Sodium and chloride regulation in freshwater and osmoconforming larvae of Culex mosquitoes
1 Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697-2525, USA and
2 Department of Biology, University of San Diego, 5998 Alcalá Park, San Diego, CA 92110, USA
*e-mail: mlpatric{at}uci.edu
Accepted July 3, 2001
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Summary |
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Key words: Culex tarsalis, Culex quinquefasciatus, sodium, chloride, ion regulation, uptake, efflux, mosquito, larva, osmoconformer.
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Introduction |
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While our understanding of ion regulation in Aedes is extensive, the eight other genera of mosquitoes that contain both freshwater and salt-tolerant species have been largely ignored. This present study is an attempt to fill that void by examining two species of the genus Culex. Species in this genus are of particular interest because recent studies show that they utilize a completely different strategy to tolerate saline environments, they osmoconform (Garrett and Bradley, 1987; Patrick and Bradley, 2000). Culex tarsalis can tolerate environmental salinities up to 70 % full-strength sea water. When held in 50 % sea water, C. tarsalis maintains hemolymph NaCl levels below that of the environment and accumulates high levels of two compatible solutes: trehalose, a disaccharide, and proline, an amino acid. This response allows hemolymph osmolality to conform to that of the environment, which eliminates the osmotic gradient for water loss and avoids the detrimental effects of high salt levels on protein function within the tissues. During an acute increase in external salinity, C. tarsalis larvae regulate their body volume by drinking the saline medium to compensate for water lost across the integument to the environment. A consequence of this drinking activity would be the incursion of a salt load into the hemolymph as ions and water are absorbed in the midgut of mosquito larvae (Kiceniuk and Phillips, 1974). Despite this, C. tarsalis larvae are able to attenuate this potential rise in hemolymph Na+ levels (Patrick and Bradley, 2000). From this work, it became apparent that osmoconforming larvae are able to regulate hemolymph NaCl levels during acute salinity changes and when acclimated to a range of salinities. We concluded that ion regulation is integral to the osmoconforming response, but little is known of the underlying mechanisms.
The goal of the present study was to compare the Na+ and Cl regulatory responses of the osmoconformer C. tarsalis with those of C. quinquefasciatus, a species restricted to freshwater habitats. We measured whole-body Na+ and Cl uptake and efflux to identify the key regulatory responses that have enabled C. tarsalis to extend its upper salinity limit and to tolerate acute salinity increases. In addition, we examined the patterns of Na+ and Cl regulation of these two species at the other end of the salinity range, water with low levels of NaCl, to determine whether there is also a disparity in their lower salinity limits.
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Materials and methods |
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Hemolymph Na+ and Cl levels during long-term and acute salinity exposure
Hemolymph Na+ and Cl concentrations were measured in both C. quinquefasciatus and C. tarsalis after 2 days in a low-NaCl medium [250 µmol l1 NaCl, 1000 µmol l1 Ca(NO3)2, 100 µmol l1 KNO3]. The low-NaCl medium was prepared from distilled water with the appropriate amount of each salt added. The 30 % seawater medium was made using Instant Ocean Salts (Aquarium Systems). Larvae were removed from the experimental medium, rinsed in distilled water and blotted dry on filterpaper disks. The larvae were placed on Parafilm and exsanguinated by making a small tear in the cuticle using fine forceps. The hemolymph was quickly collected using 1.0 µl microcapillary tubes (Drummond Microcaps). Hemolymph Cl concentrations were determined using a colorometric assay based on the liberation of thiocyanate from mercuric thiocyanate to form mercuric chloride (Patrick and Bradley, 2000). Hemolymph samples (1 µl) were diluted with 1 ml of distilled water, and 100 µl of a 1:1 solution of 13 mmol l1 mercuric thiocyanate:0.5 mol l1 ferric nitrate was added (Zall et al., 1956). Samples and Cl standards were read spectrophotometrically at an absorbance of 480 nm.
Hemolymph Cl levels were also measured in both species following acute transfer from 30 % to 50 % sea water (250 mmol l1 NaCl, 5 mmol l1 Ca2+, 5 mmol l1 K+). In our previous study (Patrick and Bradley, 2000), we had examined hemolymph Na+ concentrations under identical conditions. Batches of larvae of both species were held for 2 days in 30 % sea water. Hemolymph samples were taken from both species held in 30 % sea water and following acute transfer to 50 % sea water. Hemolymph was sampled 1 h prior to transfer (N=5 samples) and at 0.5, 1, 2, 4, 6 and 24 h post-transfer (N=48 samples at each time).
Na+ and Cl fluxes in different environmental salinities
Na+ and Cl uptake
To quantify unidirectional Na+ and Cl uptake, we used the radioisotopes 22Na and 36Cl. Preliminary tests determined the appropriate specific activity of the medium and flux time to ensure that a high activity of each isotope could be detected in the larvae. Na+ and Cl uptake experiments were performed separately but followed similar protocols.
Rates of Na+ and Cl uptake were measured in the two species in the following five treatments: tapwater, 2- and 7-day exposure to low-NaCl medium, 2-day exposure to 30 % sea water and 10 h post-transfer from 30 % to 50 % sea water.
Two hours prior to initiation of flux measurements, 68 larvae were transferred to a well of a cell culture plate (Costar, 24 wells). Holding medium (2 ml) was added to each well. To initiate the experimental flux period, the medium was removed, the larvae and well were rinsed twice with 2 ml of distilled water; 1.2 ml of appropriate experimental medium was then added to each well. Isotope (22Na or 36Cl) was either added to a batch of experimental medium prior to adding to the wells or a sample of the stock isotope solution was pipetted into the well immediately following addition of the medium. The specific activities of each isotope were as follows: 74 Bq µmol1 for both the tapwater and low-NaCl treatment groups, 0.49 kBq µmol1 for the 30 % seawater group and 0.30 kBq µmol1 for the 50 % seawater groups. A 50 µl sample of the medium was taken after 5 min, initiating the flux period. Finally, a 50 µl sample of the medium was taken approximately 10 h later, at the end of the flux period. Larvae were then transferred, via a plastic Pasteur pipette, to a medium-sized weighing boat containing approximately 25 ml of experimental medium (without isotope). Larvae were rinsed in this medium for at least 30 s then blotted on a filterpaper disk. Individual larvae were weighed and transferred to a 6 ml plastic scintillation vial containing 200 µl of distilled water. Each larva was macerated, and 5 ml of scintillation cocktail (Ecolume, ACS) was added to each vial. Media and larvae samples were assayed for radioactivity using a liquid scintillation counter (Beckman). Experimental medium samples were diluted appropriately and assayed for Na+ and Cl concentration.
Rates of Na+ and Cl uptake (Jin; nmol mg1 h1), as measured by the appearance of radioactivity in individual larvae, were calculated from the following equation:
| (1) |
where cpmlarva is the whole-body activity of the isotope, m is the mass of the larva, t is time and SAH2O is the mean specific activity of the medium (cts min1 nmol1) with regard to the isotope in question.
Na+ and Cl efflux
Rates of Na+ and Cl efflux were measured in the following five treatments: tapwater and 2-day exposure to low-NaCl medium (250 µmol l1 NaCl, 1000 µmol l1 Ca2+, 100 µmol l1 K+), 2-day exposure to 30 % sea water, and 2 and 4 h post-transfer from 30 % to 50 % sea water.
The day prior to Na+ and Cl efflux experiments, batches of larvae (N=816) were placed in 5 ml beakers with 2 ml of the appropriate holding medium. The activities of 22Na or 36Cl added to each container were as follows: 37 kBq for the 250 µmol l1 NaCl acclimation groups, 74 kBq for the tapwater groups, 370 kBq for the 30 % sea water and 50 % sea water transfer groups. Larvae were held in the isotope loading baths for a minimum of 12 h. Two hours prior to experimentation, larvae were removed from the loading baths, rinsed twice in 50 ml of the appropriate holding medium and blotted on filterpaper disks. When individual larvae had been weighed, they were then transferred either to a 1.5 ml centrifuge tube containing 1 ml of the appropriate experimental medium (Na+ efflux series) or to a well of a cell culture dish to which 2 ml of the experimental medium had been added (Cl efflux series). This initiated the flux period. When the flux period ended, the larvae were removed from the centrifuge tube or well, rinsed twice in distilled water and blotted on a filterpaper disk. Hemolymph samples were collected from each larva as before. Hemolymph samples were then diluted in 3 ml (Na+ efflux) or 2 ml (Cl efflux) of distilled water. For Na+ efflux analysis, the diluted hemolymph samples (3 ml) and medium samples (1 ml) were assayed for radioactivity of 22Na using a counter. The hemolymph samples were then assayed for Na+ concentrations using atomic absorption spectrophotometry. For Cl efflux experiments, 1 ml of the diluted hemolymph or undiluted medium sample was added to a scintillation vial containing 5 ml of scintillation cocktail. Samples were then assayed for ß radioactivity. The remaining 1 ml of diluted hemolymph sample was assayed for Cl concentration, as described above.
The rates of efflux (Jout; nmol mg1 h1) as measured by the appearance of radioactivity in the medium, were calculated by:
| (2) |
where cpmH2O is the activity of the isotope in the medium and SAlarva is the initial specific activity of the hemolymph of the larva (cts min1 nmol1).
Freshwater Na+ and Cl uptake kinetics
The rates of Na+ and Cl uptake in larvae of C. tarsalis and C. quinquefasciatus raised and held in Irvine tapwater and exposed for 2 days to 250 µmol l1 NaCl were measured at six different NaCl concentrations ranging from 250 to 8000 µmol l1 (concentrations of other salts were held constant) to determine whether transport was carrier-mediated and saturable (i.e. conforming to MichaelisMenten first-order kinetics). These kinetic experiments were performed in a defined freshwater medium [250, 500, 1000, 2000, 4000 or 8000 µmol l1 NaCl, 1000 µmol l1 Ca(NO3)2, 100 µmol l1 KNO3, pH 7.5]. Kinetic media were made up in batches with the appropriate NaCl concentration. For the Na+ uptake measurements, a sample of the 22Na stock solution was added to each well to reach the appropriate specific activity: 44.4 Bq µmol1 for 250 µmol l1 NaCl, 22.2 Bq µmol1 for 500 and 1000 µmol l1 NaCl, 11.1 Bq µmol1 for 2000 µmol l1 NaCl and 5.55 Bq µmol1 for 4000 and 8000 µmol l1 NaCl. For the Cl uptake studies, the experimental media were prepared using diluted 36Cl stock at specific activities similar to those in Na+ kinetic media.
The relationship between [NaCl]e and Na+ uptake was examined using MichaelisMenten analysis for first-order one-substrate kinetics. Values of Jmax (the maximum uptake rate) and apparent Km (the [ion] at which uptake is 50 % of Jmax) were calculated using the following equation:
| (3) |
MichaelisMenten analysis could not be performed on Cl uptake as it did not exhibit saturation kinetics over the NaCl concentration range tested.
Statistical analyses
All data are reported as means ± S.E.M. Comparisons among groups were performed using analysis of variance (ANOVA) (overall P0.05) with multiple comparisons (Scheffes test) if ANOVA proved significant. The data for hemolymph Na+ and Cl- concentrations for the tapwater treatment groups of C. quinquefasciatus and C. tarsalis were taken from the results of Patrick and Bradley (Patrick and Bradley, 2000) and used to compare statistically with the values for the low-NaCl holding treatment. Jmax and Km values from the MichaelisMenten kinetic analyses of Na+ uptake were compared by inspection of 95 % confidence intervals. If they did not overlap, they were considered different.
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Results |
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Discussion |
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Exposure to high salinity revealed that C. tarsalis was able to regulate hemolymph Na+ and Cl levels while C. quinquefasciatus was not. When the external salinity was raised from 30 % to 50 % sea water, an increase that reverses the diffusion and osmotic gradients for the larvae, C. tarsalis experienced a smaller increase in hemolymph Cl concentration relative to the considerable rise in C. quinquefasciatus (Fig. 1). This pattern in the regulation of hemolymph Cl parallels previous observations for hemolymph Na+ levels during an identical acute salinity increase (Patrick and Bradley, 2000). These results suggest that C. tarsalis, although an osmoconformer in higher salinity, regulates hemolymph NaCl at levels well below that of the environment (i.e. hyporegulates). In both studies, the potential rise in hemolymph Na+ and Cl concentrations was attenuated in C. tarsalis and, in fact, a slight decrease occurred after 4 h in 50 % sea water. In contrast, C. quinquefasciatus experienced substantial increases in concentrations of both ions, probably contributing to their death at this salinity.
During the initial few hours following transfer to 50 % sea water, C. tarsalis briefly stimulate their drinking rate to maintain a constant body volume in the face of osmotic water loss and, consequently, experience a salt load (Patrick and Bradley, 2000). The patterns in hemolymph ion levels indicate that the mechanisms of dealing with this salt load were effective by hour 4 and that, by 24 h, new and slightly higher [Na+] and [Cl] equilibria had been reached within the hemolymph. On the basis of these observations, it might be presumed that the regulatory mechanisms for Na+ and Cl in C. tarsalis larvae are identical. However, examination of unidirectional uptake and efflux indicates otherwise.
Our results show that, upon transfer to 50 % sea water, C. tarsalis modulated its Na+ efflux (Fig. 3A) and Cl uptake (Fig. 2B) components in a manner that attenuated the rise in hemolymph Na+ (Patrick and Bradley, 2000) and Cl concentrations (Fig. 1). We observed that C. tarsalis larvae had a significantly higher Na+ efflux rate (Fig. 3A) and lower Cl uptake rate (Fig. 2B) than C. quinquefasciatus larvae during the initial 2 h and 10 h post-transfer, respectively. Thus, despite the similar time course and pattern of changes in levels of the two major hemolymph ions, two separate mechanisms are functioning to control Na+ and Cl concentrations.
The greater rate of Na+ efflux observed in C. tarsalis (Fig. 3A) would function to eliminate excess Na+ that had entered the larvae as a result of the greater influx rate of this ion (Fig. 2A). The trend in Na+ efflux reflects the rapidity of the compensatory response to external salinity changes and that the modulation of efflux must be separate from the Na+ uptake mechanism. An increase in the number of Na+ transporters and/or an increase in the activity of the existing transporters must occur to increase the rate of Na+ elimination, although the latter mechanism is more plausible given the rapidity of the response (2 h).
The transport of Na+ out of the larvae is against a concentration gradient. The electrical gradient existing across the body wall of the mosquito larvae is not known. Given the hemolymph and water Na+ concentrations, an electrical gradient of greater than +16 mV (hemolymph-positive) would be required for Na+ to be passively distributed when C. tarsalis larvae are held in 50 % sea water. To date, transepithelial potential (TEP) has not been measured in mosquito larvae held in media of varying salinity, but previous studies on hyporegulating, euryhaline crustaceans and fish held in 100 % seawater medium report TEP values ranging between +10 to +25 mV (Kirschner, 1997; ODonnell, 1997). Hypo-osmoregulating brine shrimp (Artemia salina) held in 50 % sea water drink the external medium, secrete excess salts and exhibit a TEP of +10 mV (Holliday et al., 1990). On the basis of these data, we cannot rule out the possibility that Na+ transport could be passive under these conditions. It would be interesting to examine the molecular moieties responsible for Na+ transport in this species and to determine whether Na+ transport is active or passive in this brackish salinity.
Following transfer to 50 % sea water, the lower rate of Cl uptake observed in C. tarsalis relative to C. quinquefasciatus (Fig. 2B) suggests that the former species has a lower Cl permeability at higher external salinities. In 50 % sea water, Cl uptake is presumed to be passive because the concentration gradient and likely electrical gradient would favor inward diffusion (inside-positive). The lower rate of influx of Cl in C. tarsalis relative to C. quinquefasciatus (Fig. 2B) could be interpreted as a reduced number of sites for Cl entry. This strategy would reduce not only the potential Cl load during exposure to high salinity but also the need to expend additional energy to transport Cl out of the hemolymph against the electrochemical gradient. This lower Cl permeability contrasts with that of Na+ in the two species, with C. tarsalis exhibiting a slightly, but not significantly, greater Na+ uptake rate than C. quinquefasciatus (Fig. 2A). Overall, the unidirectional fluxes of Na+ were at least twice as great as the Cl fluxes, indicating a greater Na+ permeability, a trend consistent among seawater vertebrates (Kirschner, 1997) and invertebrates (Smith, 1969; ODonnell, 1997).
On the basis of our current findings and previous studies, we can propose possible mechanisms for the patterns of Na+ and Cl regulation in brackish water. When C. tarsalis larvae are acutely transferred to 50 % sea water, a salt load is incurred from drinking and/or entry across the anal papillae, but with a reduced influx of Cl. This ion load is rapidly counteracted (i.e. within 2 h) by the significant increase in Na+ and Cl extrusion at the anal papillae against their respective concentration, and most probably electrical, gradients.
In a recent study (Patrick and Bradley, 2000), we reported that C. tarsalis larvae rapidly increase their rate of drinking to regulate body volume during an acute increase in salinity. The midgut has been indicated as the site of water absorption and transport of most of the ions in the ingested medium (Kiceniuk and Phillips, 1974). With regard to the external body surface, it has been suggested that the anal papillae are the region most permeable to water (Wigglesworth, 1933b) and ions (Wigglesworth, 1933a; Koch, 1938; Stobbart, 1967). On the basis of these studies, the increase in Na+ and Cl uptake observed when both species are held in 50 % sea water (Fig. 2) could occur at one or both of these two sites, with C. tarsalis larvae possessing a reduced Cl permeability (relative to C. quinquefasciatus), indicated by a lower influx rate, in the midgut and/or anal papillae. This is particularly intriguing since C. tarsalis must experience a greater Cl load in the midgut because of the significantly greater intake of the external medium (Patrick and Bradley, 2000).
Possible sites for the excretion of the salt load from the hemolymph are the hindgut and anal papillae. Previously, Garrett and Bradley (Garrett and Bradley, 1984b) examined the morphology of key osmoregulating tissues in the larvae of Culiseta inornata, an osmoconformer and ion regulator like C. tarsalis. These authors examined the ultrastructure of the Malpighian tubules, the hindgut and the anal papillae of larvae and did not report any morphological changes associated with varying environmental salinity. The authors did suggest that the anal papillae could be the site of ion transport in osmoconforming larvae. Several morphological and radioisotopic studies have provided evidence that the anal papillae are the site for ion regulation in freshwater mosquito larvae (Wigglesworth, 1938; Ramsay, 1953; Sohal and Copeland, 1966; Stobbart, 1967). Treherne (Treherne, 1954) determined that in freshwater Aedes aegypti most Na+ exchange occurs through the anal papillae, with a very small percentage occurring across the gut. In addition, Phillips and Meredith (Phillips and Meredith, 1969) showed that the anal papillae function to regulate hemolymph Na+ and Cl to lower levels in Aedes campestris that had been acclimated to Ringers solution and transferred acutely to 430 mmol l1 NaCl. Garrett and Bradley (Garrett and Bradley, 1984a) ruled out the hindgut as the site of ion regulation in osmoconforming larvae (Culiseta inornata) held in saline media because neither in vivo nor in vitro preparations of the rectum produced hyperosmotic urine.
Na+ and Cl regulation in dilute media
The present study is the first to characterize freshwater Na+ and Cl regulation in two species belonging to the same genus (Culex) but possessing very different osmoregulatory abilities (Patrick and Bradley, 2000). The similarity in the hemolymph NaCl concentrations in the two species held in tapwater (4 mmol l1 NaCl) and for 2 days at 0.25 mmol l1 NaCl (Table 1) suggests that Na+ and Cl regulation might be similar in these two species. However, the unidirectional Na+ and Cl fluxes (Fig. 2, Fig. 3) and kinetic analysis (Fig. 4; Table 2) indicate that this is not the case. Acclimation to a medium with one-sixteenth of the NaCl levels found in tapwater did not alter rates of ion efflux in either species, but C. tarsalis exhibited a significantly higher rate of Na+ loss than C. quinquefasciatus (Fig. 3). A higher Na+ permeability across the body at higher salinity, as indicated by a greater Na+ loss, is common among euryhaline organisms (Croghan and Lockwood, 1968; Taylor and Harris, 1986; Patrick et al., 1997). Despite the fact that this laboratory colony of C. tarsalis has been maintained in fresh water for several years, it does not exhibit a reduced Na+ permeability similar to that of C. quinquefasciatus. Because of the higher rates of Na+ loss, C. tarsalis larvae require a greater rate of transport of Na+ from the environment to maintain hemolymph NaCl balance. MichaelisMenten kinetic analyses of Na+ uptake (Fig. 4C; Table 2) showed that C. tarsalis had a Jmax double that of the freshwater-restricted C. quinquefasciatus (Fig. 4C; Table 2). In addition, C. tarsalis did not upregulate Na+ uptake (Fig. 4C) following 2 days in low-NaCl medium. Consequently, this species did not maintain Na+ uptake at a rate comparable with the value measured in tapwater (Fig. 2A, Fig. 4C). If C. tarsalis larvae were held for a longer period in low-NaCl water, hemolymph Na+ balance could not be maintained unless the efflux component was reduced to a level comparable with the decrease in Na+ uptake.
Other studies of the ion-regulatory abilities of euryhaline and freshwater animals have yielded similar results. For example, Taylor and Harris determined that the euryhaline amphipod Corophium curvispinum, found in brackish water, is excluded from invading freshwater habitats with NaCl levels lower than 0.5 mmol l1 because it cannot maintain ion balance (Taylor and Harris, 1986). Sutcliffe reported that the brackish-water amphipod Gammarus duebeni has a high Na+ permeability and could maintain ion balance only in water with a NaCl concentration greater than 1 mmol l1 (Sutcliffe, 1967). In contrast, other closely related amphipods successfully inhabit low-NaCl waters by reducing their ion permeability and demand for active uptake of Na+. Similar patterns of high Na+ turnover rates have been found in the euryhaline fish Fundulus heteroclitus, a species that is able to tolerate freshwater medium but can achieve a balance only at 1 mmol l1 Na+ and 1.7 mmol l1 Cl (Patrick et al., 1997). As a consequence, it is unable to invade ion-poor bodies of water that other freshwater species of Fundulus can inhabit (such as Fundulus diaphanus) (Griffith, 1972).
While C. tarsalis has trouble maintaining ion balance in low-NaCl water, C. quinquefasciatus is equipped to survive water of lower ion concentrations by being able to stimulate Na+ absorption (Fig. 4A). By substantially increasing maximum transport capacity (Jmax) (Fig. 4A; Table 2), the freshwater obligate species maintained Na+ uptake at a rate comparable with the tapwater value, despite the large reduction in concentration of external ions available for transport. The upregulation of Jmax of C. quinquefasciatus (Fig. 4A; Table 2) could be attributed to the expression of more Na+ transporters. The time frame in which these changes took place (24 h versus more than 12 h) would provide insight into how the changes in Na+ absorption in C. quinquefasciatus are regulated (i.e. at the genetic or transporter level).
Although Jmax values were quite different in C. tarsalis and C. quinquefasciatus, affinity values were similar (Table 2). Previously, it has been argued that truly freshwater animals reduce the energy required for maintaining ion balance by first reducing integumental permeability to ions and then reducing both the Km and Jmax of the ion-uptake mechanism (Shaw, 1961). Indeed, C. quinquefasciatus has a lower Na+ efflux and Jmax for Na+ uptake relative to C. tarsalis; however, these two species share a similar Km for Na+ uptake (Table 2), a finding that contrasts with studies of freshwater and euryhaline amphipods (Sutcliffe, 1967; Taylor and Harris, 1986), chironomids (Wright, 1975), other crustaceans (Shaw, 1961) and teleosts (Patrick et al., 1997). The Km values determined in the present study for Culex mosquito larvae and also by Stobbart (Stobbart, 1965) for the freshwater obligate mosquito A. aegypti (Km=0.55 mmol l1 NaCl) are higher than the range typically reported for truly freshwater animals (typically <0.4 mmol l1 NaCl) (Shaw, 1961; Wright, 1975; Taylor and Harris, 1986; Patrick et al., 1997). This suggests that mosquitoes cannot increase the affinity of Na+ transport to survive lower-NaCl water despite the fact that mosquito larvae have been found to complete their development in distilled water (Wigglesworth, 1938; Ramsay, 1953). Stobbart (Stobbart, 1965) argued that an enhanced Na+ uptake mechanism could be achieved without an increase in affinity via the recruitment of more transporters, a pattern similar to that observed in C. quinquefasciatus held in 0.25 mmol l1 NaCl water after only 2 days (Fig. 4A; Table 2). Perhaps the Km of Na+ uptake is plastic, but changes require long-term exposure (over several generations) to low-ion medium.
Unlike Na+ uptake, the Cl uptake mechanism of the two species appears to be similar. In both cases, we see a lack of saturable uptake kinetics and a lack of response to low-NaCl water (Fig. 4B,D). These results signify completely independent mechanisms of Na+ and Cl transport in both C. tarsalis and C. quinquefasciatus. In addition, Cl uptake rates were quite low relative to Na+ uptake (Fig. 2A,B, Fig. 4), a trait matched by the low Cl efflux (Fig. 3). This situation differs from that in A. aegypti larvae, in which Na+ and Cl uptake are saturable processes with similar kinetic properties (Stobbart, 1971a; Stobbart, 1971b). Is the unsaturable pattern of Cl uptake in C. tarsalis and C. quinquefasciatus characteristic of all Culex species? These disparities in Cl uptake patterns among the mosquito genera go beyond the differences described for Na+ uptake mechanisms (i.e. Jmax) and suggest that there must be completely different moieties capable of active transport.
The underlying mechanisms by which Na+ and Cl are transported have been examined, using radioisotopic and electrophysiological methods, in freshwater and salt-tolerant mosquitoes belonging to the osmoregulating genus Aedes. Stobbart (Stobbart, 1959; Stobbart, 1960; Stobbart, 1965; Stobbart, 1967; Stobbart, 1971a; Stobbart, 1971b) thoroughly examined the freshwater-restricted A. aegypti and reported that Na+ and Cl uptake from the external medium via the anal papillae is independent, active and involves an intimate coupling to the excretion of acidic and basic equivalents, respectively (i.e. H+ and HCO3). For larvae in sea water, the nature of ion excretion by the additional rectal segment found in several salt-tolerant Aedes species (A. campestris, A. detritus, A. taeniorhychus and A. dorsalis) involves the active transport of Na+, Mg2+ and Cl (Bradley, 1987). However, in both the freshwater and seawater models, the primary source of energy driving these processes has yet to be identified.
In the freshwater, seawater and euryhaline vertebrates and invertebrates studied to date, Na+/K+-ATPase plays a role in the models proposed for ion transport (Gilles and Delpire, 1997; Kirschner, 1997; ODonnell, 1997). In both freshwater and seawater organisms, numerous studies have reported correlations between Na+/K+-ATPase activity and ion-transport rates when environmental salinity is varied, thus supporting the role of Na+/K+-ATPase in ion regulation (Holliday et al., 1990; McCormick, 1995; Piermarini and Evans, 2000; Postel et al., 2000). Both models are far from complete, and the freshwater model has recently been challenged by the identification of H+-ATPase (V-type) in osmoregulating tissues, including those of insects. The V-ATPase is central to the alkalization of the insect midgut (Klein, 1992; Zhuang et al., 1999) and the secretion of KCl-rich fluid by the Malpighian tubules of insects (Maddrell and ODonnell, 1992; Beyenbach et al., 2000). Filippova et al. (Filippova et al., 1998), using an antibody for the ß-subunit of the V-ATPase, examined freshwater-acclimated C. quinquefasciatus and reported that the H+-ATPase was located throughout the gut and the Malpighian tubules of the larvae. They did not examine the anal papillae, a key site for ion regulation in mosquito larvae. Zhuang et al. (Zhuang et al., 1999) found that antibodies for the E-subunit of V-ATPases co-localized with portasomes, which are small particles found on ion-transporting tissue and believed to be the V1 part of the H+ V-ATPase. Garrett and Bradley (Garrett and Bradley, 1984b), in examining the ultrastructure of osmoregulating tissues of the euryhaline osmoconformer Culiseta inornata, reported the presence of portasomes lining the epithelium of the anal papillae. Taken together, the above findings suggest that H+-ATPase may be present on the anal papillae of osmoconforming larvae. Whether an H+-ATPase or a Na+/K+-ATPase is driving Na+ and Cl transport in either the rectum of osmoregulating larvae (e.g. A. taeniorhynchus) or the anal papillae of osmoconforming species (e.g. C. tarsalis) during a salinity challenge has not been addressed. We are currently examining this question in the hope of advancing the understanding of ion regulation in osmoconforming mosquito larvae which was, until this present study, a mystery.
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Acknowledgments |
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