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

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withdrawn from the tracheoles that supply oxygen to flight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water to be withdrawn from the highly permeable tracheolar

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

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,

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

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

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

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

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-

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-

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

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

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