Keywords: aquaporin; insect respiration; Malpighian tubule; tracheolar cell; tracheoles.. Water movement across cell membranes is facilitated through proteins of the MIP major intrinsic
Trang 1Mosquito ( Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays
Laurence Duchesne1, Jean-Franc¸ois Hubert1, Jean-Marc Verbavatz2, Daniel Thomas1
and Patricia V Pietrantonio3
1
UMR CNRS 6026, Interactions Cellulaires et Mole´culaires, Universite´ de Rennes I, Rennes, France;2Service de
Biologie Cellulaire, CEA, Saclay, France;3Department of Entomology, Texas A&M University, College Station, TX, USA
Previous results showed that mRNA encoding a putative
aquaporin (AQP) (GenBankaccession number AF218314)
is present in the tracheolar cells associated with female
Aedes aegyptiMalpighian 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)6to 150· 10)6mÆs)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 workby Wigglesworth and others showed that
trachea breakup 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)
Trang 2form 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
Hg2+[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/
Pi) containing 50 mMEGTA 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
for 5 min with NaCl/Picontaining 0.1% (v/v) Tween and
2% (v/v) normal goat serum (PBSTG) on ice, and then
treated with 12 lgÆmL)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/Piat 4C 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· 20 min in PBSTG and incubated
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 4C 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 Trinitron color video monitor (Sony) and imported into PHOTOSHOPTM(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 inXenopus 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 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2 mMNaHCO3, 10 mMHepes/NaOH, pH 7.4) at
18C 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 mM HgCl2 The oocyte volume (V ) was calculated at each time point relative to volume at the initial observation (V0) The osmotic water permeability coefficient (Pf, in 10)6mÆs)1) was calculated from the oocyte surface area (S¼ 0.045 cm2), the initial volume (V0¼ 9 · 10)4
cm3), the molecular volume of water (Vw¼ 18 cm3/mol), and the initial rate of oocyte swelling d(V/V0)/dt, by means
of the equation:
Pf ¼ V0 dðV=V0Þ=dt=½S Vw ðosmin osmoutÞ
Trang 3where osmoutis 176 mmolÆkg)1and osminis 58 mmolÆkg)1 For glycerol transport assays, oocytes were transferred in
an iso-osmotic solution in which 140 mM 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:
P0gly ¼ ½dðV=V0Þ=dt ðV0=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 mM Tris/HCl, pH 7.4, 1 mM dithio-threitol) containing 2% (w/v) n-octyl b-D-glucopyranoside for 12 h at 4C 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 15C Velocity sedimentation on sucrose gradients
Linear 2–20% (w/v) sucrose density gradients were prepared from 2% and 20% sucrose stocksolutions (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 top of gradients and ultracentrifugation was performed at
100 000 g for 16 h at 5C Calibration curves for the determination of the apparent sedimentation coefficient were constructed using cytochrome c (S20,w¼ 1.7 S), BSA (S20,w¼ 4.3 S) and IgG (S20,w¼ 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 with DAPI SCN, Stellate cell nucleus; PCN, principal cell nucleus 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 lackof signal in the tracheal cells (TrC, tracheal cell nucleus, short arrows) and the weaksignal in tracheoles (compare with A) (C) Light micrograph
of the same tissue as in (B) showing the tracheolar cells.
Trang 4serum (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/Piand
washed in NaCl/Pi 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-(2–6-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 mMHgCl2 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 ofAeaAQP
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 AeaS1R primers and cloned into pXbG-ev1 as described
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.
Trang 5(Fig 5B) In oocytes expressing AeaAQP, OAPs, a
land-markof 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.
Trang 6a 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
melanogastergenome 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 melanogasterDRIP 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 AeaAQP in oocytes led to a 10-fold
increase in osmotic membrane water permeability, similar to
that of AQPcic (this study) Le Cahe´rec et al [24] reported a
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 AEFL at
residues 28–31, the presence of the NPARS motif
charac-teristic of orthodox aquaporins at the second NPA motif, and the lackof a GLYY motif in loop C which is present in aquaglyceroporins [22]
Water transport by AeaAQP was inhibited by mercury ions (Hg2+), 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 peakof diuresis is maintained high from
3 h to 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 Skoulakis, CAIMS, TAMU, allowed the use of the fluorescence microscope and camera Cade McDowell assisted with histochemistry, Georgette Bonnec with Xenopus oocyte preparation, and Claire Heichette with permeability measurements.
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