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: xavier.belles@ibe.upf-csic.es 14

and mdolors.piulachs@ibe.upf-csic.es) 15

16

17

18

19

20

Running title: An ovarian aquaporin from Blattella germanica 21

22

23

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

53

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56

3

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

4

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

7

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

8

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

9

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

*