identification and functional characterization of an...
TRANSCRIPT
1
Identification and functional characterization of an ovarian aquaporin from the 1
cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) 2
3
Alba Herraiz*, François Chauvigné**, Joan Cerdà**, Xavier Bellés*1, Maria-Dolors 4
Piulachs*1 5
6
* Institut de Biologia Evolutiva (CSIC-UPF), and LINC-Global, Passeig Marítim de la 7
Barceloneta 37-49. 08003 Barcelona, Spain. 8
** Laboratory of Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de 9
Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta 37-49. 08003 Barcelona, 10
Spain. 11
12
13 1 To whom correspondence should be addressed (e-mail: [email protected] 14
and [email protected]) 15
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Running title: An ovarian aquaporin from Blattella germanica 21
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Key words: BgAQP, water channel, urea transport, drought, oogenesis, panoistic ovary, 24
insect, Drosophila. 25
26
Abbreviations used: AQP, aquaporin; BgAQP, Blattella germanica aquaporin; BIB, 27
Big brain; cRNA, Capped RNA; DRIP, Drosophila integral protein; dsRNA, double 28
stranded RNA; MBS, modified Barth’s culture medium; NPA motifs, Asp-Pro-Ala 29
motifs; ORF, open reading frame; PRIP, Pyrocoelia rufa integral protein; qRT-PCR, 30
quantitative real time PCR; REST, relative expression software tool. 31
32
2
SUMMARY 33
Aquaporins (AQPs) are membrane proteins that form water channels, allowing rapid 34
movement of water across cell membranes. AQPs have been reported in species of all 35
life kingdoms and in almost all tissues, but little is known about them in insects. Our 36
purpose was to explore the occurrence of AQPs in the ovary of the phylogenetically 37
basal insect Blattella germanica (L.) and to study their possible role in fluid 38
homeostasis during oogenesis. We isolated an ovarian AQP from B. germanica 39
(BgAQP) that has a deduced amino acid sequence showing six potential transmembrane 40
domains, two NPA motifs, and an ar/R constriction region, which are typical features of 41
the AQP family. Phylogenetic analyses indicated that BgAQP belongs to the PRIP 42
group of insect AQPs, previously suggested to be water-specific. However, ectopic 43
expression of BgAQP in Xenopus laevis (Daudin) oocytes demonstrated that this AQP 44
transports water and modest amounts of urea, but not glycerol, which suggests that 45
PRIP group of insect AQPs may have heterogeneous solute preferences. BgAQP was 46
shown to be highly expressed in the ovary, followed by the fat body and muscle tissues, 47
but water-stress did not modify significantly the ovarian expression levels. RNA 48
interference (RNAi) reduced BgAQP mRNA levels in the ovary but the oocytes 49
developed normally. The absence of an apparent ovarian phenotype after BgAQP RNAi 50
suggests that other functionally redundant AQPs that were not silenced in our 51
experiments might exist in the ovary of B. germanica. 52
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INTRODUCTION 57
Cell membrane allows water diffusion, but simple diffusion cannot explain the high 58
permeability found in various tissues involved in fluid transport, where water movement 59
occurs under a very low Arrhenius activation energy (Solomon, 1968). The discovery of 60
water channels, or aquaporins (AQPs), by Peter Agre and colleagues in late 1980’s 61
answered the question of how can water can cross the lipid bilayer in such a rapid 62
manner. The first AQP discovered, CHIP28 (later named AQP1), was isolated from 63
human red blood cells (Denker et al., 1988). Since then, AQPs have been isolated from 64
species belonging to all life kingdoms, including unicellular (archaea, bacteria, yeast 65
and protozoa) and multicellular (plants and animals) organisms (King et al., 2004; 66
Maurel et al., 2008). 67
AQPs are membrane proteins belonging to the Major Intrinsic Proteins (MIP) 68
superfamily that share a common structure comprising 6 transmembrane domains 69
(TM1-TM6) connected by five loops (A-E), and cytoplasmic N- and C- termini. AQPs 70
contain two Asp-Pro-Ala (NPA) motifs located in steric contiguity with the 71
aromatic/arginine (ar/R) constriction region involved in proton exclusion and channel 72
selectivity (de Groot et al., 2003; Murata et al., 2000). The ar/R constriction site is 73
defined by four residue positions (56, 180, 189, and 195; human AQP1 numbering), 74
which in water-selective AQPs are Phe56, His180, Cys189 and Arg195 (human AQP1) (de 75
Groot et al., 2003). The AQP polypeptide chain is formed by two closely related halves 76
that may have arisen by gene duplication (Zardoya, 2005). In the cell membrane, each 77
AQP is folded into a right-handed α-barrel, with a central transmembrane channel 78
surrounded by the six full-length transmembrane helices and the two NPA-containing 79
loops, B and E. This conformation in the plasma membrane is known as the hourglass 80
model (Jung et al., 1994). Some AQPs, known as aquaglyceroporins, transport other 81
non-charged solutes such as glycerol and urea in addition to water (Gomes et al., 2009; 82
Rojek et al., 2008; Törnroth-Horsefield et al., 2010). Responsible for the substrate 83
selectivity are the amino acids forming the ar/R constriction (His is replaced by the 84
smaller amino acid Gly in aquaglyceroporins) (Beitz et al., 2006), and also five other 85
residues (P1-P5) located on the side-chains at the neighbourhood of the ar/R 86
constriction (Froger et al., 1998). The transport function of many AQPs can be inhibited 87
by mercurial sulfhydryl-reactive compounds, such as HgCl2, which block the water pore 88
(Hirano et al., 2010; Preston et al., 1993). 89
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While there is a considerable body of knowledge on mammalian AQPs, studies 90
on insect AQPs are still much limited. Phylogenetic analysis based on 18 insect 91
genomes revealed the presence of AQP orthologs in all of them (Campbell et al., 2008). 92
However, only 15 AQPs from different insects have been functionally characterized in 93
terms of substrate selectivity (Campbell et al., 2008; Goto et al., 2011; Kataoka et al., 94
2009a; Kataoka et al., 2009b; Liu et al., 2011; Philip et al., 2011). The first insect AQP 95
functionally characterized was AQPcic, from the filter chamber of Cicadella viridis 96
(L.), and was shown to be water-selective (Le Caherec et al., 1996). Since then, water-97
transporting AQPs have also been isolated from Aedes aegypti (L.) (Duchesne et al., 98
2003), Rhodnius prolixus Stal (Echevarria et al., 2001), Drosophila melanogaster 99
Meigen (Kaufmann et al., 2005), Polypedilum vanderplanki Hinton (Kikawada et al., 100
2008), Acyrthosiphon pisum (Harris) (Shakesby et al., 2009), Bombyx mori L. (Kataoka 101
et al., 2009a), Grapholita molesta (Busck) (Kataoka et al., 2009b), Eurosta solidaginis 102
Fitch (Philip et al., 2011), Anopheles gambiae Giles (Liu et al., 2011) and Belgica 103
antarctica Jacobs (Goto et al., 2011). Only two insect AQPs were shown to transport 104
glycerol and urea in addition to water: AQP-Bom2 and AQP-Gra2, from B. mori and G. 105
molesta, respectively (Kataoka et al., 2009a; Kataoka et al., 2009b). Finally, D. 106
melanogaster Big Brain AQP transports monovalent cations in the epidermal precursor 107
regions of developing larvae (Yanochko and Yool, 2002). 108
The aim of this study was to investigate the presence of AQPs in the ovary of the 109
cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) and to study their 110
possible role in water homeostasis during oogenesis and vitellogenesis. B. germanica 111
has panoistic ovaries, which is the less modified insect ovarian type (Büning, 1994). 112
During oocyte maturation, basal oocytes increase in size due to the incorporation of 113
vitellogenin and other yolk precursors (Belles et al., 1987; Ciudad et al., 2006; Martín et 114
al., 1995), and also water (Telfer, 2009). Moreover, although B. germanica is well 115
adapted to xeric environments (Appel, 1995), water-stress readily leads to oocyte 116
resorption. These circumstances make this cockroach species a good model to study 117
ovarian AQPs. Finally, insect AQPs that have been described so far were from 118
holometabolans or from phylogenetically distal hemimetabolans (hemipterans and 119
phthirapterans, within the paraneopterans), and therefore B. germanica, which is a 120
phylogenetically basal insect within the polyneopterans, is of evolutionary interest. 121
122
MATERIAL AND METHODS 123
5
Insects 124
Specimens of B. germanica (L.) were obtained from a colony reared in the dark at 30 ± 125
1°C and 60–70% r.h. in non aseptic environment. Under these conditions, the cockroach 126
fat body and ovary harbour the endosymbiont bacteroid Blattabacterium cuenoti 127
(Lopez-Sanchez et al., 2009). Sixth instar nymphs or freshly ecdysed adult females were 128
selected from the colony and used at appropriate ages. All dissections and tissue 129
sampling were carried out on carbon dioxide-anaesthetized specimens. Tissues used in 130
the experiments were from female specimens as follows: entire ovary, fat body 131
abdominal lobes, levator and depressor muscles of tibia, digestive tract from the 132
pharynx to the rectum (Malphigian tubules excluded), isolated Malpighian tubules, and 133
colleterial glands. After the dissection, the tissues were frozen on liquid nitrogen and 134
stored at -80ºC until use. 135
136
Cloning and sequencing 137
The sequence of a partial cDNA encoding a putative B. germanica AQP (544 bp) was 138
obtained from an EST library available in GenBank (Accession number: FG128078.1). 139
This fragment was amplified from cDNA synthesized from 3-day-old adult ovaries 140
using specific oligonucleotide primers and conventional RT-PCR. The resulting 141
fragment was cloned into pSTBlue™-1 vector (Novagen Madrid, Spain) and sequenced. 142
To clone the full-length cDNA, 5'- and 3'-RACE (Invitrogen, Paisley, UK) were used 143
according to the manufacturer's instructions. The PCR products were analyzed by 144
agarose gel electrophoresis, cloned into pSTBlue™-1 vector and sequenced. The 145
sequence, that showed to be an AQP homologue, was named BgAQP (Accession 146
number FR744897). 147
148
Comparison of sequences and phylogenetic analysis 149
Putative insect AQP sequences were retrieved from GenBank. Protein sequences were 150
aligned with that obtained for B. germanica, using CLUSTALX (v 1.83). Poorly aligned 151
positions and divergent regions were removed by using GBLOCKS 0.91b 152
(http://molevol.ibmb.csic.es/Gblocks_server/) (Castresana, 2000). The resulting 153
alignment was analyzed by the PHYML 3.0 program (Guindon and Gascuel, 2003) 154
based on the maximum-likelihood principle with the amino acid substitution model. The 155
data was bootstrapped for 100 replicates using PHYML. The sequences used in the 156
phylogenetic analysis were as follows. XP_002429480.1 (Pediculus humanus L.), 157
6
AAL09065.1 (PrAQP1, Pyrocoelia rufa Olivier), FJ489680 (EsAQP1, E. solidaginis), 158
NP_610686.1 (DmPRIP, D. melanogaster), XP_001865728.1 (Culex quinquefasciatus 159
Say), XP_001656932.1 (AaAQP2, A. aegypti), BAF62090.1 (PvAQP1, P. 160
vanderplanki), AB602340 (BaAQP1, B. antarctica), XP_319585.4 (AgAQP1, A. 161
gambiae), NP_001153661.1 (B. mori), XP_968342.1 (TcPRIP, T. castaneum (Herbst)), 162
XP_001607929.1 (NvPRIP, N. vitripennis (Walker)), XP_394391.1 (A. mellifera), 163
XP_972862.1 (TcDRIP, Tribolium castaneum), Q23808.1 (AQPcic, C. viridis), 164
BAG72254.1 (Coptotermes formosanus Shiraki), XP_002425393.1 (P. humanus), 165
ABW96354.1 (Bemisia tabaci (Gennadius)), XP_624531.1 (AmDRIP, Apis mellifera 166
L.), (NvDRIP, Nasonia vitripennis), AAA81324.1 (DmDRIP, D. melanogaster), 167
AAA96783.1 (BfWC1, Haematobia irritans (L.)), ABV60346.1 (LlAQP, Lutzomyia 168
longipalpis (Lutz & Neiva)), XP_319584.4 (A. gambiae), AF218314.1 (AaAQP1, A. 169
aegypti), XP_001865732.1 (C. quinquefasciatus), BAD69569.1 (AQP-Bom1, B. mori), 170
BAH47554.1 (AQP-Gra1, G. molesta), NP_001139376.1 (A. pisum), NP_001139377.1 171
(A. pisum), XP_396705.2 (AmBIB, A. mellifera), XP_001604170.1 (NvBIB N. 172
vitripennis), XP_314890.4 (A. gambiae), XP_001649747.1 (AaAQP3, A. aegypti), 173
XP_001866597.1 (C. quinquefasciatus), XP_314891.4 (A. gambiae), AAF52844.1 174
(DmBIB, D. melanogaster), XP_968782.1 (TcBIB, T. castaneum), XP_970791.1 (T. 175
castaneum), XP_970912.1 (T. castaneum), XP_001121899.1 (A. mellifera), 176
XP_001601231.1 (N. vitripennis), XP_624194.1 (A. mellifera), XP_001601253.1 (N. 177
vitripennis), NP_001106228.1 (AQP-Bom2, B. mori), BAH47555.1 (AQP-Gra2, G. 178
molesta), BAF62091.1 (PvAQP2, P. vanderplanki), XP_001650169.1 (AaAQP5, A. 179
aegypti), XP_001850887.1 (C. quinquefasciatus), XP_318238.4 (A. gambiae), 180
NP_611812.1 (D. melanogaster), NP_611813.1 (D. melanogaster), NP_611811.3 (D. 181
melanogaster), XP_001650168.1 (AaAQP4, A. aegypti), XP_554502.2 (A. gambiae), 182
NP_611810.1 (D. melanogaster), XP_001121043.1 (A. mellifera), XP_001603421.1 (N. 183
vitripennis), and XP_970728.1 (T. castaneum). 184
185
Structural predictions 186
Topographical analyses to determine transmembrane regions were carried out with the 187
programs TMHMM (www.cbs.dtu.dk/services/TMHMM/) (Krogh et al., 2001) and 188
SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi) (Letunic et al., 2009). 189
Phosphorylation sites and kinases were determined using NetPhos 2.0 and NetPhosK 190
1.0 programs (Blom et al., 1999; Blom et al., 2004), respectively, on ExPASy 191
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Proteomic Tools (http://expasy.org/tools). Predictions of tridimensional structure were 192
carried out with the 3D-JIGSAW Protein Comparative Modelling Server 193
(http://bmm.cancerresearchuk.org/~3djigsaw/) (Bates et al., 2001), and analyzed using 194
PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC. 195
196
Functional expression in Xenopus laevis oocytes 197
The open reading frame (ORF) of BgAQP cDNA was cloned into the pSTBlue™-1 198
vector and subcloned into the EcoRV/SpeI sites of the pT7Ts expression vector (Deen et 199
al., 1994). Capped RNA (cRNA) was synthesized in vitro with T7 RNA Polymerase 200
(Roche) from XbaI-linearized pT7Ts vector containing the BgAQP cDNA. The 201
isolation and microinjection of stage V-VI oocytes of X. laevis was carried out as 202
previously described (Deen et al., 1994). For water permeability experiments, oocytes 203
were injected with either 50 nl of RNase-free water (negative control) or 50 nl of water 204
solution containing 10 ng cRNA of BgAQP. For glycerol and urea uptake experiments, 205
oocytes were injected with 25 ng cRNA of BgAQP, human AQP1 (negative control) or 206
human AQP3 (positive control). Human AQP1 and AQP3 were kindly provided by P. 207
Deen (Department of Physiology, Radboud University Nijmegen Medical Centre, 208
Nijmegen, The Netherlands). 209
210
Swelling assays 211
Osmotic water permeability (Pf) was measured from the time course of oocyte swelling 212
in a standard assay (Deen et al., 1994). Water- and cRNA-injected X. laevis oocytes 213
were transferred from 200 mOsm modified Barth’s culture medium (MBS; 0.33 mM 214
Ca(NO3)2, 0.4 mM CaCl2, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM Hepes, 215
0.82 mM MgSO4, pH 7.5) to 20 mOsm MBS at room temperature. Oocyte swelling was 216
followed by video microscopy using serial images at 2 sec intervals during the first 20 217
sec period. The Pf values were calculated taking into account the time-course changes in 218
relative oocyte volume [d (V/Vo)/dt], the molar volume of water (Vw = 18 cm3/ml) and 219
the oocyte surface area (S), using the formula Vo [d (V/Vo)/dt]/ [SVw (Osmin-Osmout)]. 220
To examine the inhibitory effect of mercury on Pf, oocytes were pre-incubated for 15 221
min in MBS containing 1 mM HgCl2 before and during the swelling assays. To 222
determine the reversibility of the inhibition, the oocytes were rinsed 2 times with fresh 223
MBS and incubated for another 15 min with 5 mM β-mercaptoethanol before being 224
subjected to swelling assays. 225
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226
Radioactive solute uptake assays 227
To determine the uptake of [3H]glycerol (60 Ci/mmol) and [14C]urea (52 mCi/mmol), 228
groups of 10 X. laevis oocytes injected with water or 25 ng cRNA encoding BgAQP, 229
human AQP1 or human AQP3, were incubated in 200 µl of MBS containing 20 µCi of 230
the radiolabeled solute (cold solute was added to give 1 mM final concentration) at 231
room temperature. After 10 min (including zero time for subtraction of the signal from 232
externally bound solute), oocytes were washed rapidly in ice-cold MBS three times, and 233
individual oocytes were dissolved in 10% SDS for 1 h before scintillation counting. 234
235
RNA Extraction and retrotranscription to cDNA 236
All RNA extractions were performed using the Gen Elute Mammalian Total RNA kit 237
(Sigma, Madrid, Spain). An amount of 400 ng from each RNA extraction was DNase 238
treated (Promega, Madison, WI, USA) and reverse transcribed with Superscript II 239
reverse transcriptase (Invitrogen, Carlsbad CA, USA) and random hexamers (Promega). 240
RNA quantity and quality was estimated by spectrophotometric absorption at 260 nm in 241
a Nanodrop Spectrophotometer ND-1000® (NanoDrop Technologies, Wilmington, DE, 242
USA). 243
244
Determination of mRNA levels with quantitative real-time PCR 245
Quantitative real time PCR (qRT-PCR) reactions were carried out in triplicate in an iQ5 246
Real-Time PCR Detection System (Bio-Rad Laboratories), using SYBR®Green (Power 247
SYBR® Green PCR Master Mix; Applied Biosystems). A control without template was 248
included in all batches. The efficiency of each primer set was first validated by 249
constructing a standard curve through four serial dilutions. The PCR program began 250
with a single cycle at 95°C for 3 min, 40 cycles at 95°C for 10 sec and 55°C for 30 sec. 251
mRNA levels were calculated relative to BgActin-5c (GenBank accession number 252
AJ862721) expression, using the Bio-Rad iQ5 Standard Edition Optical System 253
Software (version 2.0). Results are given as copies of mRNA per 1,000 copies of 254
BgActin-5c mRNA. 255
256
Induction of water stress 257
B. germanica females were maintained with food and water from day 0 to day 3 of adult 258
life. Then, a group of specimens was water-deprived, while the control group received 259
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water ad libitum. Water-deprived and control specimens were studied at 24 h intervals 260
until day 7 of adult life. 261
262
RNAi experiments 263
A dsRNA (dsBgAQP) was prepared encompassing a 188 bp region starting at 264
nucleotide 771 of BgAQP sequence. The fragment was amplified by PCR and cloned 265
into the pSTBlueTM-1 vector. As control dsRNA (dsMock), we used a 307 bp sequence 266
from Autographa californica (Speyer) nucleopolyhedrovirus (GenBank accession 267
number K01149, from nucleotide 370 to 676). The preparation of the dsRNAs was 268
performed as previously described (Ciudad et al., 2006). Freshly emerged females from 269
last (6th) nymphal instar were injected into the abdomen with 3 µg of dsBgAQP in a 270
volume of 1 µl. Control specimens were injected with the same volume and dose of 271
dsMock. After the imaginal moult, one mature male (6- to 8-days-old) per female was 272
added to the rearing jars, in order to bring about mating. 273
274
Statistics 275
Data are expressed as mean ± standard error of the mean (s.e.m.). Statistical analysis of 276
gene expression values was carried out using the REST-2008 program (Relative 277
Expression Software Tool V 2.0.7; Corbett Research) (Pfaffl et al., 2002). This program 278
calculates changes in gene expression between two groups, control and sample, using 279
the corresponding distributions of Ct values as input. Values of Pf, Pgly and radioactive 280
solute uptake were statistically analyzed in an unpaired Student’s t-test. 281
282
RESULTS 283
Cloning and sequence characterization of BgAQP from Blattella germanica 284
The sequence of a partial cDNA encoding a B. germanica AQP was cloned from 285
ovarian tissue of 3-day-old adult females by using specific primers based on an 286
expressed sequence tag (EST) deposited in GenBank (accession number FG128078.1). 287
This EST nucleotide sequence was derived from male and female whole organism. 288
Consecutive 5'- and 3'-RACE experiments were carried out in order to obtain the full-289
length cDNA, which was named BgAQP (GenBank accession number: FR744897). The 290
BgAQP cDNA has 1,838 bp and contains an ORF encoding a polypeptide of 277 amino 291
acids (nucleotide positions 95-925) with an estimated molecular weight of 29,541 Da 292
and an isoelectric point of 5.86 (Figure S1). Hydrophobicity and tridimensional 293
10
predictions indicate that BgAQP has 6 putative transmembrane domains, five 294
connecting loops, and cytoplasmic N- and C-termini, which are typical AQP features. 295
The second (B) and fifth (E) loops contain the highly conserved NPA motifs (residues 296
93-95 and 209-211). The amino acids forming the ar/R constriction region (Phe73, His197 297
and Arg212) and the P1-P5 residues (Thr133, Thr213, Ala217, Tyr229 and Trp230), which are 298
conserved in water-selective AQPs, were present in BgAQP. In the amino acid 299
sequence, a consensus motif for N-linked glycosylation [NX(S/T)] in loop C (Asn35), as 300
well as seven potential phosphorylation sites (Thr48, Thr53, Ser207, Ser220, Thr255, Thr257 301
and Ser260) and one protein kinase C-specific site (Ser216), were also identified (Figure 302
S1). 303
304
Phylogenetic analysis of BgAQP 305
As the fat body and the ovary of B. germanica harbour the endosymbiont bacteroid 306
Blattabacterium cuenoti, we first evaluated whether the putative AQP cDNA cloned 307
was really originated from the host cockroach ovary. BLAST analyses against the 308
genome of B. cuenoti (Lopez-Sanchez et al., 2009), revealed that the bacteroid does not 309
have any gene sequence matching the nucleotide sequence of BgAQP, which indicates 310
that the cloned cDNA came from B. germanica. Then, in order to place BgAQP in an 311
evolutionary context within insects, we carried out a phylogenetic analysis of 312
representative insect AQPs available in public databases. The amino acid alignment of 313
these sequences suggested that BgAQP was more related to a putative AQP from the 314
phthirapteran P. humanus (XP_002429480) and the AQP homolog from the coleopteran 315
P. rufa (AAL09065.1). Subsequently, following maximum-likelihood analyses, we 316
obtained the tree depicted in Figure 1. This tree shows the same general topology 317
published by Campbell et al. (2008), who defined four main clusters of insect AQPs: 318
PRIP (from Pyrocoelia rufa Integral Protein), DRIP (from Drosophila Integral Protein), 319
BIB (from neurogenic gene Drosophila big brain), and a fourth cluster of unclassified 320
AQPs. In our tree, BgAQP falls into the well supported (96% bootstrap value) PRIP 321
cluster, whereas the DRIP cluster appears as the sister group of PRIP, and the BIB 322
cluster appears as the sister group of DRIP+PRIP. Finally, the fourth node of 323
unclassified AQPs appears as the sister group of DRIP+PRIP+BIB. 324
Figure 2 depicts an amino acid alignment of BgAQP with the four aquaporins of 325
the PRIP node that have been functionally characterized: EsAQP1 (Philip et al., 2011), 326
AgAQP1 (Liu et al., 2011), PvAQP1 (Kikawada et al., 2008) and BaAQP1 (Goto et al., 327
11
2011). The percentage of identity of BgAQP with PvAQP1, AgAQP1, BaAQP1 and 328
EsAQP1, all four from dipteran species, falls between 38% and 41%. All the amino acid 329
sequences show the typical P1-P5 and ar/R residues conserved in water-selective AQPs, 330
but they lack the Cys upstream of the second NPA motif considered as the mercurial 331
sensitive-site of some vertebrate AQPs. 332
333
Water and solute permeability of BgAQP expressed in X. laevis oocytes 334
To study whether the BgAQP effectively transports water, the cDNA was ectopically 335
expressed in X. laevis oocytes. Forty-eight hours after cRNA microinjection, oocytes 336
were transferred to a hypo-osmotic medium and oocyte swelling determined. These 337
experiments indicated that water permeability of oocytes expressing 10 ng BgAQP 338
cRNA was 18-fold higher than that of control (water-injected) oocytes (Figure 3A). 339
Despite that BgAQP does not show a Cys residue preceding the second NPA motif, pre-340
incubation of oocytes with 1 mM HgCl2 reduced significantly (p < 0.05) the Pf of 341
BgAQP-expressing oocytes, and this inhibition was partially reversed with the reducing 342
agent β-mercaptoethanol (Figure 3A). 343
To investigate if BgAQP is a water-selective AQP, as other members of the 344
PRIP group of insect AQPs, glycerol and urea permeability of oocytes expressing 345
BgAQP were analyzed by radioactive solute uptake assays. For these experiments, we 346
used oocytes expressing 25 ng BgAQP cRNA, as well as negative and positive control 347
oocytes expressing human AQP1 or AQP3, respectively, to discriminate better if 348
BgAQP was able to transport solutes. The results confirmed that BgAQP-expressing 349
oocytes did not transport glycerol significantly (Figure 3B). However, urea was 350
incorporated at slightly but significantly (p < 0.05) higher levels in oocytes expressing 351
BgAQP than in water-injected oocytes or in oocytes expressing human AQP1 (negative 352
control). These data indicated that BgAQP can transport urea, although in much lower 353
amounts than human AQP3 (positive control) (Figure 3C). 354
355
mRNA expression of BgAQP1 in the ovary during adult development 356
First of all, although the BgAQP was isolated from ovarian tissue, we were interested in 357
assessing whether it was ovary-specific. Thus, we analyzed by qRT-PCR the BgAQP 358
mRNA expression in ovary, fat body, muscle, digestive tract, Malpighian tubules and 359
colleterial glands collected from 3-day-old adult females. Results indicated that the 360
highest levels of BgAQP mRNA were observed in ovaries; lower expression was 361
12
detected in fat body and muscle tissues, whereas it was almost undetectable in 362
colleterial glands, digestive tract or Malpighian tubules (Figure 4A). 363
Then, we studied the expression pattern of BgAQP in the ovary. Thus, total 364
RNA was isolated from pools of four to six ovary pairs collected from animals at 365
selected ages and stages, and mRNA levels were analyzed by qRT-PCR. Then, we 366
determined the expression of BgAQP in the ovaries during the transition from 6th 367
nymphal instar to adult, and thereafter during the 7 days of the first gonadotrophic cycle 368
of the adult. Results (Figure 4B) showed that BgAQP transcript levels are moderate (ca. 369
35 copies of BgAQP per 1,000 copies of BgActin-5c) during the last two days of the 6th 370
nymphal instar. Then they increase ca. 2-fold in freshly emerged adults, and decrease 371
thereafter (5-20 copies of BgAQP per 1,000 copies of BgActin-5c) between day 1 and 372
day 7, in the stage of late chorion formation (D7lc) (Figure 4B). 373
In order to study whether BgAQP could play a role in the ovary under water 374
stress, we conducted a water-deprivation experiment. Adult females of B. germanica 375
were provided with food and water ad libitum during the first three days of adult life, 376
and water was then removed. Under these conditions, the females underwent basal 377
oocyte resorption from day 5. Using this experimental design, we measured ovarian 378
BgAQP mRNA levels by qRT-PCR on days 4, 5, 6 and 7 of the first gonadotrophic 379
cycle in control and water-stressed adult specimens. Although the results (Figure 4C) 380
showed a slight increase of BgAQP1 mRNA levels in water-stressed specimens at day 4 381
and 6, the differences were not statistically significant. 382
383
RNAi experiments 384
To investigate the role of BgAQP, the expression of BgAQP was silenced by RNAi and 385
the phenotype determined. For these experiments, dsBgAQP was injected in freshly 386
emerged 6th instar female nymphs, and controls were injected with dsMock in the same 387
conditions. The objective was to depress the peak of BgAQP mRNA occurring in 388
ovaries of freshly emerged adults (Figure 4B), which might be functionally important. 389
Therefore, BgAQP mRNA levels were measured in treated and control specimens just 390
after the imaginal moult. Results showed that BgAQP mRNA levels in dsBgAQP-391
treated specimens were 12.19 ± 1.22 copies per 1,000 copies of BgActin-5c, whereas in 392
dsMock-treated they were 55.34 ± 8.17 copies per 1,000 copies of BgActin-5c (Figure 393
4D), which indicates that BgAQP mRNA levels were reduced ca. 80% by the RNAi. 394
Statistically, according to REST-2008 (Pfaffl et al., 2002), BgAQP was down-regulated 395
13
in ovaries from dsBgAQP-treated specimens by a mean factor of 0.223 (p < 0.0001). 396
The duration of the 6th nymphal instar was 8 days, either in dsBgAQP-treated as in 397
controls, and the subsequent, imaginal moult proceeded normally. After the imaginal 398
moult, we added to the rearing jars a mature male per female, and we studied the first 399
reproductive cycle of the females. The number of days until the formation of the first 400
ootheca was 7, either in dsBgAQP-treated as in controls (dsMock-treated), and most of 401
the mated females formed the ootheca correctly (10 out of 11 in dsBgAQP-treated and 7 402
out of 7 in controls). All formed ootheca were viable, and the duration of 403
embryogenesis was similar in controls and treated, ca. 18 days. Concerning the number 404
of nymphs hatched per ootheca, the values were 40.71 ± 4.07 nymphs in dsMock-405
treated females (n = 7), and 36.36 ± 9.24 in dsBgAQP-treated females (n = 10), 406
differences that were not statistically significant. 407
408
DISCUSSION 409
We have isolated and functionally characterized a novel AQP from the ovary of the 410
cockroach B. germanica, which has been named BgAQP. Its deduced amino acid 411
sequence shows the characteristic structural features of AQPs. BgAQP has the ar/R 412
constriction and P1-P5 residues typically conserved in water-specific AQPs (Beitz et al., 413
2006; Froger et al., 1998). Accordingly, BgAQP effectively transported water when 414
expressed in X. laevis oocytes, and this transport was inhibited by mercury chloride 415
(HgCl2) and recovered with β-mercaptoethanol, as it occurs for other AQPs (Yukutake 416
et al., 2008). Site-directed mutations on human AQP1 have revealed that the Cys189, 417
located three amino acids upstream of the second NPA in loop E, is the mercury-418
sensitive site (Preston et al., 1993). However, it has been shown that the absence of 419
Cys198 does not prevent inhibition of water flux by mercury (Daniels et al., 1996; 420
Tingaud-Sequeira et al., 2008; Yukutake et al., 2008), which implies that sensitivity of 421
AQPs to mercurial compounds may involve other residues in addition to Cys189. 422
BgAQP lacks a Cys residue near the second NPA box, but it has a Cys residue shortly 423
downstream of the first NPA in loop B (Cys98). This residue could be a good candidate 424
as the site for mercurial inhibition in BgAQP since it locates in a similar spatial position 425
than Cys189 in AQP1. Other insect AQPs lacking a Cys residue on loop E, such as 426
PvAQP1 or EsAQP1, are also mercury-sensitive and similarly to BgAQP they have a 427
candidate Cys in loop B (Duchesne et al., 2003; Kikawada et al., 2008; Philip et al., 428
2011). 429
14
In our study, radioactive solute uptake assays showed that BgAQP is not 430
permeable to glycerol but it is able to transport low amounts of urea. To date, two insect 431
AQPs from lepidopteran larvae show glycerol and urea uptake: AQP-Bom2 and AQP-432
Gra2, which have been classified as typical aquaglyceroporins (Kataoka et al., 2009a; 433
Kataoka et al., 2009b). BgAQP cannot be classified as an aquaglyceroporin because 434
does not transport glycerol, does not show the residues in the ar/R constriction region 435
conserved in aquaglyceroporins, and is phylogenetically distant from AQP-Bom2 and 436
AQP-Gra2. Our data therefore indicate that BgAQP is the first reported insect AQP 437
from the PRIP group that shows water and urea permeability. This feature has only been 438
described so far for some teleost AQP paralogs related to mammalian AQP8 (Tingaud-439
Sequeira et al., 2010), which is water and ammonia permeable (Litman et al., 2009). 440
The structural basis of BgAQP for water and urea permeability, while excluding that of 441
glycerol, is yet unknown as it occurs for teleost Aqp8s (Cerdà and Finn, 2010; Tingaud-442
Sequeira et al., 2010), and needs further investigation. Nevertheless, the permeability of 443
BgAQP to urea may be physiologically relevant. A metabolic complementation for 444
nitrogen metabolism appears to exist between B. germanica and their endosymbiont 445
bacteroid B. cuenoti through the combination of a urea cycle (host) and urease activity 446
(endosymbiont) (Lopez-Sanchez et al., 2009). Interestingly, the endosymbionts localize 447
in the fat body and in the ovary, just where BgAQP is consistently expressed, and where 448
urea must be presumably transferred from the host tissues to the endosymbiont 449
bacteroids. Our findings suggest that BgAQP might play a role in this mechanism for 450
the transport of urea. 451
The phylogenetic analyses reported by Campbell et al. (2008) grouped insect 452
AQPs into four groups: DRIP, PRIP, BIB, and non-classified AQPs, the latter being the 453
sister group of the other three. Parallel studies carried out by Kambara et al. (2009), 454
followed by Goto et al. (2011), resulted in a tree with a topology similar to that reported 455
by Campbell et al. (2008), although the main nodes were not nominated but simply 456
numbered. However, the PRIP and DRIP nodes of Campbell et al. (2008) are recovered 457
in the “Group 1” of Kambara et al. (2009) and Goto et al. (2011). The tree obtained in 458
our study shows the same topology of these studies, and consistently clusters the 459
BgAQP sequence into the PRIP node. This node appears functionally heterogeneous 460
given that it contains a member that transports water and urea, such as BgAQP, and 461
other members that are apparently water-selective, namely EsAQP1 (Philip et al., 2011), 462
AgAQP1 (Liu et al., 2011), PvAQP1 (Kikawada et al., 2008) and BaAQP1 (Goto et al., 463
15
2011). However, the ability of PvAQP1 and AgAQP1 to transport urea has not been 464
investigated (Kikawada et al., 2008; Liu et al., 2011), whereas other PRIP members 465
have not been functionally studied at all. Therefore, the possibility that other members 466
of this node can transport urea, as BgAQP does, remains to be determined. 467
Expression analyses indicated that BgAQP mRNA was accumulated in fat body, 468
muscle and ovarian tissues, although the highest transcript levels were observed in the 469
ovary. Another AQP primarily expressed in the ovary has been described in the 470
American dog tick, Dermacentor variabilis (Say) (Holmes et al., 2008), which has been 471
speculated that may function in lipid metabolism and/or water transport. However, no 472
experimental studies have been carried out with this AQP, beyond the functional 473
expression in X. laevis oocytes. In the yellow fever mosquito, A. aegypti, 6 AQPs have 474
been characterized, and one of them, namely AaAQP2, is strongly expressed in ovaries 475
(as well as in the Malpighian tubules and the midgut) (Drake et al., 2010). Nevertheless, 476
specific knockdown of AaAQP2 through RNAi, although significantly decreased the 477
corresponding transcript levels, did not give any noticeable phenotype (Drake et al., 478
2010). In teleosts, a functional AQP1-related water channel has been described in 479
oocytes where it plays a role during oocyte hydration prior to ovulation (Fabra et al., 480
2005; Zapater et al., 2011). During vitellogenesis, basal oocytes of B. germanica 481
increase in size exponentially due to the incorporation of yolk precursors and water 482
(Belles et al., 1987; Ciudad et al., 2006; Martín et al., 1995), and AQPs may be involved 483
in oocyte water balance during this process. Concerning the expression pattern in the 484
ovary, BgAQP mRNA levels show a peak in freshly emerged adult females, which 485
suggest that most of the BgAQP mRNA necessary for the first reproductive cycle, is 486
produced just after the imaginal moult, and then their translation is regulated according 487
to the protein needs. 488
In an attempt to investigate the functions of BgAQP in the ovary of B. 489
germanica, we monitored its expression in water-stressed females. The hypothesis was 490
that water deprivation would modify the expression of BgAQP in the ovary, since one 491
the earliest consequences of water stress in B. germanica is the resorption of the oocytes 492
(our unpublished results). However, despite the fact that ovaries from water-stressed 493
females started basal oocyte resorption 48 h after water deprivation, no significant 494
differences were observed in the levels of BgAQP mRNA in the ovary between control 495
and water-stressed females, although a tendency to increase the levels in the latter group 496
was observed. It is known that AQPs are regulated post-translationally by 497
16
phosphorylation-dependent gating and especially by trafficking of the protein between 498
the plasma membrane and the intracellular storage vesicle (Törnroth-Horsefield et al., 499
2010). Therefore, the effects of water stress on BgAQP may not be evident at the 500
mRNA level. 501
As a further approach to investigate the role of BgAQP in the ovary, we 502
performed RNAi experiments. B. germanica is very sensitive to RNAi (Belles, 2010), 503
and expression of membrane-associated proteins has been successfully silenced (Ciudad 504
et al., 2006). In the present study, the timing of the treatment was designed to depress 505
the peak of BgAQP mRNA expression occurring in freshly emerged adults, and, indeed, 506
the dsBgAQP treatment effectively reduced this peak by 80% in the ovary. However, no 507
phenotypic differences were observed between RNAi-treated and controls. Neither in 508
terms of the duration of the gonadotrophic cycle or the embryogenesis period, nor in the 509
quality of the imaginal moult or the oothecae formed, or the offspring. These results are 510
reminiscent of those obtained on A. aegypti (see above), where RNAi of AaAQP2, a 511
mosquito AQP strongly expressed in the ovary, did not result in phenotypical effects 512
(Drake et al., 2010). The most parsimonious explanation for these results is that there 513
must be other ovarian AQPs in B. germanica not affected by our RNAi experiments that 514
play functions redundant with BgAQP. Indeed, an insect as successful as B. germanica, 515
arguably well adapted to xeric environments (Appel, 1995), is likely to have robust and 516
redundant mechanisms to regulate water balance. 517
518
ACKNOWLEDGEMENTS 519
We are grateful to Javier Igea for his help with the phylogenetic analyses. Financial 520
support from the Spanish Ministry of Science and Innovation (projects BFU2008-00484 521
to M-D. Piulachs, CGL2008-03517/BOS to X. Bellés, and AGL2010-15597 to J. 522
Cerdà), Generalitat de Catalunya (2005 SGR 00053), and LINC-Global is gratefully ack 523
nowledged. A. Herraiz received a pre-doctoral research grant (JAE-LINCG program) 524
from CSIC. F. Chauvigné was supported by a postdoctoral fellowship from Juan de la 525
Cierva Programme (Spanish Ministry of Science and Innovation). Thanks are also due 526
to David L. Denlinger and Shin G. Goto for providing us with the sequence of BaAQP1 527
that was used in our phylogenetical analyses.528
17
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Yukutake, Y., Tsuji, S., Hirano, Y., Adachi, T., Takahashi, T., Fujihara, K., 677 Agre, P., Yasui, M. and Suematsu, M. (2008). Mercury chloride decreases the water 678 permeability of aquaporin-4-reconstituted proteoliposomes. Biol Cell 100, 355-63. 679 Zapater, C., Chauvigné, F., Norberg, B., Finn, R. N. and Cerdà, J. (2011). 680 Dual neofunctionalization of a rapidly evolving aquaporin-1 paralog reveals constrained 681 and relaxed traits controlling channel function during meiosis resumption in marine 682 teleosts. Mol. Biol. Evol. in press. 683 Zardoya, R. (2005). Phylogeny and evolution of the major intrinsic protein 684 family. Biol Cell 97, 397-414. 685 686 687
688
21
LEGENDS OF CURRENT FIGURES 689
690
Fig. 1: Maximum-likelihood phylogenetic tree showing the position of BgAQP 691
(encircled), from Blattella germanica, with respect to other insect AQPs. Scale bar 692
represents the number of amino acid substitutions per site. Numbers at the nodes are 693
bootstrap values (only bootstrap values >50 are shown). The sequences are indicated by 694
the initials of the scientific name of the species, followed by the accession number and, 695
in parenthesis, the acronym commonly used, if any. Complete binomial names of the 696
species are indicated in Material and Methods. The PRIP, DRIP and BIB nodes are 697
indicated. BgAQP falls in the PRIP node. Also indicated are the aquaglyceroporins 698
(Glp) AQP-Bom2, from Bombyx mori, and AQP-Gra2 from Grapholita molesta, in the 699
node of non-classified AQPs. 700
701
Fig. 2: Amino acid sequence alignment of BgAQP from Blattella germanica with the 702
four aquaporins of the insect PRIP node that have been functionally characterized: 703
BaAQP1 from Belgica antarctica (AB602340), which transports water but not glycerol 704
or urea (Goto et al., 2011), PvAQP1 from Polypedilum vanderplanki (BAF62090.1), 705
which transports water but not glycerol (urea transport was not studied) (Kikawada et 706
al., 2008), and AgAQP1 from Anopheles gambiae (XP_319585.4) and EsAQP1 from 707
Eurosta solidaginis (FJ489680), which transport water (Liu et al., 2011; Philip et al., 708
2011). Fully conserved amino acids are shaded in black, whereas conserved 709
substitutions are shaded in grey. The transmembrane domains of BgAQP1 are 710
underlined, and the NPA motifs are highlighted in green. The amino acids typically 711
conserved in water-selective AQPs, in the ar/R constriction region (de Groot et al., 712
2003) as well as in the positions P1 to P5 (Froger et al., 1998), are shaded in yellow and 713
blue color, respectively. 714
715
Fig. 3: Water and solute permeability of BgAQP expressed in X. laevis oocytes. A) 716
Osmotic water permeability (Pf) of oocytes injected with water (controls) or expressing 717
10 ng BgAQP cRNA. Water permeability was inhibited with 1 mM HgCl2 and reversed 718
with 5 mM β-mercaptoethanol (BME) (n = 12 oocytes). B-C) Radioactive glycerol (A) 719
and urea (B) uptake by control oocytes and oocytes expressing 25 ng cRNA encoding 720
BgAQP, human AQP1 (hAQP1) or human AQP3 (hAQP3) (n = 10-12 oocytes). In each 721
22
histogram, different letters at the top of the columns indicate significant differences (t-722
test, p < 0.001). 723
724
Fig. 4: Expression of BgAQP. A) Expression in different tissues from 3-day-old 725
females [Ov: ovary; FB: Fat body; Mu: Muscle; DT: Digestive tract; MT: Malpighian 726
tubes; CG: Colleterial glands]. B) Expression in the ovary during the last two days of 727
6th nymphal instar (days 7 and 8), and during the first gonadotrophic cycle (days 0 to 728
7LC). [N: nymph; D: day; LC: late choriogenesis stage]. C) Expression in the ovary of 729
water-stressed females. Females were water-deprived on day 3 of adult age, and ovaries 730
were dissected on day 4, 5, 6 or 7, in order to measure BgAQP mRNA levels. Control 731
females were maintained with water ad libitum. D) Expression in dsMock- and 732
dsBgAQP-treated specimens in RNAi experiments. The asterisk indicates that the 733
difference between both is statistically significant (p < 0.0001). Treatments were carried 734
out on freshly emerged 6th (last) instar nymphs, and measurements in freshly emerged 735
adult. Data represent number of copies per 1,000 copies of BgActin-5c, and are 736
expressed as the mean ± s.e.m. (n = 3). 737
738
23
LEGEND OF SUPPLEMENTARY FIGURE 739
740
Fig. S1: Structural features of BgAQP. A) Nucleotide sequence and the deduced 741
amino acid sequence. Domains are underlined; NPA motifs are in grey; circles 742
represent putative phosphorylation sites; double circle represents putative PKC 743
regulation site; a putative N-glycosylation site is indicated in green; in yellow are the 744
amino acids forming the ar/R constriction; in blue the P1-P5 water-selective AQPs 745
conserved sites; in red the putative polyadenylation signal. 746
747
100
59
91
63
97
66
54
81
93
71
86
59
67
100
93
87
96
100
100
51
63
Ba, AB602340 (BaAQP1)8393
100
83
51
78
100
96
100
66
100
53
0.2
Tc, XP_968342.1 (TcPRIP)
Es, FJ489680 (EsAQP1)Dm, NP_610686.1 (DmPRIP)
Cq, XP_001865728.1
Aa, XP_001656932.1 (AaAQP2)
Ag, XP_319585.4 (AgAQP1)
Pv, BAF62090.1 (PvAQP1)
Bm, NP_001153661.1
Bg, FR744897 (BgAQP)
Ph, XP_002429480.1
Pr, AAL09065.1 (PrAQP1)
Nv, XP_001607929.1 (NvPRIP)Am, XP_394391.1
Cq, XP_001865732.1
Aa, AF218314.1 (AaAQP1)
Ag, XP_319584.4Ll, ABV60346.1 (LlAQP)
Dm, AAA81324.1 (DmDRIP)Hi, AAA96783.1 (BfWC1)
Bm, BAD69569.1 (AQP-Bom1)Gm, BAH47554.1 (AQP-Gra1)
Am, XP_624531.1 (AmDRIP)Nv, XP_001607940.1 (NvDRIP)
Cf, BAG72254.1Ph, XP_002425393.1
Cv, Q23808.1 (AQPcic)
Bt, ABW96354.1
Tc, XP_972862.1 (TcDRIP)
Ap, NP_001139376.1
Ap, NP_001139377.1
Bm, NP_001106228.1(AQP-Bom2)Gm, BAH47555.1 (AQP-Gra2)
Pv, BAF62091.1 (PvAQP2)
Ag, XP_318238.4
Aa, XP_001650169.1 (AaAQP5)
Cq, XP_001850887.1
Dm, NP_611812.1
Dm, NP_611813.1
Dm, NP_611811.3
Aa, XP_001650168.1 (AaAQP4)
Ag, XP_554502.2Dm, NP_611810.1
Am, XP_001121043.1Nv, XP_001603421.1
Tc, XP_970728.1
Am, XP_624194.1Nv, XP_001601253.1
Nv, XP_001601231.1
Am, XP_001121899.1
Tc, XP_970791.1Tc, XP_970912.1
Am, XP_396705.2 (AmBIB)Nv, XP_001604170.1 (NvBIB)
Ag, XP_314890.4
Aa, XP_001649747.1 (AaAQP3)
Cq, XP_001866597.1Ag, XP_314891.4
Dm, AAF52844.1 (DmBIB)
Tc, XP_968782.1 (TcBIB)
PRIP
DRIP
BIB
Glp
BaAQP1 PvAQP1 AgAQP1 EsAQP1 BgAQP
---MTMKYTLGANELSAKT-HNLWKSILAEFIG--IFILNFFSCAACTQAAFKTGIDTYTRIT---MAFKYSLGADELKGKTGTSLYKAIFAEFFG--IFILNFFGCAACTHAK---------------NTMGYSLGTEELSSKS-SGLWRALLAEFVGTCIFILNFFACAACTHAN-------------MPQKFDYSLGLHELKSKE-HRLWQALVAEFLG--NFLLNFFACGACTQPE------------MAGFNVQERLGMEDLKR----GIWKALLAEFLG--NLLLNYYGCASCIAWTKDTPESVTPENE
BaAQP1 PvAQP1 AgAQP1 EsAQP1 BgAQP
ILANGTDSPEIVAENIFANDLTLIALAFGLSVFMAAMTIGHISGCHINPAVTVGLLAAGKVSV------------------GDEVLIALAFGLSVFMAAMTIGHVSGCHINPAVTFGLLAAGKISL------------------GDKTLISLAFGLSVFMVVMSIGHISGGHINPAVTAGMLAAGKVSL-------------------GGTFKALAFGLAVFIAITVIGNISGGHVNPAVTIGLLVAGRVTVHVENP------------RANHVLVALTFGLVIMAIVQSIGHVSGAHVNPAVTCAMLITGNIAI
BaAQP1 PvAQP1 AgAQP1 EsAQP1 BgAQP
LRAVFYIVAQCAGAAAGVASLNALVSGVAGAGPRGLGHTSLSMGVSEFQGLGFEFFLGFVLVLIRAIFYVLAQCVGSVAGTASLAVLTNGTEIA--IGIGHTQLNPTVSVYQGLGFEFFLGFILILIRALMYVAAQCAGAVAATTALDVLIPKSFQN---GLGNTGLKEGVTDMQGLGFEFFLGFVLVLLRAVCYIIFQCLGSIAGTAAIRTLIDEEYYG---GLGHTHLAPNITELQGLGIEFFLGLVLVLIKGFLYIIAQCIGSLAGTAVLKAFTPNGTQG---KLGATELGEDVLPIQGFGVEFMLGFVLVI
BaAQP1 PvAQP1 AgAQP1 EsAQP1 BgAQP
VVFGVTDENKPDSRFIAPLAIGLTVTLGHLGTVSYTGSSMNPARTFGTALITGNWEHHWIYWACVVGVCDENKPDSRFIAPLAIGLTVTLGHLGVVTYTGSSMNPARSFGTAFITGDWENHWVYWLCVFGVCDENKPDSRFVAPLAIGLTVTLGHLGVVEYTGSSMNPARSFGTAFVTDSWAHHWIYWATVFGALDANKPDSRFTAPLAIGLSVTLGHLGTIRYTGASMNPARTLGTAFAVHNWDAHWVYWIVVFGVCDANRPEFKGFAPLVIGLTITLGHLAALSYTGSSMNPARTLGSAVVSGIWSDHWVYWL
BaAQP1 PvAQP1 AgAQP1 EsAQP1 BgAQP
GPILGGVAAALLYVLAFAAPEIDSHA-----PEKYRQV-QTDDKEMRRLNA-----------GPIAGGIAASLLYSIFFSAPDIEVHR-----SDKYRQVTQNDDKELRTLSA-----------GPILGGVTAALLYCQLFKAP-TASDA-----SERYRTS-ADDKEVMLNLMKHAALEESVITTGPIMGGIAAALIYTQIIEKPLVHTAVKVIEVSEKYRTH--ADDREMRKLDSTRDYA------GPILGGCSAGLLYKYVLSAAPVETTT-------EYSPVQLKRLDKKKEEDGMP---------
0
100
200
300
400
500
600
700
800
cont
rol
BgA
QP
+HgC
l2
+BM
E
Swelling assayAb
a
c
d
P’f(
µm
/sec
)
0
20
40
60
80
100
120
140
cont
rol
BgA
QP
hAQ
P1
hAQ
P3
[3H] glycerol uptakeB
aa a
b
pmol
/ooc
yte
0
10
20
30
40
50
60
70
80
90
cont
rol
BgA
QP
hAQ
P1
hAQ
P3
[14C] urea uptakeC
pmol
/ooc
yte
a
b
a
c
A
0
Rel
ativ
eex
pres
sion
5
1
2
3
4
Ov FB Mu DT MT CG
B
Rel
ativ
eex
pres
sion
7 8 0 1 2 4 6 7LC0
20
40
60
80Last nymphal
instarAdult
Age (days)
Water-stressedControl
Rel
ativ
eex
pres
sion
4 5 6 70
4
8
12
16
Age (days)
C
0
10
20
30
40
50
60
Rel
ativ
eex
pres
sion
dsMock dsBgAQP
D
*