ABSTRACT
The exchange of labelled calcium between the external medium and the whole body was investigated in the larva of Aedes aegypti (L.) using a closed, two-compartmental model. The transport system for the uptake of Ca2+ was found to be saturable and obeyed Michaelis-Menten kinetics. The efficiency of the inward transport of calcium from dilute solutions was markedly reduced by starvation or by ruthenium-red, a selective inhibitor of Ca2+ activated ATPase, indicating that this transport system is energy dependent. Unlike transport systems for the major monovalent ions, the Ca2+ transport system is not located in the anal papillae, since removal of these organs resulted in enhanced Ca2+ fluxes. While over 95 % of the calcium in the larva appeared to be distributed in the extracellular haemolymph, only 16 % of the total calcium was readily exchangeable with the external medium; thus the majority of the calcium is apparently bound to haemolymph constituents. The results suggest that calcium pumps consisting of Ca2+ activated ATPases play an important role in the absorption of Ca2+ from dilute solutions in the gut and its reabsorption from the urine in the rectum.
INTRODUCTION
The ability of the larva of the freshwater mosquito Aedes aegypti (L.) to maintain its principal electrolytes against a steep concentration gradient between the haemolymph and the external medium has been demonstrated by several investigators (Koch, 1938; Wigglesworth, 1938; Ramsay, 1953; Stobbart, 1959, 1960, 1965, 1967, 1971). It has been shown that the mechanisms for keeping the concentrations of chloride, sodium and potassium far above those in the external medium include independent transport and reabsorption processes that occur in organs such as the anal papillae, the rectum and the Malpighian tubules. Earlier work in this field has been comprehensively discussed by Treherne (1954) and Stobbart (1965, 1967).
While previous studies on the exchange of ions between the larva and the external medium have focused on monovalent ions such as Na+, K+ and Cl−, which play a major role in osmoregulation, the exchange of other ions has not been extensively studied in this species. Among the bivalent ions, the study of Ca2+ merits prime consideration in view of the important role of this ion in the regulation of various functions in living organisms, including insects (Berridge & Prince, 1972). The purpose of the present study was to investigate the processes of calcium exchange between the larva and the external medium using labelled 45Ca, and to characterize some of the mechanisms involved in the transport of Ca2+ from the external medium to the larva.
MATERIALS AND METHODS
Mosquito larvae
The ROCK (Rockefeller) strain of Aedes aegypti was reared in an environmental chamber at a temperature of 27–30 °C at 80–85% relative humidity. Filter paper, upon which eggs had been deposited less than a month previously, was cut into strips, each containing about 200 eggs. Each of several such strips was submerged in 800 ml of distilled water and 10 ml of liver powder suspension [10 g (1 tablespoon) per 1000 ml of water] in individual white enamel photographic pans as larvae were needed. An equal amount of liver suspension was added to each pan on day 1 and 20–25 ml on days 2, 3 and 4.
For studies of larvae without anal papillae, the papillae were ‘pinched off’ with a pair of forceps approximately 18 h before transport experiments had begun. It was assumed that complete healing of the wound had been achieved during this time period.
Measurement of calcium uptake
This series of experiments was designed to investigate whether calcium ions enter the larva by simple diffusion, by a saturable transport mechanism or by a combination of both. The inward movement by diffusion would be expected to be directly related to the external calcium concentrations, whereas a non-linear relationship would be expected when the inward calcium movement involves a saturable transport.
The uptake of calcium by individual larvae was measured under steady state conditions over a wide range of calcium concentrations in the external medium. Early 4th instar larvae were placed in small beakers at a density of approximately 3 larvae/mL Each beaker contained a known concentration of CaCl2, ranging between 0·01 and 50 mm, in deionized water. The larvae were left in the beaker for 2h, after which a tracer amount of 45CaC12 (N.E.N., Boston, MA) was added to each beaker. Duplicate aliquots were used to measure the calcium concentration and 45Ca specific radioactivity (SA). After 1 h exposure to the isotope, larvae were removed rapidly, immersed briefly in deionized water, blotted, and transferred in groups of three into scintillation vials containing 0·1 ml of deionized water. The larvae were then crushed with a glass rod and their radioactivity was measured in a scintillator spectrometer (Beckman LS-200) after the addition of 10ml scintillation fluid (Aquasol, N.E.N.). The rate of 45Ca uptake was expressed as c.p.m. larva−1 h−1. The initial rate of calcium uptake (nmol larv−1 h−1) was determined after the 45Ca uptake during the first hour had been divided by the SA of 45Ca in the external medium.
Measurement of calcium fluxes
Early 4th instar larvae were placed in small beakers containing 0·1 mm-CaC2 and other test substances as specified. The larvae (3 per ml in 10 ml) were left in the solution for 24 h without food. It was assumed that steady state conditions were approached within this time period. A tracer amount of 45CaCl2 was then added to each beaker and duplicate aliquots taken for the measurements of 45Ca concentration (c.p.m. ml−1) and SA. At predetermined time intervals between 0·5 and 30 h following the addition of the isotope, groups of three larvae were removed for the measurement of 45Ca uptake. The larvae were washed, blotted and crushed in a scintillation vial for the determination of their radioactivity as already described.
Assay of 45Ca specific activity
Calcium concentration in the external medium was determined in 10 μl aliquots. The sample was diluted with deionized water to a final concentration of 10– 50 μM and the calcium content was determined by atomic absorption (Perkin Elmer model 372). An air-acetylene flame was used and the integration time of each sample was set to 2 s. The full scale standard was a 50 μM solution of CaCl2. Radioactivity was assayed in duplicate 10 μl aliquots using a scintillation spectrometer and the SA is expressed as c.p.m. nmol calcium−1. When total calcium was determined in the larva, groups of 10 larvae were rapidly washed with deionized water. The larvae were then homogenized in a Brinkman polythron for 30 s. The amount of calcium in the homogenate was determined by atomic absorption, as already described, and the amount per larva was calculated. An aliquot of the homogenate was taken for the measurement of radioactivity when applicable and the SA was determined as c.p.m. nmol−1 for the calcium in the larva.
RESULTS
Calcium uptake
The uptake of calcium increased with increasing calcium concentrations in the external medium in a manner which suggested the presence of a saturable transport mechanism. Furthermore, when net calcium uptake was studied in larvae which with left for 24h in deionized water without food (starved larvae), there was a marked decrease in mean values for calcium uptake while the relationship between uptake and calcium concentration in the medium was still consistent with the combination of nonsaturable and saturable transport kinetics (Fig. 1A, B). The nonsaturable component was estimated as the clearance of calcium from the external medium into the larva at the extremely high concentrations above 10 mm. The nonsaturable clearance was determined as 0· 048 μl larva−1 in both starved and fed larvae (Fig. 1). The saturable transport component was obtained after the nonsaturable component had been substracted from the value for the total uptake (Fig. 1C, D). The saturatable process was analysed according to the method of Lineweaver & Burk (1934) in both starved and fed larvae. Analysis of the data from fed larvae resulted in an affinity constant (Km) of 0· 43 mm and a maximum transport velocity (Vmax) of 0·2 nmol h−1 larva−1 whereas the 24 h starved larvae had a Km of 0·35 mm and Vmax of 0·12 nmol h−1 (Fig. 2).
Calcium fluxes
Untreated control larvae
The fractional rate constants for the influx (Kin) and efflux (Kout ) of calcium were determined during a 30h period without food at a concentration of 0·1 mm calcium in the external medium, and should therefore represent the condition of starved larvae. When values for the quantity of 45Ca in the larva (*QL) were divided by the concentration of 45Ca (c.p.m. ml-1) in the external medium (*QM) and plotted against time, the resulting data could be fitted quite closely to an exponential function of time whose asymptote is Kin/(Kin + Kout) and whose approach to its asymptote is determined by Kin + Kout (equation 1). Nonlinear regression analysis of the data from untreated larvae produced the function shown in Fig. 3. In these larvae, the mean value for Kin was 3·35 × 10−4ml h−1 larva−1, which corresponds to an entry of 0 ·0335 nmol calcium h−1 into the average larva when the concentration of calcium in the external medium is 0·1 mm. The mean value for Kout was 0·0266 h−1 indicating that 0·0266 of the exchangeable calcium pool of the larva is released into the medium each hour. The combined value for (Kin + Kout) which determines the approach of *QL/*QM to its asymptote can also be obtained by linear regression after rearrangement of equation (1) as follows:
Analysis of the data according to equation (2) above resulted in a straight line with a slope of − (Kin + Kout). This presentation allows a readier appreciation of the agreement between the data and equation (l), and also demonstrates the increasing uncertainty the data as the asymptotic value is approached (Fig. 4).
Under steady state conditions, when the amount of calcium entering the larva per unit time is expected to be equal to the amount leaving the larva in the same time period, the fraction representing 0·0266 of the exchangeable calcium pool should be equal to 0·0335 nmol h−1 and the size of the exchangeable calcium pool in the average larva would be calculated as 0·0335/0·0266 = 1·26 nmol. The total amount of calcium in the average larva (2·3 mg) was found to be 7·6 nmol. Thus, the calcium pool that is readily exchangeable with calcium in the external medium represents only about 16% of the total calcium in the larva.
Effect of ruthenium-red
When ruthenium-red, a selective inhibitor of Ca2+ activated ATPase (Watson, Vincenzi & Davis, 1971), was present in the external medium at a concentration of 0-1 mm, the uptake of 45Ca was much slower than in untreated larvae (Fig. 3). This slower uptake resulted from both a decrease in Kin and an increase in Kout compared to the corresponding values in untreated larvae (Table 1). In addition, the average amount of total calcium was 5·9 nmol larva−1, significantly lower than the mean value of 7·6 nmol larva−1 found for the control larvae. It is assumed that the calcium loss took place during the 24h period before the tracer was introduced; the larvae were therefore tested under new steady state conditions.
The curve representing 45Ca uptake in larvae whose anal papillae were ‘pinched off’ (Fig. 3) was clearly different from the curve obtained from untreated larvae. The
Removal of anal papillae
value of 2·21 × 10−3 ml h−1 larva−1 for Kin was over sixfold higher than the Kin of 3·35 × 10− 4ml h−1 in the untreated larvae, whereas the value for Kout was only 3·5-fold higher (Table 1). These differential changes resulted in a more rapid accumulation of 45Ca in the ‘papillae-less’ larva than in the intact untreated larva. The average amount of total calcium in the papillae-less larvae was 9·8 nmol larva−1, a value significantly higher than the 7·6 nmol larva−1 in the untreated larvae, indicating a no steady state where the quantity of calcium in the larva is higher in comparison to the steady-state conditions of control larvae.
DISCUSSION
The present results demonstrate that larvae of Aedes aegypti have a saturable transport system for the uptake of Ca2+, which obeys Michaelis-Menten kinetics. This system is able to move Ca2+ inwards from dilute solutions and appears to be energy-dependent, since its efficiency is markedly decreased during starvation. At the external calcium concentration of 0·1 mm, the effect of 24 h starvation accounted for a reduction of about 35 % in the rate of calcium uptake by the saturable system from 0·042 to 0·027 nmol h−1 larva−1. This marked decrease appears to be due to a reduction in the number of ‘calcium carriers’ as is evident from the substantial decrease in the value for Vmax (Fig. 2). The small change in the affinity for calcium (Km) is not likely to be related to the decrease in calcium uptake.
A saturable transport system for sodium has been demonstrated in Aedes aegypti larvae (Stobbart, 1965). The system for sodium transport appeared to be located in the anal papillae since most of the exchange of labelled sodium occurred through these organs. Similarly, transport systems for other monovalent ions have been shown to be located in the anal papillae (Koch, 1938; Wigglesworth, 1933, 1938) and the one for chloride is also known to be saturable (Stobbart, 1967). The present results indicate however that the intact papillae are not necessary for the inward movement of calcium. The finding that removal of the anal papillae resulted in even higher rates of calcium fluxes (Fig. 3, Table 1) indicates that these organs may act as partial barriers to the movement of calcium. Wigglesworth (1933) has shown that divalent ions, including Ca2+, do not diffuse into the cells of the anal papillae as do monovalent ions when presented in the external medium. Nevertheless, the present experiments do not rule out a possible role for the anal papillae in the processes which regulate calcium uptake from dilute solutions. It is possible, for example, that the larvae compensate for removal of the papillae by increased uptake of fluid and salts in the gut. Stobbart (1960) showed that papillae-less larvae could bring about a net uptake of sodium, presumably through the gut.
The calcium fluxes are presented here as transfer constants according to the theoretical relationship for a two compartmental system (Riggs, 1963). The influx, Kin, is expressed in terms of calcium clearance from 1 ml of the external medium whereas the efflux, Kout, is expressed as a fraction of the exchangeable calcium pool in the larva. The exchangeable pool in the average 2·3 mg larva was calculated as 1·25 nmol calcium and represented only 16 % of the total calcium pool in the larva. The anatomical location of this exchangeable pool is presumably limited to the haemolymph. Assuming that the haemolymph constitutes about 62 % of the body weight (Stobbart, 1965), this amount (1·25 nmol) of exchangeable calcium would be distributed in 1·44 μl (2·3 × 0·62) haemolymph and the concentration of the readily exchangeable calcium in the haemolymph would be 0·87 mm. This value appeared to be much lower than values reported for haemolymph calcium in other aquatic insects which ranged from 11 to 23mm in endopterygotes (Sutcliffe, 1962). A preliminary determination taemolymph calcium in the present study resulted in a mean concentration of t 0·3 mm, indicating that over 95 % of the calcium in the larva is distributed in the extracellular haemolymph. Since only 16% of the total calcium was found here to be readily exchangeable with the external medium, the remaining 84 % apparently represents bound calcium which is not readily exchangeable.
The average Kin in the untreated larva (3·35 × 10−4 ml h−1) corresponds to a calcium entry of 0·0335 nmolh−1 from a 0·1 mm-CaCl2 solution. This value appears to be in good agreement with the mean value of 0 · 032nmolh−1 obtained for the calcium uptake from a 0·1 mm solution in the starved larva (Fig. 1). Thus, in the present experiments, the initial rate of calcium uptake appears to represent the unidirectional inward movement or calcium influx, because the ‘back movement’ of the tracer during the first hour of measurement is practically negligible. The average Kout in untreated larvae (0·0266 h−1) corresponds to a half-life of 26 h for the turnover of the exchangeable calcium pool. Similarly, Kout values in ruthenium-red treated larvae and in larvae without anal papillae corresponded to half-lives of 10·3 h and 7·4 h respectively. The finding that ruthenium-red brings about a new steady-state with higher Kou and lower Kin suggests that calcium pumps consisting of calcium-activated ATPases play an important role in two independent processes: one is the absorption of calcium from dilute solutions into the larva and the other is the prevention of calcium loss to the external medium. The sites of calcium entry into the larva or its loss to the medium have not been determined in the present study, but preliminary results with ligatures indicate that the entry of calcium ions is most likely to occur through the gut whereas the loss is most likely to occur in the urine. It is possible, therefore, that both the gut and the rectum contain calcium pumps which are sensitive to ruthenium-red, and which enhance the entry of calcium into the haemolymph. The calcium pump in the gut would act to transport calcium from the drinking fluid to the haemolymph whereas the calcium pump in the rectum would act to reabsorb calcium which has been excreted by the Malpighian tubules in the urine.
ACKNOWLEDGEMENTS
The help of Dr Herbert L. Meltzer of New York State Psychiatric Institute in the determinations of calcium concentrations by atomic absorption is gratefully acknowledged.