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: ,
/>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
 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. 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.
Ó FEBS 2003 A. aegypti aquaporin forms orthogonal arrays (Eur. J. Biochem. 270) 427
References
1. Mill, P.J. (1985) Structure and physiology of the respiratory
system. In Comprehensive Insect Physiology, Biochemistry and
Pharmacology (Kerkut, G.A. & Gilbert, L.I., eds), pp. 517–593.
Pergamon Press, Oxford, UK.
2. Mill, P.J. (1998) Trachea and tracheoles. In Microscopic Anatomy
of Invertebrates, Vol. 11. Insecta, pp. 303–336. (Harrison, F. W. &
Locke, M., volume eds; Harrison, F. W. & Ruppert, E. E., treatise,
eds).Wiley-Liss,Inc,NewYork,USA.
3. Sla
´
ma, K. (1999) Active regulation of insect respiration. Ann.
Entomol. Soc. Am. 92, 1–2.
4. Wigglesworth, V.B. (1983) The physiology of insect tracheoles.
Adv. Insect Physiol. 17, 85–148.
5. Wigglesworth, V.B. & Lee, W.M. (1982) The supply of oxygen to
the flight muscles of insects: a theory of tracheole physiology.
Tissue Cell 14, 501–518.
6. Wigglesworth, V.B. (1965) Respiration. The Principles of Insect

Physiology, pp. 317–368. Methuen Ltd., London, UK.
7. Park, J.H. & Saier, M.H. Jr (1996) Phylogenetic characterization
of the MIP family of transmembrane channel proteins. J. Membr.
Biol. 153, 171–180.
8. Hatakeyama, S., Yoshida, Y., Tani, T., Koyama, Y., Nihei, K.,
Ohshiro, K., Kamiie, J I., Yaoita, E., Suda, T., Hatakeyama, K.
& Yamamoto, T. (2001) Cloning of a new aquaporin (AQP10)
abundantly expressed in duodenum and jejunum. Biochem.
Biophys. Res. Commun. 287, 814–819.
9. Walz,T.,Hirai,T.,Murata,K.,Heymann,J.B.,Mitsuoka,K.,
Fijiyoshi, Y., Smith, B.L., Agre, P. & Engel, A. (1997) The
three-dimensional structure of aquapoin-1. Nature (London) 387,
624–627.
10. Borgnia, M., Nielsen, S., Engel, A. & Agre, P. (1999) Cellular and
molecular biology of the aquaporin water channels. Annu. Rev.
Biochem. 68, 425–458.
11. Van Os, C.H., Kamateeg, E J., Marr, N. & Deen, P.M.T. (2000)
Physiological relevance of aquaporins: luxury or necessity.
Pflu
¨
gers Arch Eur. J. Physiol. 440, 513–520.
12. Verkman, A.S. & Mitra, A.K. (2000) Structure and function of
aquaporin water channels. Am. J. Physiol. 278, F13–F28.
13. Bai, C., Fukuda, N., Song, Y., Ma, T., Matthay, M.A. &
Verkman, A.S. (1999) Lung fluid transport in aquaporin-1 and
aquaporin-4 knockout mice. J. Clin. Invest. 103, 555–561.
14.Nielsen,S.,King,L.S.,Christensen,B.M.&Agre,P.(1997)
Aquaporins in complex tissues. II. Subcellular distribution in
respiratory and glandular tissues of rat. Am. J. Physiol. 273,
C1549–C1561.

15. Hasegawa, H., Ma, T., Skach, W.R., Matthay, M.A. & Verkman,
A.S. (1994) Molecular cloning of a mercurial-insensitive water
channel expressed in selected water transporting tissues. J. Biol.
Chem. 269, 5497–5500.
16. Yang, B., van Hoek, A.N. & Verkman, A.S. (1997) Very high
single channel water permeability of Aquaporin-4 in Baculovirus-
infected cells and liposomes reconstituted with purified Aqua-
porin-4. Biochemistry 36, 7625–7632.
17. Yang, B., Brown, D. & Verkman, A.S. (1996) The mercurial
insensitive water channel (AQP4) forms orthogonal arrays in
stably transfected Chinese hamster ovary cells. J. Biol. Chem. 271,
4577–4580.
18. Rash, J.E., Yasumura, T., Hudson, C.S., Agre, P. & Nielsen, S.
(1998) Direct immunogold labeling of aquaporin-4 in square arrays
of astrocyte and ependymocyte plasma membranes in rat brain
and spinal cord. Proc. Natl. Acad. Sci. USA 95, 11981–11986.
19. Verbavatz, J.M., Ma, T., Gobin, R. & Verkman, A.S. (1997)
Absence of orthogonal arrays in kidney, brain and muscle from
transgenic knockout mice lacking water channel aquaporin-4.
J. Cell Sci. 110, 2855–2860.
20. Beuron, F., Le Cahe
´
rec, F., Guillam, M.T., Cavalier, A., Garret,
A., Tassan, J.P., Delamarche, C., Schultz, P., Mallouh, V.,
Rolland, J.P., Hubert, J.F., Gouranton, J. & Thomas, D. (1995)
Structural analysis of a mip family protein from the digestive tract
of Cicadella viridis. J. Biol. Chem. 270, 17414–17422.
21. Le Cahe
´
rec,F.,Bron,P.,Verbavatz,J.M.,Garret,A.,Morel,G.,

Cavallier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert,
J.F. (1996) Incorporation of proteins into (Xenopus) oocytes by
proteoliposome microinjection: functional characterization of a
novel aquaporin. J. Cell Sci. 109, 1285–1295.
22. Pietrantonio, P.V., Jagge, C., Keeley, L.L. & Ross, L.S. (2000)
Cloning of an aquaporin-like cDNA and in situ hybridization in
adults of the mosquito Aedes aegypti (Diptera: Culicidae). Insect
Mol. Biol. 9, 407–418.
23. Lagre
´
e, V., Froger, A., Deschamps, S., Hubert, J.F., Delamarche,
C., Bonnec, G., Thomas, D., Gouranton, J. & Pellerin, I. (1999)
Switch from an aquaporin to a glycerol channel by two amino
acids substitution. J. Biol. Chem. 274, 6817–6819.
24. Le Cahe
´
rec, F., Deschamps, S., Delamarche, C., Pellerin, I.,
Bonnec, G., Guillam, M T., Thomas, D., Gouranton, J. &
Hubert, J F. (1996) Molecular cloning and characterization of an
insect aquaporin functional comparison with aquaporin 1. Eur.
J. Biochem. 241, 707–715.
25. Preston, G.M., Jung, J.S., Guggino, W.B. & Agre, P. (1994)
Membrane topology of aquaporin CHIP: analysis of functional
epitope-scanning mutants by vectorial proteolysis. J. Biol. Chem.
269, 1668–1673.
26. Laemmli, U.K. (1970) Cleavage of the structural proteins during
the assembly of the head of bacteriophage T4. Nature (London)
227, 680.
27. Lagre
´

e, V., Froger, A., Deschamps, S., Pellerin, I., Delamarche,
C., Bonnec, G., Gouranton, J., Thomas, D. & Hubert, J.F. (1998)
Oligomerization state of water channels and glycerol facilitators.
Involvement of loop E. J. Biol. Chem. 273, 33949–33953.
28. Verbavatz, J.M., van Hoek, A.N., Ma, T., Sabolic, I., Valenti, G.,
Ellisman, M.H., Ausiello, D.A., Verkman, A.S. & Brown, D.
(1994) A 28kDa sarcolemmal antigen in kidney principal cell
basolateral membranes: relationship to orthogonal arrays and
MIP26. J. Cell Sci. 107, 1083–1094.
29. Zampighi, G.A., Kreman, M., Boorer, K.J., Loo, D.D., Bezanilla,
F., Chandy, G., Hall, J.E. & Wright, E.M. (1995) A method for
determining the unitary functional capacity of cloned channels
and transporters expressed in Xenopus laevis oocytes. J. Membr.
Biol. 148, 65–78.
30. Roudier, N., Bailly, P., Gane, P., Lucien, N., Gobin, R., Cartron,
J P. & Ripoche, P. (2002) Erythroid expression and oligomeric
state of the AQP3 protein. J. Biol. Chem. 277, 7664–7669.
31. Aerts, T., Xia, J.Z., Slegers, H., De Block, J. & Clauwaert, J.
(1990) Hydrodynamic characterization of the major intrinsic
protein from the bovine lens fiber membranes. Extraction in
n-octyl-beta-D-glucopyranoside and evidence for a tetrameric
structure. J. Biol. Chem. 265, 8675–8680.
32. Smith, B.L. & Agre, P. (1991) Erythrocyte Mr 28,000 transmem-
brane protein exists as a multisubunit oligomer similar to channel
proteins. J. Biol. Chem. 266, 6407–6415.
33. Bron, P., Lagre
´
e, V., Froger, A., Rolland, J.P., Hubert, J.F.,
Delamarche, C., Deschamps, S., Pellerin, I., Thomas, D. & Haase,
W. (1999) Oligomerization state of MIP proteins expressed in

Xenopus oocytes as revealed by freeze-fracture electron-micro-
scopy analysis. J. Struct. Biol. 128, 287–296.
34. Shi, L., Skach, W.R. & Verkman, A.S. (1994) Functional
independence of monomeric CHIP28 water channels revealed by
expression of wild-type mutant heterodimers. J. Biol. Chem. 269,
10417–10422.
35. Huang, Y., Tracy, R., Walsberg, G.E., Makkinje, A., Fang, P.,
Brown, D. & van Hoek, A.N. (2001) Absence of aquaporin-4
428 L. Duchesne et al.(Eur. J. Biochem. 270) Ó FEBS 2003
water channels from kidneys of the desert rodent Dipodomys
merriami merriami. Am.J.Physiol.280, F794–F802.
36. Neely, J.D., Amiry-Moghaddam, M., Ottersen, O.P., Froehner,
S.C., Agre, P. & Adams, M.E. (2001) Syntrophin-dependent
expression and localization of Aquaporin-4 water channel protein.
Proc. Natl. Acad. Sci. USA 98, 14108–14113.
37. Song, Y., Jayaraman, S., Yang, B., Matthay, M.A. & Verkman,
A.S. (2001) Role of aquaporin water channels in airway fluid
transport, humidification and surface liquid hydration. J. Gen.
Physiol. 117, 573–582.
38. Ma, T., Fukuda, N., Song, Y., Matthay, M.A. & Verkman, A.S.
(2000) Lung fluid transport in aquaporin 5-knockout mice. J. Clin.
Invest. 105, 93–100.
39. Verkman, A.S., Matthay, M.A. & Song, Y. (2000) Aquaporin
water channels and lung physiology. Am. J. Physiol. 278, L867–
L879.
40. Daniels, M.J., Mirkov, T.E. & Chrispeels, M.J. (1994) The plasma
membrane of Arabidopsis thaliana contains a mercury-insensitive
aquaporin that is a homolog of the tonoplast water channel
protein tip. Plant Physiol. 106, 1325–1333.
41. Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. &

Baraban, J.M. (1994) Molecular characterization of an
aquaporin cDNA from brain: candidate osmoreceptor and
regulator of water balance. Proc. Natl Acad. Sci. USA 91, 13052–
13056.
42. Wigglesworth, V.B. (1938) The regulation of osmotic pressure and
chloride concentration in the haemolymph of mosquito larvae.
J. Exp. Biol. 15, 235–245.
43. Wigglesworth, V.B. (1938) The absorption of fluid from the tra-
cheal system of mosquito larvae at hatching and moulting. J. Exp.
Biol. 15, 248–254.
44. Clements, A.N. (1992) The Biology of Mosquitoes. Chapman &
Hall, New York, USA.
45. Pietrantonio, P.V., Jagge, C. & McDowell, C. (2001) Cloning and
expression analysis of a 5HT7-like serotonin receptor cDNA from
mosquito Aedes aegypti female excretory and respiratory systems.
Insect Mol. Biol. 10, 357–370.
46. Nielsen, S., Chou, C L., Marples, D., Christensen, E.I., Kishore,
B.K. & Knepper, M.A. (1995) Vasopressin increases water per-
meability of kidney collecting duct by inducing translocation of
aquaporin-CD water channels to plasma membrane. Proc. Natl.
Acad. Sci. USA 92, 1013–1017.
Ó FEBS 2003 A. aegypti aquaporin forms orthogonal arrays (Eur. J. Biochem. 270) 429