This study describes the expression patterns of P-type Na+/K+-ATPase and V-type H+-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene,both ATPases are expressed in all of the osmoregulatory tissues of larvae(midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach,Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na+/K+-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na+/K+-ATPase, apical V-type H+-ATPase) and ion-transporting (V-type H+-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations,whereas the adult stomach has uniform expression of basolateral P-type Na+/K+-ATPase and apical V-type H+-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H+-ATPase (apically and cytoplasmically) whereas P-type Na+/K+-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na+/K+-ATPase (basal) and V-type H+-ATPase(apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na+/K+-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na+/K+-ATPase and V-type H+-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

Osmoregulatory challenges of the yellow fever mosquito, Aedes aegypti, change with each developmental stage. The aquatic larval form,which resides in fresh water, faces a continual gain of water from drinking and also via osmotic flux across the body surface from the environment. Concurrently, larvae suffer diffusional losses of the predominant hemolymph ions, sodium and chloride, to the dilute external medium. Once the mosquito pupates and undergoes eclosion to an adult, the osmoregulatory challenges are intermittent and disparate in nature. Under non-feeding conditions, the adult mosquito is primarily concerned with conserving body water to avoid a reduction in body volume and concentration of the body ions. When the adult male or female takes a nectar meal this presents a hemolymph water load/ion dilution situation whereas a blood meal taken by a female presents both a water and salt load(Clements, 2000).

To deal with these insults to hemolymph homeostasis, larval and adult mosquitoes are equipped to rapidly respond and restore water and ion balance. Freshwater larvae eliminate excess water load by producing copious, dilute urine through the coordinated activity of the Malpighian tubules and hindgut,whereas the four anal papillae surrounding the anal opening are the primary sites of Na+ and Cl- absorption. Through these mechanisms of ion and water regulation hemolymph composition remains stable(Bradley, 1994). When adults ingest nectar, elimination of the water load is initiated within seconds of feeding and occurs without a significant loss of ions. This process, as in the larva, involves the Malpighian tubules and hindgut and results in rapid urine production and excretion. When a female takes a blood meal, excess salts(Na+, K+ and Cl-), which are absorbed across the stomach, are rapidly eliminated by Malpighian tubules/hindgut activity(Clements, 2000).

In order for the larva and adult to perform the above described osmoregulatory functions, ions must be transported against their electrochemical gradients. Primary urine production by the Malpighian tubules of larval A. aegypti is the consequence of active K+ and Na+ secretion (Cl- movement is considered to occur passively) from the hemolymph into the tubule lumen, with water following down its osmotic gradient (Clements,2000). Prior to being excreted, ions are actively absorbed from the urine by the rectal epithelium back into the hemolymph(Ramsay, 1950). In the adult female, excess Na+, K+, Cl- and water from a blood meal are secreted from the hemolymph into the Malpighian tubules, the active process being the movement of cations against their electrochemical potentials (Beyenbach,1995).

But what is the proximal source of energy driving this active transport in the different osmoregulatory organs of mosquitoes? For many years, insect physiologists examining ion transport have focused on the V-type H+-ATPase and have consider it the major player in establishing favourable, ion-motive gradients. This V-type H+-ATPase has been characterized in Malpighian tubules of adult A. aegypti by electrophysiological studies (Beyenbach et al., 2000) and in larvae by immunlocalization assays(Filippova et al., 1998). With regards to other osmoregulatory organs, this pump has been localized(Filippova et al., 1998; Zhuang et al., 1999) and characterized in vitro (Boudko et el., 2001) as the generator of larval midgut alkalinity (pH 11)whereas in the adult stomach, V-type H+-ATPase-overexpressing cells correlate with the distribution of malaria parasite oocysts in both Aedes aegypti and Anopheles gambiae(Cociancich et al., 1999). By contrast P-type Na+/K+-ATPase, long considered to be driving ion transport in many osmoregulatory systems, such as the thick ascending loop of vertebrate nephrons(Greger and Kunzelmann, 1990)and the gills of various aquatic invertebrates(Lucu and Towle, 2003; Onken et al., 2003), has not garnered as much attention in mosquito ion transport systems despite the fact that ouabain-sensitive P-type Na+/K+-ATPase activity has been reported in adult A. aegypti Malpighian tubules(Hegarty et al., 1991) and in the midgut of Anopheles stephensi(MacVicker et al., 1993). So the question remains as to the relative importance of these two ATPases in driving ion transport in each of the osmoregulatory tissues of larval and adult A. aegypti.

This present study takes a first step in addressing this issue by surveying all of the tissues relevant to osmoregulation in A. aegypti larvae(gastric caeca, midgut, Malpighian tubules, rectum, anal papillae) and adults(stomach, Malpighian tubules, anterior intestine, rectum) in order to determine V-type H+-ATPase and P-type Na+/K+-ATPase distribution and expression patterns, at the protein and gene levels using immunohistochemistry and reverse-transcriptase PCR assays. Our findings, in conjunction with previous morphological and physiological studies, provides a foundation for understanding the relative importance of V-type H+-ATPase and P-type Na+/K+-ATPase in the disparate osmoregulatory challenges faced by A. aegypti at two stages of development.

Experimental animals

A colony of Aedes aegypti (Linnaeus) was established in S. S. Gill's laboratory at UC Riverside, CA, USA. Larvae were hatched and held in dechlorinated tapwater in large rectangular metal pans. Larvae were fed daily a 2:1 ground liver powder:dry yeast mixture. For this study, fourth instar larvae were collected and placed in a smaller pan with food. For the adult females examined in this study, pupae were collected from the larval pans and placed in small cardboard containers (50 per container). Adult mosquitoes eclosed from the pupal stage and were supplied with a cotton baton soaked with a sugar solution. They were maintained at 27°C, 70% relative humidity and a 12 h:12 h light:dark cycle.

Tissue collection for immunohistochemistry and RT-PCR

To obtain tissues for immunohistochemistry (whole-mount and paraffin embedded) and RNA isolation for reverse transcriptase polymerase chain reaction (RT-PCR) assay, fourth instar larvae were rinsed and dissected in ice-cold phosphate-buffered saline (PBS). The entire larval gut (gastric caeca, midgut, Malpighian tubules, hindgut, anal papillae) was removed and the peritrophic membrane and contents thereof were removed. For whole-mount immunohistochemistry, the entire gut was transferred to 4% paraformaldehyde(PFA)/PBS solution and fixed overnight at 4°C. For paraffin embedding,whole larvae were used with the heads removed and several incisions were made in the cuticle along the body to ensure proper fixation in 4% PFA/PBS solution at 4°C overnight. Tissues for RNA isolation, the midgut (including gastric caeca), Malpighian tubules, hindgut, and the anal papillae were separated and transferred into cryovials on dry ice. Tissues from 15-20 larvae were combined in these tubes.

Adult female mosquitoes used in this experiment were 5-7 days post-eclosion. They were identified and separated from males after CO2 anesthesia. For whole-mount immunohistochemistry, the entire gut including the stomach, Malpighian tubules, anterior hindgut and rectum were dissected out in ice-cold PBS and fixed in 4% PFA/PBS solution overnight at 4°C. After rinsing in PBS, an incision along the longitudinal plane of the stomach was made. For tissues to be embedded in paraffin, the head, wings,legs and the last abdominal segment were removed and small tears in the abdominal cuticle were made to ensure penetration of the fixative. The thorax/abdomen was then fixed overnight in 4% PFA/PBS at 4°C. For RNA sampling, females were dissected in ice-cold PBS and the anterior midgut/stomach, Malpighian tubules, anterior intestine and the rectum were separated and transferred to cyrovials in dry ice. Tissues from 20-25 adult females were combined.

Immunohistochemistry of P-type Na+/K+-ATPase and V-type H+-ATPase in larval and adult female mosquitoes

To localize P-type Na+/K+-ATPase protein, the monoclonal antibody, `a5', raised against the α-subunit of avian P-type Na+/K+-ATPase in mice by Dr Douglas Fambrough, was employed. This antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB). This antibody was developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242,USA. To localize V-type H+-ATPase, a polyclonal serum antibody raised against the B subunit of the V-type H+-ATPase of Culex quinquefasciatus was employed(Filippova et al., 1998).

For whole-mount immunohistochemistry, gut tissues were thoroughly rinsed in PBS followed by a methanol dehydation/rehydration series. Tissues were rinsed with PBS then blocked for 2 h at room temperature with PBS/0.1% Triton X-100(PBT) including 2% bovine serum albumin (BSA). Tissues were incubated overnight at 4°C in a 5 μg ml-1 solution of the primary antibody a5 (avian Na+/K+-ATPase) and a 1:1000 dilution of polyclonal serum antibody to the V-type H+-ATPase made with PBT/1% BSA. An equivalent solution with supernatant NS-1 myeloma solution(supplied by DSHB) and rabbit preimmune serum (1:1000 dilution) served as a control for both antibodies. To remove unbound antibody, tissues were rinsed several times with PBT/1% BSA/2% normal goat serum (NGS). Next, tissues were incubated for 2 h at room temperature with Cy3-labeled goat anti-mouse secondary antibody and Cy5-labeled goat anti-rabbit secondary antibody(Jackson Immuno Research, CO, USA) at a 1:2000 dilution in PBT/1% BSA/2% NGS. The Cy3 anti-mouse and Cy5 anti-rabbit secondary antibodies were developed to have minimal cross reactivity with other species in the incubation medium. Preliminary tests indicated that concurrent incubation of α5 and V-type H+-ATPase antibodies and two secondary antibodies did not result in cross reactivity. After further rinsing with PBT/1% BSA/2% NGS, the tissues were mounted on slides using 90% glycerol/4% N-propyl gallate. The slides were stored at -20°C in the dark.

The thorax/abdomen of larvae or adult female mosquitoes were rinsed thoroughly in PBS then put through an ethanol dehydration series. The tissues were incubated in 100% ethanol followed by 70% ethanol/30% xylene solution,then 30% ethanol/70% xylene for 1 h each and finally 100% xylene overnight at room temperature. The next day, finely chopped paraffin chips (Paraplast Plus™) were added to the vials to approximately 50% of total volume. After 3-4 h, the xylene was changed and samples were incubated overnight at room temperature. The vials were then placed in an oven at 55-60°C. After the paraffin melted, half of the volume was replaced with melted paraffin and incubated at 55-60°C for 2-3 h. Next, the entire volume was replaced with fresh melted paraffin several times. The larval tissues and melted paraffin were then transferred to an embedding mold and allowed to solidify.

Sections (8 μm thick) of larval and adult tissue were made then mounted onto poly-lysine-coated slides and allowed to dry. Sections were dewaxed in 100% xylene, put through an ethanol rehydration series and finally washed with distilled water then transferred to PBT. Next the slides were blocked with PBT/2% BSA for 1-2 h at room temperature. Sections were incubated with the primary antibodies for V-type-ATPase and P-type Na+/K-ATPase (in PBT/1% BSA) at 4°C overnight. The next day, sections were washed with PBT/1% BSA/2% NGS and incubated with Cy3- and Cy5-labeled secondary antibodies for 2-3 h followed by rinsing with PBT/1% BSA/2% NGS. Finally the sections were mounted in glycerol/gelatin/Tris pH 7.4, with coverslips on top and stored at -20°C in the dark.

Whole mounts and tissue sections were examined by scanning confocal microscopy with a helium/neon laser (Zeiss LSM510 Axioplan 2; located in the Center of Advanced Microscopy and Microanalysis, University of California,Riverside). The tissues were analyzed with a 10×, 40× and 100× objectives. All images were imported into Adobe Photoshop for assembly and labelling.

Sequencing

cDNA libraries were made from isolated midgut and Malpighian tubules of fourth instar larvae and adult female Aedes aegypti as previously described (Jin et al., 2003). Analysis of random clones from these cDNA libraries identified a number of V-type H+-ATPase subunits, and P-type Na+/K+-ATPase. These clones were fully sequenced in both directions, and the nucleotide sequences deposited in GenBank.

RNA isolations and RT-PCR

Total RNA was extracted from the adult female stomach using TRIzol reagent(Invitrogen, Carlsbad, CA, USA) and further purified using RNeasy mini kit clean up protocol (Qiagen, Valencia, CA, USA). For adult Malpighian tubules,anterior hindgut, rectum and larval Malpighian tubules, hindgut, and anal papillae, total RNA was isolated using the RNeasy mini kit animal tissue protocol (Qiagen). RNA concentrations were determined (GeneQuant II spectrophotometer, Pharmacia Biotech, Piscataway, NJ, USA).

cDNA was reverse transcribed from equal amounts of total RNA from the larval and adult tissues using Superscript II First Strand cDNA synthesis kit(Invitrogen) with oligo hexamers, and included no-RT controls for each tissue. Primers for P-type Na+/K+-ATPase subunit and V-type H+-ATPase B subunit gene expression were designed to lie in adjacent exons based on the cDNA sequences for A. aegypti and the Softberry exon prediction program (RNASPL; www.softberry.com)so as to minimize genomic DNA amplification. 18s rRNA gene expression was also determined for each tissue as a control. All PCR assays were designed with an annealing temperature of 55°C. PCR products were run on a 1.5%agarose/ethidium bromide gel and documented using a Fotodyne system (Hartland,WI, USA).

Immunohistochemistry

Larva

Whole mounts of gastric caeca of larval A. aegypti indicated a regionalization of P-type Na+/K+-ATPase and V-type H+-ATPase with the former expressed only in the proximal (i.e. region close to junction with midgut) two-thirds of each caecum, and the latter having a higher expression in the distal third(Fig. 1A). Paraffin sections showed P-type Na+/K+-ATPase expression on the basal membrane of cells located in the proximal region whereas V-type H+-ATPase was located on the apical membrane throughout the length the caeca. In the distal third of gastric caeca, the V-type H+-ATPase was expressed on both the apical and basal membranes(Fig. 1B-D).

Expression levels and location of the two ATPases changed along the entire length of larval midgut. In the most anterior segment of the midgut, just posterior to the junction with the gastric caeca, only P-type Na+/K+-ATPase was evident(Fig. 1E,G). In the most posterior region of the midgut there was also high P-type Na+/K+-ATPase expression(Fig. 1F). In both locations,P-type Na+/K+-ATPase was expressed basally(Fig. 1G,I) whereas in the mid-anterior midgut region, it was expressed on the apical membrane(Fig. 1H). V-type H+-ATPase expression was highest in the anterior midgut region(Fig. 1E) where it was located on the basolateral membrane (Fig. 1H). In the posterior midgut V-type H+-ATPase was found on the apical membrane (Fig. 1I).

The distribution of the ATPases in the Malpighian tubules was dependent on cell type and region (Fig. 2). The V-type H+-ATPase was expressed only in the principal cells along the entire length of the tubules(Fig. 2A,C,E). It was localized primarily to the apical membrane of the principal cells however there appeared to be cytosolic expression in the distal portion of the Malpighian tubules(Fig. 2H) but not in the proximal region (Fig. 2G). The stellate cells expressed only P-type Na+/K+-ATPase and at a very high level relative to the neighbouring principal cells(Fig. 2A,D). However, stellate cells were observed only in the distal two thirds of each tubule(Fig. 1F, Fig. 2D) whereas the proximal third of the tubule contained only principal cells(Fig. 1F, Fig. 2B,C). Additionally,P-type Na+/K+-ATPase expression in the principal cells of the proximal region was higher relative to the distal principal cells that had very little, if any, P-type Na+/K+-ATPase expression as seen in both the whole mounts (Fig. 2B,D) and sections of the tubules(Fig. 2G,H). The proximal principal cells expressed P-type Na+/K+-ATPase on the basal membrane (Fig. 2G).

In the anterior intestine (or anterior hindgut) of the larval A. aegypti there was no P-type Na+/K+-ATPase labeling and a very low level of V-type H+-ATPase(Fig. 2F). By contrast, the rectum had high levels of P-type Na+/K+-ATPase expression (Fig. 2F) localized to the basal region (Fig. 2I)whereas V-type H+-ATPase was not readily observed in the rectal epithelium, and there was only non-specific labeling of the fecal content(Fig. 2I).

Sections of the anal papillae showed the presence of both ATPases, with P-type Na+/K+-ATPase expressed on the basal membrane(lumen) and V-type H+-ATPase on the apical membrane facing the environment (Fig. 2J,K).

Adult female

Both ATPases were present in the adult female stomach, in the same cells but in different regions (Fig. 3A-D). In an optical longitudinal section through the posterior region of the stomach, the P-type Na+/K+-ATPase was seen to be located laterally in the cell (Fig. 3A) whereas the V-type H+-ATPase was found within the cytoplasm (Fig. 3B). Paraffin cross sections of the stomach also indicated P-type Na+/K+-ATPase to be located laterally in the cells, all the way up to the apical region, in addition to being expressed on the basal membrane. V-type H+-ATPase expression was restricted to the apical region of the stomach epithelium (Fig. 3C,D). The entire stomach epithelium was surveyed but there were no obvious regional differences except that perhaps there was a relatively higher expression level for both ATPases in the posterior region of the stomach (anterior region not shown).

The adult Malpighian tubules exhibited the same distribution of the ATPases as described above for the larvae. The adult tubules showed high expression of the V-type H+-ATPase in the principal cells(Fig. 3F), with the highest expression on the apical membrane but cytosolic labeling was also evident(Fig. 3H, Fig. 4A,B). There was no observable V-type H+-ATPase labeling in the stellate cells(Fig. 3F). Instead stellate cells, found in the distal region, had high expression of P-type Na+/K+-ATPase relative to the adjacent principal cells(Fig. 3E). In the proximal third of the Malpighian tubules, P-type Na+/K+-ATPase labeling was higher in the principal cells relative to the distal population(Fig. 3E,G) and was localized to the basal membrane (Fig. 3H). Again, as in the larva, stellate cells(Fig. 1F, Fig. 2B,C) were not observed in the proximal thirds of the Malpighian tubules in the adults(Fig. 3G).

The hindgut of the adult female (Fig. 4) differed in the distribution of the ATPases relative to the larval tissue (Fig. 2). The anterior hindgut of the adult, which is a thicker epithelium than the larval counterpart, had high expression of both ATPases. V-type H+-ATPase was expressed on the apical membrane and P-type Na+/K+-ATPase was localized basolaterally(Fig. 4A,B,E,F). The adult female rectal epithelium had very low expression of both ATPases(Fig. 4C,D) but there was very intense labeling of the rectal pads (Fig. 4A-D). The apical aspect of these structures expressed V-type H+-ATPase whereas the internal aspect of the pad had even higher expression of P-type Na+/K+-ATPase(Fig. 4A-D).

RT-PCR

The genes for P-type Na+/K+-ATPase and V-type H+-ATPase were expressed in all of the larval(Fig. 5A) and adult tissues(Fig. 5B).

Sequences of V-type H+-ATPase and Na+/K+-ATPase subunits

The full-length and EST sequences reported in this paper have been deposited in the GenBank database. Full-length sequences are for V-type H+-ATPase A subunit (accession no AF008922), 110 kDa integral subunit mRNA (AF173554), and c (small) subunit (AF008924). ESTs submitted are for V-type H+-ATPase B subunit (CB690686), C subunit (CB690794), E subunit (CB751596), H subunit (CB690773), proteolipid (AI648290), 110 kDa(CB690738), 110 kDa* (CB751597), 110 kDa long form (CB690834) and for the subunit of P-type Na+/K+-ATPase (CB690713).

Expression of the genes coding for V-type H+-ATPase and P-type Na+/K+-ATPase was evident in all the key osmoregulatory tissues of larval and adult A. aegypti(Fig. 5). However, at the protein level, as assessed by immunolocalization techniques, we found intriguing patterns of distribution of these two ATPases with regards to the region of organ, cell type, or membrane location (Figs 1, 2, 3 and 4). Our results provide new information on the relative importance of V-type H+-ATPase and P-type Na+/K+-ATPase in the ion regulatory mechanisms in the less-studied tissues of larval and adult mosquitoes (gastric caeca,hindgut, anal papillae) but also raises questions regarding the current models of ion transport in those organs that have been the focus of many studies(Malpighian tubules, midgut). These findings indicate that both ATPases are contributing to the extraordinary osmoregulatory abilities of this insect.

Malpighian tubules

Our observations of differential expression of the two ATPases in the two cell types and the location of these cells types along the length of the Malpighian tubules suggest a divergence in function of the principal cells in the distal and proximal regions and a role for the stellate cells in the ion transport processes of the distal segment.

The distally positioned principal cells exhibited high expression of V-type H+-ATPase (Fig. 2A,E,H, Fig. 3F),confirming previous immunohistochemical assays of both adult(Weng et al., 2003) and larval(Filippova et al., 1998) forms of A. aegypti. Studies employing bafilomycin A1, a potent inhibitor of V-type H+-ATPase, confirmed that this pump, localized to the brush border of the apical membrane of principal cells(Fig. 2H), is a major energy source for the secretory processes of A. aegypti Malpighian tubules(Beyenbach et al., 2000; Weng et al., 2003). The V-type H+-ATPase generates an electromotive potential by pumping protons from the cell into the tubule lumen(Wieczorek et al., 1999; Beyenbach et al., 2000), thus providing the energy to drive the secretion of cations viaNa+, K+/nH+ antiporters(Petzel, 2000; Weng et al., 2003). The expression of V-type H+-ATPase throughout the cytoplasm of the distal principal cells could be that of the dissociated V1 subunit(Sumner et al., 1995),cytoplasmic pools of V-type H+-ATPase being recruited to the apical membrane, or those pumps associated with the vesicular membranes enclosing concretion bodies. With regards to the latter possibility, concretion bodies were previously determined to be located only in the principal cells and to contain high concentrations of divalent cations (Ca2+,Mg2+, Mn2+) and phosphorous(Bradley et al., 1990). Perhaps V-type H+-ATPase plays a role in facilitating the transport or making available for transport (i.e. by acidification) these cations within the concretion bodies, indicating a dual function for the H+ pump in the distal tubule.

Several features of stellate cells observed in this study point to a role in ion transport function. These cells, found only in the distal, secretory region of the Malpighian tubules (Fig. 2A-E, Fig. 3E-G),exhibited high expression of P-type Na+/K+-ATPase(Fig. 2A,D, Fig. 3E) and no V-type H+-ATPase (Fig. 2E, Fig. 3F), a pattern of expression that contrasts with the principal cells in the same distal segment(Fig. 2A,D,E, Fig. 3E,F). We could not confirm the membrane location of P-type Na+/K+-ATPase,however, we presume this pump to be localized to the basal region of stellate cells, where the membrane is more elaborate with extensive foldings(Mathew and Rai, 1976; Bradley et al., 1982; Yu and Beyenbach, 2004) and mitochondria (Satmary and Bradley,1984b).

The distal location of stellate cells does suggest a secretory function,however, the role of the Na+ pump in ion secretion in insect Malpighian tubules, including mosquitoes, has been ambiguous because of conflicting results utilizing the P-type Na+/K+-ATPase inhibitor ouabain. Using adult A. aegypti Malpighian tubules, Hegarty et al. (Hegarty et al., 1991)reported the presence of ouabain-sensitive fluid secretion, whereas other studies reported that ATPase activity(Weng et al., 2003) and membrane electrical properties (Williams and Beyenbach, 1984) were ouabain insensitive. Interestingly,Torrie et al. (Torrie et al.,2004) identified an ouabain transporter (organic anion transporting polypeptide; oatp) that colocalizes with P-type Na+/K+-ATPase in Drosophila melanogasterMalpighian tubules. This protein is the first step in the ouabain elimination pathway and serves to minimize ouabain concentrations and its inhibitory effects on P-type Na+/K+-ATPase that resides on the basal membrane of D. melanogaster tubules(Torrie et al., 2004). Furthermore, highly homologous oatp genes have been identified in both the Anopheles (Torrie et al., 2004) and Aedes genomes (data not shown), which indicates that these ouabain transporters are conserved within the dipterans. If expression of this ouabain transporter protein is confirmed and found to colocalize with P-type Na+/K+-ATPase to the stellate cells of A. aegypti Malpighian tubules, then the ouabain insensitivity noted in previous mosquito studies(Weng et al., 2003; Williams and Beyenbach, 1984)can be explained. Our observations of A. aegypti tubules, together with the discovery of this ouabain transport system in another dipteran Drosophila (Torrie et al.,2004), suggest P-type Na+/K+-ATPase and stellate cells play a critical role in the secretory function of mosquito Malpighian tubules.

Another intriguing aspect of stellate cell morphology that lends support for a greater functional role was their slender, elongated, cellular extensions that share a substantial area of contact with up to four adjacent principal cells (Fig. 2D,E, Fig. 3E,F). Although this was also noted in a previous scanning electron microscopy study(Satmary and Bradley, 1984a),stellate cells, until now, were dismissed as being vital to secretory function because they constitute only between 16-26% of the total cell number of tubules (Satmary and Bradley,1984b; Cabrero et al.,2004), and the dimensions of their cellular extensions and contact area with principal cells were underestimated utilizing light microscopy(Yu and Beyenbach, 2004). Instead, we propose that stellate cells themselves, or the interface between stellate and principal cells, could be the site for Cl- transport,a major aspect of the ion secretory mechanism that remains unresolved.

Currently, there are two proposed routes for transepithelial Cl-secretion in adult Aedes Malpighian tubules: a transcellular pathway through stellate cells and a paracellular shunt between adjacent principal cells (Beyenbach, 2003a; Beyenbach, 2003b; Massaro et al., 2004; Yu and Beyenbach, 2001; Yu and Beyenbach, 2004). Evidence supporting a transcellular Cl- pathway is based upon the characterization of two types of Cl- channels on the apical membrane of stellate cells from adult A. aegypti tubules(O'Connor and Beyenbach, 2001)and, more recently, leucokinin receptors localized to the stellate cell population in adult Anopheles gambiae tubules(Radford et al., 2004). Previous work had confirmed that Cl- secretion is stimulated by leucokinin in A. aegypti tubules(Pannabecker et al., 1993; Yu and Beyenbach, 2001; Yu and Beyenbach, 2004),however, the effect of leucokinin on these Cl- channels in stellate cells has not been examined so as to confirm whether this is the primary site of Cl- secretion. Our observation of high P-type Na+/K+-ATPase expression in stellate cells, in addition to the above described features of stellate cells(O'Connor and Beyenbach, 2001; Radford et al., 2004), lend support for a leucokininsensitive, Na+-driven, transcellular Cl- transport. Obviously, a basal Cl- transport mechanism needs to be identified in the stellate cell. One possible candidate is the bumetanidesensitive Na+/K+/2Cl-cotransporter, which has been electrophysiologically characterized to the basal aspect of principal cells in both A. aegypti(Hegarty et al., 1991; Scott et al., 2004) and Drosophila Malpighian tubules(Ianowski and O'Donnell,2004). Note that in both cases, it is thought that Cl-is recycled back to the hemolymph via Cl- channels. A second candidate is a Na+-driven Cl-/HCO3- exchanger (NDAE1) that colocalizes with P-type Na+/K+-ATPase to the basal membrane of D. melanogaster Malpighian tubules(Sciortino et al., 2001).

Evidence for a paracellular Cl- shunt in adult AedesMalpighian tubules comes from transepithelial diffusional Cl-potentials that imply a single barrier for the Cl- shunt, such as a septate junction (Pannabecker et al.,1993; Yu and Beyenbach,2001; Yu and Beyenbach,2002; Yu and Beyenbach,2004) and leucokinin-stimulated Cl- conductance in the distal portion of adult A. aegypti tubules that is independent of the presence of stellate cells, thus negating a role for stellate cells in Cl- conductance (Yu and Beyenbach, 2004). With regards to the latter study, we assert that it would be extremely difficult, if not impossible, to isolate a segment of the distal tubule without some portion of one or more stellate cells being present because of their uniform distribution thoroughout the distal region and their long, cellular extensions that interdigitate between the principal cells. Thus there is uncertainty as to whether the transcellular stellate cell route or a principal-stellate cell paracellular route for the Cl-secretion was functioning in those segments believed to be devoid of stellate cells. An examination of the ultrastructure of the principal-principal cell and principal-stellate cell paracellular regions and the effect of leucokinin on these paracellular junctions may help to resolve this issue. From the above discussion, there is still much to ascertain regarding Cl-transport, not only in adult tubules but in larval A. aegypti, which has garnered little attention.

Evidence of functional segmentation of A. aegypti Malpighian tubules came from the observation of the proximal segment consisting only of principal cells (Fig. 2B,C, Fig. 3G) that expressed V-type H+-ATPase and P-type Na+/K+-ATPase on the apical and basal membrane, respectively(Fig. 2G, Fig. 3H). Cabrero et al.(Cabrero et al., 2004),examining alkaline phosphatase expression in several genera of dipterans,confirmed the absence of stellate cells in both Anopheles and Aedes mosquito lower tubules and suggested a role in reabsorption for this enzyme found only in this segment of dipteran Malpighian tubules. The lower, proximal segment of both D. melanogaster and Rhodnius prolixus has been determined to be involved in the reabsorption of K+ from the tubule lumen(Maddrell and Phillips, 1975; Maddrell, 1978; O'Donnell and Maddrell, 1995; Haley and O'Donnell, 1997; Rheault and O'Donnell, 2001). Interestingly, in D. melanogaster tubules, stellate cells are found in greatest number in the main, secretory segment whereas in the lower,resorptive segment, there are far fewer(Sözen et al., 1997), a pattern we also observed in A. aegypti tubules(Fig. 2A-E, Fig. 3E-G). The function of the proximal region of the mosquito tubules has yet to be studied but it is possible that one or both ATPases could be driving the proposed resorptive activities. Indeed, in other insect Malpighian tubules, resorption is an energy requiring process (O'Donnell and Maddrell, 1995; Haley and O'Donnell, 1997), however, the specific ATPase driving resorptive activity has yet to be identified. Evidence of V-type H+-ATPase activity in the resorptive segment of D. melanogaster tubules comes from the ability to acidify lumenal fluid(O'Donnell and Maddrell,1995). Studies employing ouabain had indicated that P-type Na+/K+-ATPase was not playing a role in resorption in Rhodnius tubules (Haley and O'Donnell, 1997) but this needs to be re-examined in light of the recent discovery of ouabain transporters in insects(Torrie et al., 2004). Further examination of both the morphology and physiology of Aedes Malpighian tubules is essential to ascertain whether we can assign both secretory and resorptive functions to these organs. Differentiation of ion regulatory function along the length of the tubules has been well characterized in several diverse taxa of insects [e.g. hymenopterans(Arab and Caetano, 2002),orthopterans (Kim and Spring,1992), hempiterans (Maddrell and Phillips, 1975)] including dipterans(Dow et al., 1994; O'Donnell and Maddrell,1995).

Our observations, described in this section, hold true for both the larval and adult Malpighian tubules, suggesting that there are no apparent morphological distinctions or differences in ATPase function between the two life stages despite having disparate osmoregulatory demands. Mosquito Malpighian tubules do not undergo autolysis during metamorphosis(Clements, 2000) or morphological rearrangements via a change in cell number or proportion of principal cells to stellate cells(Satmary and Bradley, 1984b). Perhaps the source of differentiation in ion transport function between larval and adult A. aegypti is a consequence of differential modulation of expression and/or regulation of the ATPases or the population of secondary ion transporters (i.e. Na+, K+, Cl- channels,antiporters and symporters) that these two ATPases energize.

Midgut

In contrast to the Malpighian tubules, the midgut differs dramatically between the larval and adult stages both in gross morphology and location of the two ATPases.

The gastric caeca, the eight blind-end sacks at the anterior aspect of larval midgut, showed regional ATPase expression differences that correlate with their physiological function as designated by their ultrastructural characteristics (Volkman and Peters,1989a; Volkman and Peters,1989b; Zhuang et al.,1999). Located in the distal third of each caecum,ion-transporting cells are thought to absorb ions from the caecal lumen(Volkmann and Peters, 1989b). It is here that we observed V-type H+-ATPase on both membranes of these cells, indicating a novel ion-transporting tissue (Fig. 1B,D). V-type H+-ATPase activity in these cells was substantiated in the measurements of a bafilomycin-sensitive, inward alkaline,pH gradient at the distal aspect of the gastric caecum of A. aegyptilarvae (Boudko et al., 2001). Additionally, morphological studies reported that the apical membrane of the ion-transporting cells possess long, slender microvilli studded with portasomes and mitochondria extending up into each microvillus (Volkmann and Peters, 1989a; Zhuang et al.,1999), traits analogous to the principal cells of Malpighian tubules and the presence of V-type H+-ATPase(Bradley and Snyder, 1989). The basement membrane of these cells, however, has not been examined for the presence of portasomes (i.e. the V1 subunit of V-type H+-ATPase), but only characterized as having extensive infoldings and associated mitochondria (Volkman and Peters, 1989a; Zhuang et al.,1999). Perhaps the apical and basal V-type H+-ATPase function under different conditions such as when the ion or pH level of ingested water varies. Indirect support for this comes from ultrastructural changes in the ion-transporting cells with increased water salinity,specifically the reduction in apical microvilli length, accumulation of mitochondria in the basal region and enhancement of basal channels (Volkmann and Peters, 1989b). This may also suggest a reversal in the direction of ion and water movement from absorption to secretion with high salinity water being ingested.

The resorbing/secretory cells that comprise the proximal two thirds of each caecum of larval A. aegypti (Volkmann and Peters, 1989a) had high expression of P-type Na+/K+-ATPase on the basal membrane and V-type H+-ATPase on the apical membrane facing the lumen(Fig. 1B-D). These cells have extensive infoldings of both the basal and apical membranes and each are populated with mitochondria. However, these organelles do not reside within the apical microvilli as in the ion-transporting cells (Volkmann and Peters,1989a). It is conceivable that one or both of the ATPases are involved in the nutrient resorption activity of these cells as amino acid and glucose uptake in insects can be Na+ or H+ driven(Hediger, 1994; Harvey and Wieczorek, 1997; Liu et al., 2003). Interestingly, when external salinity is varied, these cells, like the ion-transporting cells, alter their ultrastructure suggesting these ion-dependent nutrient transport systems could be modulated (Volkmann and Peters, 1989b).

Our present examination of the main portion of the larval midgut found variation in the membrane location of both ATPases. This first ever look at P-type Na+/K+-ATPase expression in A. aegyptilarval midgut revealed high expression of this pump throughout this tissue(Fig. 1E-I). As a consequence,our findings introduce further complexity to the current osmoregulatory and nutrient absorption models for this tissue. Previous examinations of A. aegypti larval midgut (Filippova et al., 1998; Gill et al.,1998; Zhuang et al.,1999; Boudko et al.,2001) had focused on the role of V-type H+-ATPase in the alkalinization process and assumed that it was the sole source of proximal energy to drive ion and nutrient transport. The main reason for this was that the model for larval mosquito midgut alkalinity was adopted from the lepidopteran midgut, which had previously been documented to be devoid of P-type Na+/K+-ATPase activity(Wieczorek et al., 1999). As we have stated above, P-type Na+/K+-ATPase function needs to be reconsidered in light of the recent discovery of ouabain transporters in dipterans (Torrie et al.,2004).

V-type H+-ATPase expression in the larval midgut was similar to patterns previously examined (Filippova et al., 1998) including studies utilizing an antibody specific to the E subunit of the V-type H+-ATPase, with localization patterns correlating with the presence of portasomes (V1 subunit of V-type H+-ATPase) (Zhuang et al.,1999), bafilomycin-sensitive pH gradients(Boudko et al., 2001) and transepithelial potentials (TEP) (Clark et al., 1999). The highest V-type H+-ATPase expression was evident in the mid-anterior region (Fig. 1E) and was localized to the basal membrane(Fig. 1G,H) where portasomes line the extensive infoldings of this membrane(Zhuang et al., 1999). It is in this segment that lumenal pH is most alkaline (pH >10)(Zhuang et al., 1999; Boudko et al., 2001), and the TEP is lumen-negative with the basement membrane hyperpolarized(Clark et al., 2000). All of this evidence points to the extrusion of protons into the hemolymph via a basal V-type H+-ATPase. In the posterior midgut, the V-type H+-ATPase switched to an apical location(Fig. 1I) and is supported by previous findings of apical membrane hyperpolarization, lumen positive TEP(Clark et al., 1999) and colocalization of the E subunit of V-type H+-ATPase and portasomes to this membrane (Zhuang et al.,1999).

In the very anterior of the larval midgut, P-type Na+/K+-ATPase localized to the basal membrane with no V-type H+-ATPase expression in these cells(Fig. 1E,G). This particular region has not been described with regards to its ultrastructure or physiology, so a potential role for the high density of basal P-type Na+/K+-ATPase cannot be put forth at this time. Recently, Onken et al. (Onken et al.,2004) reported an ouabain-sensitive transepithelial voltage in the anterior midgut region of larval A. aegypti and proposed that P-type Na+/K+-ATPase was localized to the basal membrane. Perhaps it was this very anterior region that Onken et al.(Onken et al., 2004)characterized because this section lacks V-type H+-ATPase. Further along the anterior midgut segment (Fig. 1H) the P-type Na+/K+-ATPase was located apically, then basally in the posterior region(Fig. 1F,I).

This intriguing flipping pattern of the two ATPases between the apical and basal membranes down the length of the gut suggests that the activity of the two pumps could be coordinated as it is in other ion transporting epithelia(Ehrenfeld and Klein, 1997). For instance, it was suggested that the source of protons for the basal V-type H+-ATPase of the anterior midgut could be the gut lumen and that transport into the cell could occur through an apically located K+/nH+ antiporter(Boudko et al., 2001). If this configuration is present, then the apical P-type Na+/K+-ATPase could function to maintain the diffusive outward K+ gradient, thereby driving K+/H+exchange on this membrane. In the posterior midgut, the basal P-type Na+/K+-ATPase could power Na+ absorption as A. aegypti larvae typically reside in Na+-poor environments and may rely upon diet as a Na+ source. Na+absorption could occur across the apical membrane through a Na+channel coupled to the V-type H+-ATPase with transport across the basal membrane via Na+/K+-ATPase(Ehrenfeld and Klein, 1997). This inward Na+ gradient could also aid in the absorption of amino acids. An amino acid transporter (KAAT1) was cloned and characterized in Manduca and was found to accept both Na+ and K+as cosubstrates (Liu et al.,2003). To truly characterize ATPase function in the larval midgut,it will be necessary to know the ionic concentrations of the lumen and midgut cells in the anterior and posterior regions. This will then help to describe the environment in which P-type Na+/K+-ATPase and V-type H+-ATPase are functioning when expressed on either membrane. This,in addition to further physiological characterization of ion transport in the midgut, will help to clarify coordinated or independent functioning of the two ATPases.

In the midgut of the adult female (also referred to as the stomach), P-type Na+/K+-ATPase and V-type H+-ATPase were expressed on the basolateral and apical membrane respectively(Fig. 3A-D) with perhaps,qualitatively, higher levels of expression of both ATPases in the posterior region (comparison not shown). This pattern correlates with the morphological features of the posterior region that are indicative of ion and water transport function (Billingsley,1990). The posterior midgut is also the region where the blood meal, taken by a female mosquito, is moved to(Nation, 2002). Columnar cells, the predominant cell type in the midgut, possess very elaborate apical and basolateral membrane systems with the latter forming a basal labyrinth consisting of extensive, deep infoldings laden with mitochondria(Clements, 2000). It is within this region we believe P-type Na+/K+-ATPase is located because of the intense and expansive labeling of this pump on the lateral aspect (Fig. 3A,C) and throughout the basal region (Fig. 3C,D). Additional evidence of P-type Na+/K+-ATPase was provided by the finding that more than 84% of ATPase activity in adult mosquito stomach is ouabain sensitive(MacVicker et al., 1993). This ATPase may serve a role in the absorption of ions and water across the adult midgut in a manner similar to that described for solute-coupled water transport in the mammalian small intestine(Larsen et al., 2002). In this model, the laterally located P-type Na+/K+-ATPase drives both inter- and transcellular water and ion absorption via the recirculation of Na+ through a basolateral Na+/K+/2Cl- cotransporter. Interestingly,expression of the P-type Na+/K+-ATPase gene in the stomach of adult female A. aegypti decreased following a blood meal(Sanders et al., 2003),perhaps as a means to attenuate the proposed solute-coupled water transport,thus avoiding excessive ion and water influx across the gut. By contrast,V-type H+-ATPase gene expression in the stomach rapidly increased following a blood meal. Concurrent with the H+ pump expression pattern was the upregulation of amino acid and sugar transporter genes(Sanders et al., 2003), which together, may suggest a H+-driven nutrient absorption mechanism in the adult female stomach. Examination of protein expression and activity of these two ATPases and the secondary transporters coupled to these ATPases is essential to help elucidate their role in the diuresis response of adult mosquitoes.

Hindgut and larval anal papillae

Although expression of P-type Na+/K+-ATPase and V-type H+-ATPase genes was evident in the larval rectum(Fig. 5A), only P-type Na+/K+-ATPase protein labeling was observed(Fig. 2F,I), suggesting that it is the primary pump energizing the resorption of Na+, K+and Cl- from the urine, as previous in vitro and in vivo investigations have detailed(Bradley, 1987; Bradley, 1994). Our finding of high expression of P-type Na+/K+-ATPase on the basal membrane of the rectum (Fig. 2F,I) is in agreement with other insect studies. P-type Na+/K+-ATPase has been found in the rectum in D. melanogaster using the same α5 antibody(Lebovitz et al., 1989) and also by ouabain-inhibitable sensitivity assays in the locust rectum(Peacock, 1981). The basolateral region of the rectal epithelium of larval A. aegypticonsists of an elaborate labyrinth that appears to extend almost to the apical region (Clements, 2000). Based on the intense P-type Na+/K+-ATPase labeling that appears to occupy the basal two-thirds of the rectal cells(Fig. 2I), we propose this pump is residing on these basal infoldings. However, the lack of V-type H+-ATPase expression in the rectal epithelium of larval A. aegypti (Fig. 2F,I)conflicts with other findings (Filippova et al., 1998) where, using the same V-type H+-ATPase antibody, high expression in the rectum of this species was reported. However,only whole-mount specimens where examined in the previous study and hence the non-specific V-type H+-ATPase labeling of the rectal contents was not detected as it was in the paraffin sections of this present study(Fig. 2I). Although the ultrastructure of the Aedes larval rectum has yet to been examined,in Culex mosquito larvae, the apical membrane of the rectal cells is extensive and studded with portasomes(Bradley, 1987) suggesting that the V-type H+-ATPase should be there.

We successfully determined that V-type H+-ATPase and P-type Na+/K+-ATPase proteins immunolocalize to the apical and basal membranes, respectively, in the anal papillae of larval A. aegypti (Fig. 2J,K). Earlier studies had pinpointed the anal papillae as the predominant organs for Na+ and Cl- uptake in freshwater mosquito larvae(Wigglesworth, 1938; Treherne, 1954; Stobbart, 1965), however,these four, cuticle-lined, saclike organs have been troublesome for the examination of ion transport processes because of their design(Bradley, 1994). More recently,with the use of self-referencing ion-selective (SeRIS) microelectrodes, Donini and O'Donnell (Donini and O'Donnell,2005) confirmed that the anal papillae of larval A. aegypti serve as the major site for Na+, Cl- and K+ uptake. With regards to the role of ATPase, Patrick et al.(Patrick et al., 2002)characterized in vivo Na+ and Cl- uptake in A. aegypti larvae and found that V-type H+-ATPase was involved in both transport processes, but in a novel way. The external application of bafilomycin A1, a specific antagonist of V-type H+-ATPase, resulted in a stimulation of Cl- uptake(Patrick et al., 2002), a pattern that departs from all other Cl- absorptive processes examined (Fenwick et al.,1999; Phillips et al.,1996) and intimates a novel H+-dependent Cl-uptake mechanism (i.e. Cl- channel). In the same experiment,bafilomycin inhibited Na+ uptake, indicating an apical V-type H+-ATPase/Na+ channel moiety as described in other freshwater animals, where the extrusion of protons sets up the inward electrodiffusive gradient for Na+(Fenwick et al., 1999; Ehrenfeld and Klein, 1997). Our present finding of apical V-type H+-ATPase expression(Fig. 2J,K), along with reports of the apical lamella being studded with portasomes and associated with mitochondria (Sohal and Copeland,1966; Meredith and Phillips,1973; Garrett and Bradley,1984) bolsters the findings of Patrick et al.(Patrick et al., 2002). The role of the basal Na+/K+-ATPase in anal papillae remains to be probed but could serve as the entry step of Na+ into the hemolymph. The extensive, deep basal infoldings and tightly associated mitochondria (Sohal and Copeland,1966; Meredith and Phillips,1973; Garrett and Bradley,1984) is a feature shared with the larval rectum (see above) and indicative of an ion transport role. One way in which this ion transporting tissue differs from others in the larva is that it is syncytial, meaning it lacks a lateral cell wall. Perhaps this unusual morphology gives rise to the novel ion transporter configurations (i.e. Cl- transport) noted previously (Patrick et al.,2002).

To date, only morphological studies have been performed on the adult mosquito hindgut. Our study is the first to examine specific ion transport proteins in these epithelia. The patterns of basolateral P-type Na+/K+-ATPase and apical V-type H+-ATPase localization in the adult hindgut (Fig. 4), in addition to previous studies(Hopkins, 1967; Tongu et al., 1969) infer that both the anterior hindgut and rectal pads are sites of ion and water movement. The rectum of adult A. aegypti consists of a thin rectal sac in which six (female) or four (male) rectal pads(Clements, 2000) protrude into the lumen (Fig. 4A-D). By contrast, the anterior hindgut is a thicker epithelium(Fig. 4A,B,E,F) relative to the rectal sac (Fig. 4A). Both the rectal pads and the anterior hindgut possess a well-developed basolateral labyrinth populated with mitochondria, a trait shared with the larval rectum and anal papillae (Clements,2000). Following a blood meal, the most notable changes occurred in the basal and lateral membrane systems of the rectal pads, intimating enhanced ion and water absorption activity(Hopkins, 1967). In Fig. 4C, the intense P-type Na+/K+-ATPase labeling on these basolateral infoldings of the rectal pads indicate that the Na+ pump must be driving these absorptive processes following a blood meal. The apical membrane of the anterior hindgut contains elaborate infoldings with mitochondria(Tongu et al., 1969) and was found to highly express the H+ pump(Fig. 4E,F). By contrast,Hopkins (Hopkins, 1967),reported that the apical membrane of the rectal pads is not elaborately infolded. We observed only a thin layer of V-type H+-ATPase expression in this region (Fig. 4C,D) thus confirming that this membrane is not extensive. Additional studies are necessary to determine what role these organs and two ATPases serve in the adult mosquito osmoregulatory activities.

Conclusion

We have established the presence of P-type Na+/K+-ATPase and V-type H+-ATPase in the key osmoregulatory tissues of larval and adult A. aegypti. The patterns of protein expression, as determined through immunolocalization techniques,suggest that both ATPases are contributing to solute transport, specificially by providing the proximal energy, in the form of favourable electrochemical gradients, to move ions and organic solutes across membranes. Now, we can identify the specific roles of P-type Na+/K+-ATPase and V-type H+-ATPase in the two life stages of the A. aegyptiby monitoring the gene and protein expression profiles of these two pumps during the mosquito's response to physiological challenges such as varying the chemical composition of the larva's watery environment or a blood meal taken by the adult female. These studies will then provide information on how the ATPases are regulated.

This research was supported by research grants AI 32572 and 48049 from the National Institutes of Health to S.S.G. and the University of California President's Postdoctoral Fellowship to M.L.P.

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