. exp. Biol. 36, 157-176. SHAW J. (1961) Sodium balance in Eriocheir sinensis (M. Edw.). T h e adaptation of the Crustacea to fresh water. J. exp. Biol. 38, 153-162. SHAW J. & SUTCLIFFE D. W. (1961) Studies on sodium balance in Gammarus duebeni Lilljeborg and Gammarus pulex pulex (L.). ft. exp. Biol. 38, 1-15. SKou J. C. (1965) Enzymatic basis for active transport of Na + and K + across cell membranes. Physiol. Rev. 45, 596-617. TASCH P. (1963) Evolution of the Branchiopoda. In Proc. Conf. Evolution Crustacea (Edited by WHXTTINGTON H. B. & ROL~ W. D. I.) pp. 145-157. Harvard University, Cambridge, Massachusetts.