Tài liệu Báo cáo khoa học: Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes docx

Mosquito ( Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes Laurence Duchesne 1 , Jean-Franc¸ois Hubert 1 , Jean-Marc Verbavatz 2 , Daniel Thomas 1 and Patricia V. Pietrantonio 3 1 UMR CNRS 6026, Interactions Cellulaires et Mole ´ culaires, Universite ´ de Rennes I, Rennes, France; 2 Service de Biologie Cellulaire, CEA, Saclay, France; 3 Department of Entomology, Texas A&M University, College Station, TX, USA Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immuno- histochemistry detected the protein, AeaAQP, also in tra- cheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 · 10 )6 to 150 · 10 )6 mÆs )1 , which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylo- genetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze-fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water- permeable homotetrameric MIP which natively can form 2D crystals. Keywords: aquaporin; insect respiration; Malpighian tubule; tracheolar cell; tracheoles. Knowledge on insect respiration is abundant in the areas of morphology of the respiratory system, its adaptations and the physics of respiration [1–3]. By contrast, the molecular mechanisms of tracheole ventilation are poorly understood. Early work by Wigglesworth and others showed that trachea break up into large (1–2 lm diameter) or small (0.2– 0.4 lm internal diameter) tracheoles arising as fine intra- cellular canals from tracheolar cells [4]. From experiments in which myrcene and kerosene were injected into trachea of Apis mellifera (Hymenoptera), Tenebrio molitor (Coleop- tera), Pieris brassicae (Lepidoptera) and Rhodnius prolixus (Hemiptera), it was concluded that there are differences in the permeability of tracheolar walls in different parts of the tracheole system of different species. This was based on differential leakage of the injected fluid. The permeability of the tracheoles in the flight muscles is much greater than elsewhere [4]. In Apis and Musca flight muscle, leakage from small tracheoles occurred from the tracheole end close to the muscle mitochondria. Water fills the tracheoles immediately after insect death and, especially when insects are at rest, water has been observed within the blind end of tracheoles in muscles or the gut wall of insects of several orders, including higher dipterans (Musca) [5]. In contrast, during periods of high-energy demand such as flight, water is withdrawn from the tracheoles that supply oxygen to flight muscles [5]. The physiological significance of this movement of fluid in tracheoles that supply oxygen to tissues with very different oxygen demands when active or at rest, such as muscle, is interpreted as a compromise between the need to conserve water and the need to obtain oxygen which is critical for all terrestrial animals [5]. During muscle activity, formation of metabolites increases osmotic pressure in the myocyte cytoplasm around the tracheolar endings, causing water to be withdrawn from the highly permeable tracheolar endings. This allows oxygen to reach the end of the tracheole that is near the muscle cell mitochondria [6]. Almost nothing is known about the insect respiratory system and its function at the molecular level or the mechanisms that regulate this movement of water in the tracheoles. Water movement across cell membranes is facilitated through proteins of the MIP (major intrinsic protein) family [7], the aquaporin (AQP) proteins in epithelial and nonepithelial cells. Eleven different aquaporins have been cloned from mammals, and others have been identified in bacteria, plants, insects, and amphibians. Two groups have been defined among the mammalian aquaporins: aquaporins, AQP0, 1, 2, 4, 5, 8 and 10, selective for water; aquaglyceroporins, AQP3, 7 and 9, with slightly less selective pores, permeated by water, glycerol and other small nonelectrolytes. The latter are also known as glycerol facilitator-like proteins [8]. The true aquaporins Correspondence to P. V. Pietrantonio, Department of Entomology, Heep Center Room 412, 2475 TAMU, College Station, TX 77843-2475, USA. Fax: + 1 979 845 6305, Tel.: + 1 979 845 9728, E-mail: p-pietrantonio@tamu.edu, http://insects.tamu.edu/new/people/faculty/pietranp.html Abbreviation: OAP, orthogonal arrays of particles; AQP, aquaporin; MIP, major intrinsic protein. (Received 21 July 2002, revised 19 November 2002, accepted 25 November 2002) Eur. J. Biochem. 270, 422–429 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03389.x form single water channels, although they arrange as tetramers in the cell membrane [9]. For aquaporin reviews, see refs [10–12]. Substantial water transport occurs in the mammalian lung [13] as well as in insect tracheoles [4]. Four aquaporins, AQP1, AQP3, AQP4 and AQP5, are present in mammalian lung [14]. AQP4 is unusual among aquaporins in that water transport is not inhibited by Hg 2+ [15], it has a high intrinsic water permeability [16], and it forms crystalline orthogonal arrays of particles (OAPs) in cell membranes, as revealed by freeze-fracture electron microscopy [17] and by immunogold labeling of tissues [18]. These OAPs are absent from AQP4 null mice [19]. The only insect aquaporin that has been studied in detail, the Cicadella viridis aquaporin (AQPcic) expressed in the filter chamber of this homopteran, is also organized as a tetramer in the membrane where it forms a regular 2D array [20,21]. The cloning from a Malpighian tubule cDNA library and the in situ localization of an aquaporin, AeaAQP, in tracheolar cells associated with the Malpighian tubules of females of the dengue vector mosquito (Aedes aegypti; Diptera, Culicidae) had been previously reported [22]. In situ localization had shown that the transcript is present in the tracheolar cells closely associated with the Malpighian tubules, both in the tracheoles and in the tracheolar cell body but not in the Malpighian tubule epithelium or other tracheolar cells supplying the digestive system. Considering its localization and the high sequence similarity to the mammalian mercury-insensitive water channel protein AQP4, and to AQPcic, it was speculated that AeaAQP may be expressed in tracheolar cells for rapid water transport [22]. Here we provide evidence that AeaAQP is indeed expressed in the tracheolar cells associated with the Malpighian tubules, where it probably forms orthogonal arrays of channels able to transport water at high rates. These findings provide a molecular mechanism to support the theory on tracheole physiology proposing that changes in tissue osmotic pressure are associated with rapid water movement in tracheoles supplying tissues with different oxygen demands during activity or rest. This movement of water from the highly permeable tracheole endings is thus physiologically important for insect respiration [4,5]. Materials and methods Whole mount immunohistochemistry A. aegypti females, 0–2 days old, were dissected for Malpighian tubules. Tissues were fixed in freshly prepared 4% (v/v) formaldehyde in phosphate buffered saline (NaCl/ P i ) containing 50 m M EGTA for 2 h with slow agitation at room temperature. The fixative was removed by 3 · 10 min washes with 70% ethanol on ice. Tissues were washed twice for5minwithNaCl/P i containing 0.1% (v/v) Tween and 2% (v/v) normal goat serum (PBSTG) on ice, and then treatedwith12lgÆmL )1 proteinase K (Sigma) at room temperature for 10 min. After a 5-min rinse with PBSTG, tissues were incubated overnight with 10% (v/v) normal goat serum in NaCl/P i at 4 °C. All subsequent steps were carried out with slow agitation at room temperature. This solution was replaced and tissues were incubated again overnight. Tissues were incubated with 1 : 500 dilutions of normal rabbit serum (Sigma) or anti-AQPcic polyclonal serum overnight [20,22]. The latter antiserum recognizes the AeaAQP in Western blots of female Malpighian tubules dissected with tracheolar cells attached [22]. Negative controls without primary antibody were also conducted. Tissues were washed 4 · 20mininPBSTGandincubated in a 1 : 750 dilution of biotinylated anti-rabbit IgG (Vector Laboratories) for 1 h. Tissues were washed 4 · 20 min in PBSTG and incubated in a 1 : 200 dilution of Texas Red- Streptavidin (Vector Laboratories) for 30 min. Tissues were washed 6 · 30 min in PBSTG. Tissues were kept in PBSTG overnight at 4 °C and mounted in Vectashield Mounting Medium with DAPI for nuclear staining (Vector Laborat- ories). Fluorescence microscopy was with a Zeiss Axiophot microscope using filters for DAPI [glass (G) 365 nm, dichroic mirror (FT) 395 nm, long path (LP) 420 nm)] and Rhodamine [band path (BP) 546 nm, FT 580 nm, LP 590 nm]. Images were obtained with a C5810 color chilled 3-chip CCD camera (Hamamatsu Photonics, K. K. Systems, Hamamatsu City, Japan), connected to the microscope and to a MacIntosh (Apple Computer, Inc.) computer with a C5810 plug-in module. Images were visualized with a TrinitronÒ color video monitor (Sony) and imported into PHOTOSHOP TM (Adobe Systems, Inc.) software in color. Files of images captured with filters for blue (DAPI) and red (Rhodamine) were merged to obtain color prints. Plasmid construction AQPcic and the glycerol facilitator protein of Escherichia coli (glpF) constructs were as in [23]. The coding region of AeaAQP was amplified from the pSPORT-AeaAQP by PCR using two primers: AeapS1F, 5¢-GGAAGATCTATGACTGAAAGCG CA-3¢; AeapS1R, 5¢-GGAAGATCTTTAAAAATCGTA AGATTCC-3¢. The PCR primers contain BglII restriction sites (bold) used to clone into the pXG-ev1 vector. Functional analysis in Xenopus oocytes AQPcic, GlpF and AeaAQP cRNA were prepared in vitro using the mCap mRNA capping kit (Stratagene) and injected into stage VI oocytes. Oocytes were incubated in buffer (82.5 m M NaCl, 2.5 m M KCl, 1 m M CaCl 2 ,1m M MgCl 2 ,2m M NaHCO 3 ,10m M Hepes/NaOH, pH 7.4) at 18 °C for 48 h. Osmotic water permeability and apparent glycerol permeability of oocytes were measured as previ- ously described [24]. For water permeability measurements, the time course of oocyte swelling in response to a threefold dilution of extracellular buffer was monitored at 15 s intervals for 2.5 min by video recording, in the presence or absence of 0.5 m M HgCl 2 . The oocyte volume (V )was calculated at each time point relative to volume at the initial observation (V 0 ). The osmotic water permeability coefficient (P f ,in10 )6 mÆs )1 ) was calculated from the oocyte surface area (S ¼ 0.045 cm 2 ), the initial volume (V 0 ¼ 9 · 10 )4 cm 3 ), the molecular volume of water (V w ¼ 18 cm 3 /mol), and the initial rate of oocyte swelling d(V/V 0 )/dt, by means of the equation: P f ¼ V 0 Â dðV=V 0 Þ=dt=½S Â V w Âðosm in À osm out Þ Ó FEBS 2003 A. aegypti aquaporin forms orthogonal arrays (Eur. J. Biochem. 270) 423 where osm out is 176 mmolÆkg )1 and osm in is 58 mmolÆkg )1 . For glycerol transport assays, oocytes were transferred in an iso-osmotic solution in which 140 m M glycerol was present. The increase in oocyte volume corresponds to the water influx accompanying the solute uptake. The volume changes were followed by video microscopy for 15 min. Apparent glycerol permeability was calculated from the equation: P 0 gly ¼½dðV=V 0 Þ=dtÂðV 0 =SÞ Membrane preparation and protein solubilization Xenopus total membranes were prepared by the method described in [25]. Xenopus oocyte membranes were incuba- ted in TB buffer (20 m M Tris/HCl, pH 7.4, 1 m M dithio- threitol) containing 2% (w/v) n-octyl b- D -glucopyranoside for 12 h at 4 °C or in the same buffer containing 1% (w/v) SDS for 12 h at room temperature. Insoluble materials were removed by centrifugation at 100 000 g for 45 min at 15 °C. Velocity sedimentation on sucrose gradients Linear 2–20% (w/v) sucrose density gradients were prepared from 2% and 20% sucrose stock solutions (v/v) in TB buffer containing 2% (w/v) n-octyl b- D -glucopyranoside or 0.1% (w/v) SDS. Solubilized proteins (1–10 lg) were layered on topofgradientsandultracentrifugationwasperformedat 100 000 g for 16 h at 5 °C. Calibration curves for the determination of the apparent sedimentation coefficient were constructed using cytochrome c (S 20,w ¼ 1.7 S), BSA (S 20,w ¼ 4.3 S) and IgG (S 20,w ¼ 7 S). After centrifugation, 20 fractions were collected from the bottom of each gradient and analyzed by SDS/PAGE [26]. Proteins of each fraction were revealed by either Coomassie Blue staining or Western blotting. Antibodies and Western blotting analysis AQPcic and AeaAQP immunodetection were performed using a polyclonal rabbit antiserum raised against the native C. viridis protein [20]. Proteins resolved by SDS/PAGE were electrotransferred to poly(vinylidene difluoride) mem- branes. The blots were first incubated with anti-AQPcic Fig. 1. Whole mount immunohistochemistry of 1–2 day-old female Malpighian tubules. In both figures one Malpighian tubule is oriented vertically along its length. (A) Control with preimmune rabbit serum and Texas Red-labeled secondary antibody; nuclei (in blue) stained withDAPI.SCN,Stellatecellnucleus;PCN,principalcellnucleus. The longer arrows point to the tracheolar cell (tc) nuclei; notice the lack of signal above the background surrounding these nuclei. (B) Tissues incubated with rabbit anti-aquaporin serum and Texas Red-labeled secondary antibody. Tracheolar cells (tc, longer arrows), which are closely associated with Malpighian tubules, show positive immuno- reactivity for this aquaporin. The brightest tracheolar cell towards the centre bottom of the figure is above the focus plane. Notice the lack of signalinthetrachealcells(TrC,trachealcellnucleus,shortarrows)and the weak signal in tracheoles (compare with A). (C) Light micrograph ofthesametissueasin(B)showingthetracheolarcells. 424 L. Duchesne et al.(Eur. J. Biochem. 270) Ó FEBS 2003 serum (1 : 1000) and the proteins were revealed as previously described [27] using the ECL detection kit (Amersham). Freeze-fracture analysis For freeze-fracture electron microscopy, defolliculated AeaAQP-injected oocytes and water-injected oocytes were fixed overnight in 2% (v/v) glutaraldehyde in NaCl/P i and washed in NaCl/P i . As described previously [19,28–30], oocyte membranes were incubated for several hours in 30% (v/v) glycerol, inserted between two copper holders, and rapidly frozen in liquid nitrogen-cooled freon at )150 °C. Samples were transferred into a freeze-fracture apparatus (Balzers, Balzers Switzerland) at )130 °C under a vacuum of 10 )7 Torr. Specimens were fractured and shadowed with % 1.5 nm platinum at an angle of 45°, followed by carbon at 90°. Tissue replicas were digested overnight in bleach. They were then washed in water, dried and observed under the electron microscope (Philips EM 400). Results Aquaporin localization Immunohistochemistry with polyclonal antibodies con- firmed the localization of aquaporin in tracheolar cells associated with the female Malpighian tubules (Fig. 1). Signal was not detected above background level in trache- olar cells associated with the midgut or tissues such as Malpighian tubule epithelium (Fig. 1B), midgut or hindgut (not shown). Negative controls with preimmune serum did not show any staining of the tracheolar cells, as expected (Fig. 1A). In addition, reverse transcriptase PCRs with specific primers for this aquaporin designed towards the 5¢ and 3¢ ends of the cDNA showed that in nonblood-fed females this aquaporin mRNA is also transcribed in the head (2–6-day- old females) and hindgut (5–10-day-old females) (not shown). Oocyte swelling assays Figure 2 shows the primers utilized and the vector con- structed to produce AeaAQP cRNA for Xenopus oocyte injection. Figure 3A shows that the swelling rate of oocytes injected with AQPcic or AeaAQP in response to a threefold dilution of the buffer medium was increased 10–15-fold compared with that of control oocytes. These increases were inhibited by preincubation of oocytes in 0.5 m M HgCl 2 .On the other hand, no significant increase in apparent glycerol permeability was measured for AeaAQP oocytes, whereas under the same conditions GlpF-expressing oocytes exhi- bited 4–6-fold increases in glycerol permeability (Fig. 3B). Oligomeric form of Aea AQP To investigate the native oligomeric state of AeaAQP protein, experiments on velocity sedimentation on sucrose gradients were performed. After swelling assays, AeaAQP from Xenopus oocyte membranes was solubilized with either the nondenaturing detergent n-octyl b- D -glucopyranoside or the denaturing detergent SDS and analysed on a linear 2–20% (w/v) sucrose density gradient. As shown in Fig. 4, Western blotting revealed that AeaAQP, when solubilized in n-octyl b- D -glucopyranoside, peaks at sedimentation frac- tions corresponding to a 6.8–7S sedimentation coefficient value. Previous hydrodynamic analyses of the aquaporins AQP0 [31], AQP1 [32], and AQPcic [20] solubilized in n-octyl b- D -glucopyranoside, as well as measurements of the amounts of bound detergent, demonstrated that 6.8S sedimentation coefficients correspond to homotetramers for these members of the aquaporin family. Owing to the high sequence homology and hydrophobicity profile simi- larity, the sedimentation coefficient of AeaAQP in Fig. 4 only fits with a homotetrameric form of the protein. In addition, when 1% of the denaturing detergent SDS was used for membrane protein solubilization, the AeaAQP sedimentation coefficient shifted from 6.8S to 2.8S, a value that corresponds to monomers (data not shown). Orthogonal arrays in oocyte membranes The ultrastructure of AeaAQP was examined by freeze- fracture electron microscopy in plasma membranes of Xenopus oocytes injected with AeaAQP cRNA (Fig. 5A) and compared with those of oocytes injected with water Fig. 2. Plasmid construction. The coding region of AeaAQP was amplified from the pSPORT-AeaAQP [22] by thermal cycling using AeaS1F and AeaS1RprimersandclonedintopXbG-ev1asdescribed in Materials and methods. Fig. 3. AeaAQP functional properties. Oocytes were injected with cRNA encoding AQPcic, E. coli GlpF or AeaAQP, or injected with water (H 2 O control oocytes). (A) Osmotic water permeability (P f ) with or without pretreatment in 0.5 m M HgCl 2 for 15 min. (B) Gly- cerol apparent permeability (P¢ gly ). For each experiment, data were obtained from 10 to 15 oocytes. The given P f and P¢ gly are the means of three to five independent experiments. Ó FEBS 2003 A. aegypti aquaporin forms orthogonal arrays (Eur. J. Biochem. 270) 425 (Fig. 5B). In oocytes expressing AeaAQP, OAPs, a land- mark of some aquaporins [19,28], were often observed (Fig. 5A, arrows). OAPs were never observed in water- injected oocytes, where only isolated intramembrane parti- cles were observed here (Fig. 5B) and in previous reports [30]. This demonstrates that, in Xenopus oocytes, AeaAQP forms OAPs, similar to those formed by AQP-0 (MIP26) or AQP4 [19,28]. Particle spacing within AeaAQP-induced OAPs was % 6.7 nm, a value comparable to previous reports on the OAP-forming aquaporins, AQP0 (6.8– 6.9 nm), AQP4 (6.8 nm) [28] and AQPcic (6.8 nm) [33]. Of interest is the observation that the density or size of intramembrane particles outside of OAPs was not markedly increased in AeaAQP-injected oocytes, suggesting that most of the protein is located within the OAPs. Discussion The experiments described confirmed that the putative water channel cDNA cloned from a female mosquito Malpighian tubule library [22] and expressed in tracheolar cells encodes an aquaporin and not an aquaglyceroporin, and forms orthogonal arrays in Xenopus oocyte membranes where it transports water at high rates. It was also demonstrated that the protein is probably arranged as a homotetramer in the membrane, similarly to other aquapo- rins [34]. These findings are significant because there has been little progress on the understanding of insect tracheole ventilation at the molecular level. Although it was previously shown that the A. aegypti aquaporin transcript is present in the tracheolar cells, and Western blots of Malpighian tubule (dissected with attached tracheolar cells) homogenates exhibited the expected size band (26 kDa) [22], here we provide further evidence of AeaAQP expression in the tracheolar cells by direct positive immunolocalization. This is important because aquaporin mRNA may not always be translated; for example, in the kidney of the desert rodent Dipodomys merriami merriami, although the mRNA for the homo- logous mammalian AQP4 is synthesized, it is not trans- lated and AQP4 is not expressed [35]. Although transcription of AeaAQP is not limited to the tracheolar cells associated with the Malpighian tubules because the mRNA is present at least in the head and hindgut, we were not able to detect AeaAQP protein above immuno- fluorescence background level in midgut, hindgut, Malpighian tubule epithelium, or tracheolar cells associ- ated with the midgut in female mosquito (not shown). These results are similar to the in situ localization results previously reported [22]. In C. viridis, AQPcic mRNA is present only in the filter chamber, where the protein forms Fig. 4. Sucrose density gradient centrifugation of AeaAQP expressed in Xenopus oocytes and revealed by Western blot analysis. Proteins were extracted from oocyte membranes with 2% of the nondenaturing detergent n-octyl b- D -glucopyranoside and then analyzed on 2–20% linear sucrose gradient. Twenty fractions were collected; top of gra- dient is on the right. The positions of marker proteins, cytochrome c (1.7S), BSA (4.3S), and IgG (7S) detected by Coomassie Blue staining of acrylamide gels, are indicated at the top. Gradient fractions were analyzed by SDS/PAGE and Western blotting using anti-AQPcic IgG. The 6.8–7S apparent sedimentation coefficient fits with a homotetra- meric quaternary structure of AeaAQP. Fig. 5. Freeze-fracture of oocytes expressing AeaAQP. Oocytes were transferred to 2.5% glutaraldehyde and prepared for freeze-fracture electron microscopy. The plasma membrane of oocytes expressing AeaAQP exhibited numerous orthogonal arrays of particles (A, arrows). These struc- tures, typical of some but not all aquaporins, were never observed in water-injected oocytes (B). Bar ¼ 100 nm. 426 L. Duchesne et al.(Eur. J. Biochem. 270) Ó FEBS 2003 a crystalline array of particles for a water-shunting system, and not detected in midgut or other organs of the digestive system [20,24]. In the respiratory system, the restricted localization of AeaAQP to the tracheolar cells and its absence from the neighboring tracheal cells (Fig. 1B) suggests a specific function in water movement in tracheolar cells and tracheoles, and/or in the functional co-ordination of the Malpighian tubules and the tracheoles supplying them. Each of the 11 mammalian aquaporins has a characteristic subcellular localization and tissue expression pattern which may indicate their physiological roles [36]. However, in most situations it is not certain if their presence is critical for organ function. Studies on knockout mice for specific aquaporins suggest that, in mammals, aquaporins are necessary for extremely high rates of active fluid transport but are more of a luxury at medium or low fluid transport rates [11]. For example, AQP1 and AQP4 knockout mice have normal survival rates, although these two proteins are abundantly expressed in normal lung, which suggested a critical role in lung function [37]. AQP5 null mice, however, have 10-fold reduced airspace-to-capillary water permeability in lung [38]. The physiological signifi- cance of redundant expression of aquaporins in some tissues of mammals such as the lung is not completely understood, and aquaporins may have other unknown functions [39]. In mammalian lung, aquaporins are a major route of osmotically driven water transport among the airspace, interstitial, and capillary compartments, but they are not required for physiologically important lung functions [39]. Six aquaporins have been identified in the Drosophila melanogaster genome by sequence similarity (www.flybase. org; release 2). However, studies on insect aquaporins are lacking, and it is not known if redundancy is also present in insect tissues. The most similar proteins to AeaAQP are the D. melanogaster DRIP product (75.4% similarity) followed by Haematobia irritants aquaporin (74.3%), AQPcic (66%), and mouse AQP4 (43.6%) [22]. In a phylogenetic alignment using a PAM 250 residue table, this group of aquaporins branches off a second group of Drosophila aquaporins containing products for genes CG7777, CG17664 and CG17662 (not shown). It appears that AeaAQP may be more functionally related to AQPcic and mammalian AQP4, of which it appears to represent an ancestral form [22]. Expression of AeaAQPinoocytesledtoa10-fold increase in osmotic membrane water permeability, similar to that of AQPcic (this study). Le Cahe ´ rec et al.[24]reporteda 15-fold increase in permeability using the same system for AQPcic, and other authors reported similar increases for mammalian AQP4 [15]. As observed in freeze-fracture analysis of oocyte membranes, most of the AeaAQP protein is located inside the orthogonal arrays of particles, as AQP4 is organized within membranes. Unfortunately, because of the low abundance of tracheolar cells associated with Malpighian tubules, the probability of obtaining a freeze- fracture image of native membranes from these cells is very low and we were not able to obtain it. AeaAQP has the structural characteristics of other aquaporins, including the conserved sequence ÔAEFLÕ at residues 28–31, the presence of the NPARS motif charac- teristic of orthodox aquaporins at the second NPA motif, and the lack of a GLYY motif in loop C which is present in aquaglyceroporins [22]. Water transport by AeaAQP was inhibited by mercury ions (Hg 2+ ), similar to insect AQPcic and in contrast with mammalian AQP4 with which they share the highest sequence similarity among mammalian aquaporins. Res- idues flanking the NPA motifs are involved in mercury insensitivity [40,41]. AeaAQP has three cysteines at positions 79, 106 and 163; Cys79 is in the vicinity of the first conserved NPA sequence in loop B [22], and, according to the currently accepted hourglass model for aquaporin function, this Cys79 residue appears to be the most likely candidate for the water pore inhibited by mercury in AeaAQP [41]. It was also proposed for AQPcic that Cys82 or Cys90 close to the NPA box that are present in loop B, or Cys132 in loop C could be the mercury-binding residues [24]. Sequence alignment of insect aquaporins and mouse AQP4 shows that the insect sequences have a Cys residue three residues ahead of the first NPA motif in loop B. It is possible that this residue is indicative of mercurial sensitivity in insect aquaporins, as AeaAQP does not have Cys in loops C or E. It has been demonstrated that the only target cysteine for mercury in AQPcic is Cys82 [27], and Cys79 is the AeaAQP homolog of Cys82 in AQPcic. The movement of fluid from the interior of tracheoles in response to high oxygen demand has been confirmed in larvae and adults of Aedes [5,42,43]. High oxygen demand by the Malpighian tubules of females may occur during intense diuresis which takes place either after a blood meal or after adult emergence. In the latter, there is a first burst of urine, which declines sharply 20 min after emergence, and subsequently the peak of diuresis is maintained high from 3hto% 24–36 h after emergence [44]. It is noteworthy that the AeaAQP immunological signal was most frequent and intense in tracheolar cells of younger females, especially in newly emerged females, and was observed more sporadi- cally in older females. This suggests that aquaporin may facilitate water removal from tracheolar cells during diuresis to facilitate oxygen supply to the Malpighian tubules. Alternatively, it is possible that the observed expression pattern is associated with water removal from tracheoles that occurs after molting [1]. A serotonin receptor has also been recently discovered in the tracheolar cells associated with Malpighian tubules in A. aegypti females [45]. It would be interesting to test if AeaAQP is simultaneously expressed in these serotonin receptor- expressing tracheolar cells and if serotonin is in any way involved in modulating water transport in tracheolar cells. Hormonal regulation of mammalian AQP1 and AQP2 has been established [46]. Acknowledgements This research was supported by La Fondation Langlois France and the National Institutes of Health/National Institute of Allergies and Infectious Diseases, USA, award number 5 R01 AI 46447 to P.V.P. Drs S. Datta and E. 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