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Function and immuno-localization of aquaporins in the Antarctic midgeBelgica antarctica

Shu-Xia Yi a, Joshua B. Benoit b,c, Michael A. Elnitsky a,d, Nancy Kaufmann e, Jeffrey L. Brodsky e,Mark L. Zeidel f, David L. Denlinger b, Richard E. Lee Jr.a,*a Miami University, Oxford, OH 45056, USAb Ohio State University, Columbus, OH 43210, USAc Yale University, New Haven, CT 06510, USAd Mercyhurst College, Erie, PA 16546, USAe University of Pittsburgh, Pittsburgh, PA 15260, USAf Beth Israel Deaconess Medical Center, Boston, MA 02215, USA

1. Introduction

The Antarctic midge, Belgica antarctica, is the southern-most,free-living insect. This freeze-tolerant, terrestrial chironomid has atwo-year life cycle and progresses through four larval instars thatare all capable of overwintering (Lee et al., 2006; Lee andDenlinger, 2006). Wingless adults emerge in summer, mate andlay eggs within a 10-day period. Larvae tolerate a wide range ofenvironmental stresses including freezing, severe desiccation, andosmotic extremes (Lee et al., 2006; Rinehart et al., 2006; Lee andDenlinger, 2006). Exposure to dehydration not only increasesdesiccation tolerance but also confers cross-tolerance to cold in thelarvae (Hayward et al., 2007; Elnitsky et al., 2008; Benoit et al.,2009).

Aquaporins (AQPs) are channel proteins that are important inthe movement of water across the cell membrane and have been

described in mammals, yeast, anurans and arthropods (Borgniaet al., 1999). AQPs and aquaglyceroporins (GLPs) function as themain passageways for water and glycerol into and out of the cell.Currently, 13 mammalian aquaporins (AQP0 to 12) have beencharacterized, and their expression is tissue and membranespecific (Agre, 2006; Verkman, 2009). Eleven members of theinsect aquaporin family have been functionally expressed (LeCaherec et al., 1996; Elvin et al., 1999; Pietrantonio et al., 2000;Echevarrıa et al., 2001; Duchesne et al., 2003; Kaufmann et al.,2005; Kikawada et al., 2008; Kataoka et al., 2009a,b) including twofrom Drosophila melanogaster (BIB, big brain protein and DRIP,Drosophila integral protein) (Yanochko and Yool, 2002; Kaufmannet al., 2005; Spring et al., 2009). Among these insect APQs, only twoGLPs, namely AQP-Bom2 (from silkworm Bombyx mori) and AQP-Gra2 (from the oriental fruit moth Grapholita molesta), wereidentified from the digestive tract (Campbell et al., 2008; Kataokaet al., 2009a,b; Tomkowiak and Pienkowska, 2010).

The role of AQPs and GLPs in freeze tolerance has been mainlyexamined clinically and industrially through the artificial expres-sion of aquaporins as a tool to increase survival of mouse oocytes(Edashige et al., 2003) and fish eggs (Hagedorn et al., 2002) during

Journal of Insect Physiology 57 (2011) 1096–1105

A R T I C L E I N F O

Article history:

Received 10 November 2010

Received in revised form 27 January 2011

Accepted 3 February 2011

Keywords:

AQPs

Belgica antarctica

Dehydration

Immuno-localization

Water channel proteins

A B S T R A C T

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes.

Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in

response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the

Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric

chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility

to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from

the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular

viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin),

AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-

like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in

any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased

in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like

proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins

in water relations and freezing tolerance in B. antarctica.

� 2011 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Zoology, Miami University, Oxford, OH

45056, USA. Tel.: +1 513 529 3141; fax: +1 513 529 6900.

E-mail address: leere@muohio.edu (R.E. Lee Jr.).

Contents lists available at ScienceDirect

Journal of Insect Physiology

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate / j in sp h ys

0022-1910/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2011.02.006

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cryopreservation and to improve viability of baker’s yeast afterfreezing (Tanghe et al., 2002). However, few studies have examinedthe role of these channel proteins in animals that are naturallyfreeze tolerant. Recently, Izumi et al. (2006) and Philip et al. (2008)demonstrated that AQPs and GLPs play a role in freezing toleranceof insects. They used mercuric chloride (HgCl2), a known inhibitorof some aquaporins (Preston et al., 1993), to block functionality ofthe water channels, which resulted in reduced cellular survivalduring freezing.

The goals of this study were (1) to explore physiological roles ofaquaporins by assessing cellular changes that occur in response towater fluctuations and cold exposure by in vitro treatment of larvaltissues with HgCl2, (2) to immuno-localize AQP2-, AQP3-, AQP4-,and DRIP-like proteins in isolated tissues and (3) to examineabundance of these proteins in response to dehydration, rehydra-tion, and freezing in B. antarctica. Our results demonstrate thepresence of aquaporins and an aquaglyceroporin (AQP3-likeprotein), and suggest they have important roles in cold anddehydration tolerance in the midge.

2. Materials and methods

2.1. Insect collection

Larvae of B. antarctica were collected from sites on Cormorantand Humble Islands near Palmer Station on the Antarctic Peninsula(648460 S, 648040 W) and shipped (�5 8C for 7 days) to Miami

University (Oxford, OH) in January and February, 2006 and 2007,and subsequently stored at 4 8C. Larvae were manually picked fromthe substrate and kept in water at 4 8C and in the dark for 24 hbefore use in experiments (Lee et al., 2006). Adults werehandpicked from the surface of the substrate after they emergedin a 4 8C-cold room at Miami University, OH.

2.2. Cold and dehydration tolerance

Ability of B. antarctica larval tissues to tolerate freezing wasevaluated according to Izumi et al. (2006) with modifications byPhilip et al. (2008). Fat body, Malpighian tubules and midgut (seeNardi et al., 2009 for anatomical description of alimentary canal)were dissected from larvae according to Teets et al. (2008). Briefly,individuals were dissected in Coast’s solution containing 100 mMNaCl, 8.6 mM KCl, 4.0 mM NaHCO3, 4.0 mM NaH2PO4�H2O, 1.5 mMCaCl2�2H2O, 8.5 mM MgCl2�6H2O, 24 mM glucose, 25 mM HEPES,and 56 mM sucrose (Coast and Krasnoff, 1988); tissues were thentransferred into a 2-ml centrifuge tube containing 0.75 ml Coast’ssolution at 4 8C for 2 h to standardize the sample. Tissue samplesthat appeared to be damaged were not used.

For cold tolerance, 0.75 ml treatment solution was added toeach tube to generate the following concentrations: (1) 1� Coast’ssolution (control); (2) 1� Coast’s solution with 0.2 mM mercuricchloride (HgCl2) to block aquaporins; and (3) 1� Coast’s solutionwith 0.2 mM HgCl2 and 2 mM b-mercaptoethanol, which inhibitsthe blocking effect of HgCl2 (Preston et al., 1992; Philip et al., 2008).

Fig. 1. Cell viability of B. antarctica tissues following exposure to 4 8C and two freezing temperatures (�10 and �20 8C). All samples were held in Coast’s solution. Values

represent means of 8 samples relative to controls held at 4 8C, which represented 100% viability. Different letters indicate significant differences between mean � SE (ANOVA;

P < 0.05).

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Samples were then moved to 4, �10, or �20 8C for 12 h followed bya 2-h recovery at 4 8C. Functional viability of the cells was assessedwith CellTiter 96 Non-radioactive Cell Proliferation Assay (Pro-mega, Madison, WI) according to the manufacturer’s instructions.Mitochondria of living cells reduced yellow 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a purpleformazan product. Tissue samples were incubated with MTT at15 8C for 4 h, and the reaction was terminated by addition ofSolubilization/Stop Solution (Promega, Madison, WI). After 1 hincubation at 37 8C, the optical density of the solution wasmeasured at 570 nm with correction at 630 nm. At completion ofthe experiment, samples were dried at 0% RH, 60 8C until constantmass and weighed to standardize samples according to the drymass. Viability for each sample was compared to the controlsample (1� Coast’s solution at 4 8C), which was considered 100%viability.

Midgut tissues for cellular dehydration experiments werequickly removed from the tube, weighed and returned todetermine their initial mass before treatment. To induce dehydra-tion and overhydration, additional treatment solutions were addedto tubes containing the samples to generate the followingconditions: (1) 4� Coast’s solution (dehydration); (2) 4� Coast’ssolution with 0.2 mM HgCl2; (3) 4� Coast’s solution with 0.2 mMHgCl2 and 2 mM b-mercaptoethanol; and (4) 1� Coast’s solution toserve as a control. Overhydration stress was accomplished bystoring the tissues in (1) 1/4 Coast’s solution; (2) 1/4 Coast’ssolution with 0.2 mM HgCl2; and (3) 1/4 Coast’s solution with0.2 mM HgCl2 and 2 mM b-mercaptoethanol; and (4) 1� Coast’ssolution to serve as a control. Tissue mass was measuredgravimetrically using an electrobalance (CAHN 35) after drawingoff surface water with a tissue. Immediately after weighing, tissueswere dried on pre-weighed aluminum pans at 90 8C, 0% RH untildry mass was constant. Water content within each sample wasdetermined as the difference between the initial or hydrated massand the dry mass. Changes in water content were determinedrelative to initial water content, set as 100%.

2.3. Stress treatments of whole insects

To test the effects of stress treatments on soluble proteinprofiles and the abundance of AQPs, we conducted dehydration,rehydration and freezing treatments on three replicates of 10larvae or adults (N = 30). Dehydration treatment was carried outfor 48 h as previously described (Hayward et al., 2007). Briefly,insects were placed on nylon gauze netting (pore Ø100 mm), whichwas then suspended near the top of 50 ml centrifuge tubescontaining 35 ml 3.16% NaCl, a solution that produced a 98.2% RHenvironment (Winston and Bates, 1960) and covered with a tight-fitting lid.

Rehydration treatment followed Lopez-Martinez et al. (2009)by exposing the dehydrated larvae or adults to 100% RH for 12 h.The rehydration treatment was used to restore water lost duringdehydration (Benoit et al., 2007). Both dehydration and rehydra-tion treatments were performed in a cold-room at 4 8C.

For freezing treatment, larvae or adults were kept in 1.5 ml testtubes and frozen at �20 8C for 24 h. Untreated controls weremaintained in water at 4 8C.

2.4. Fluorescence immuno-cytochemistry of whole mount tissues

Larval and adult tissues including the central nervous system(brain and ventral nerve cord), salivary glands, alimentary tract(foregut, midgut and hindgut), Malpighian tubules, fat body,trachea, integument and muscles were dissected in Coast’ssolution. Whole mount immuno-cytochemistry was used tofluorescently immuno-localize aquaporins with their correspond-

ing antibodies (Duchesne et al., 2003). Tissues were fixed in freshlyprepared 4% (v/v) paraformaldehyde in PBS (175 mM NaCl,1.86 mM NaH2PO4, 8.41 mM Na2HPO4, pH 7.2) containing50 mM EGTA for 2 h on a shaker at room temperature. Afterremoving the fixative by 3� 10 min washes with 70% ethanol onice, tissues were washed 2� 5 min with PBS containing 0.1% (v/v)Tween 20 and 2% (v/v) normal goat serum (PBSTG) (VectorLaboratories, Burlingame, CA) on ice, and then treated with 12 mg/ml proteinase K (Sigma, St. Louis, MO) for 10 min at roomtemperature. Following a 5-min rinse with PBSTG, tissues wereincubated overnight with 10% (v/v) normal goat serum in PBS at4 8C. After changing the solution once and incubating againovernight, tissues were incubated at 4 8C overnight with thefollowing primary antibodies: rabbit anti-actin (1:100 dilution inPBSTG), anti-AQP2 (1:100), anti-AQP3 (1:50), anti-AQP4 (1:400),anti-DRIP (1:500). Negative controls were obtained using rabbitpre-immuno serum (1:100) in place of the primary antibodies orby omitting a primary antibody in the procedure. All primaryantibodies were purchased from Sigma, except for rabbit anti-DRIPantiserum and pre-immuno serum which were prepared and usedas described by Kaufmann et al. (2005) and Spring et al. (2007).Tissues were washed 4� 25 min in PBSTG and incubated withbiotinylated anti-rabbit IgG (Vector Laboratories) in a dilution of1:750 for 1 h. After washing 4� 25 min in PBSTG, tissues wereincubated with Texas Red-Streptavidin (Vector Laboratories) in adilution of 1:200 for 30 min. Tissues were washed 6� 25 min inPBSTG and then mounted in Vectashield mounting medium withDAPI for nuclear staining (Vector Laboratories). Fluorescencemicroscopy was carried out using an Olympus BH-2 microscope (Yiand Lee, 2003). Images were visualized with a camera linked to a

Fig. 2. Water content of the midgut tissue after incubation in dehydrating and

overhydrating media. Control, Coast’s solution; dehydration, 4� Coast’s solution;

overhydration, 1/4 Coast’s solution; mercuric chloride, solution with 0.2 mM HgCl2;

b-mercaptoethanol, solution with 0.2 mM HgCl2 and 2 mM b-mercaptoethanol. (A)

All samples held under dehydrating conditions except control and (B) all samples

held under overhydrating conditions except control. Each sample represents the

mean � SE of 30 midguts.

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computer and processed with Spot Advanced software (Yi et al.,2007).

2.5. Protein preparation and SDS–PAGE

Larvae or adults were homogenized in 150 mM NaCl, 10 mMTris–HCl (pH 7.2), 0.1% sodium deoxycholate (an anionic detergentuseful for extraction of membrane receptors and other plasmamembrane proteins) and protease inhibitors (5 mg/ml aprotinin,5 mg/ml antipain, 5 mg/ml leupetin, and 1 mM PMSF) (Goel et al.,2006; Yi et al., 2007). After sonication and incubation on ice for30 min, the homogenate was centrifuged twice at 16,000 � g for25 min at 4 8C to remove unsolubilized fragments. The resultingsupernatant was collected as soluble proteins. Protein concentrationwas determined using Bio-Rad protein assay reagent (Bio-Rad,Hercules, CA) and BSA as a standard. Fifty micrograms of proteinsample was mixed with Laemmli sample buffer containing 5% b-mercaptoethanol, incubated at 95 8C for 3 min, and then analyzed bySDS–PAGE on a 4–15% gradient gel (Bio-Rad). Precision Plus ProteinStandard Kit (Bio-Rad) was used as a reference for molecular mass.

2.6. Immuno-blotting

Following electrophoresis, proteins were transferred to acellulose membrane (Bio-Rad) and western blotting was done asdescribed (Yi et al., 2007). Non-specific antigens were blocked with10% non-fat milk in western wash buffer at 4 8C overnight.Membranes were incubated with corresponding primary antibodiesin a 5% non-fat milk solution at room temperature for 2 h, using thefollowing dilutions: rabbit anti-actin (1:400), anti-DRIP (1:4,000),anti-AQP2 (1:500), anti-AQP3 (1:200), anti-AQP4 (1:1,000), and goatanti-rabbit and anti-mouse IgG-HRP conjugates. After 3� 15-min

washes in western wash buffer, membranes were incubated for 2 hwith secondary antibody (goat anti-rabbit or anti-mouse IgG-HRPconjugates) diluted 1:1000 in western wash buffer. Immuno-reactive bands were visualized by a 2-min incubation in ECLdetection reagents (Amersham Biosciences, Piscataway, NJ), ex-posed to an autograph film and developed.

2.7. Statistical analysis

Differences in cold survival and changes in water content wereanalyzed using ANOVA followed by Tukey’s post hoc test.Percentage data were arcsine-transformed prior to analysis.

3. Results

3.1. Cold tolerance

Freezing tolerance we observed for larval tissues was similar tothat reported in previous studies with this insect (Lee et al., 2006;Teets et al., 2008; Benoit et al., 2009). Cell viability among control,HgCl2-treated and b-mercaptoethanol treatment at 4 8C did notdiffer statistically (Fig. 1A, D, and G; ANOVA, P > 0.05), indicatingthat these treatments had little effect on cellular viability of fatbody, Malpighian tubules and midgut. When the tissues wereexposed to �10 8C, viability of the fat body was reduced by 30%compared to controls (Fig. 1B), while viability of midgut andMalpighian tubule cells were reduced by only 15% (Fig. 1E and H).Mercuric chloride is a known inhibitor of some aquaporins(Preston et al., 1993; Izumi et al., 2006), while b-mercaptoethanolcounteracts the blocking effect of this compound (Izumi et al.,2006; Philip et al., 2008). When aquaporins were blocked by HgCl2

at �10 8C, cell viability was significantly reduced in the fat body,

Fig. 3. Negative controls of larval tissues incubated with rabbit preimmuno-serum in place of primary antibody (A–D) or without primary antibody (E–G). (A and E) Fat body;

(B) salivary gland; (C) midgut; (D) hindgut and Malpighian tubules; (F) midgut and Malpighian tubules; and (G) trachea. �, negative. Nuclei stained with DAPI appear as blue

spots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Malpighian tubules and midgut (Fig. 1B, E, and H; ANOVA,P < 0.05). When b-mercaptoethanol was added to the solution, thenegative effects of HgCl2 were alleviated (Fig. 1B, E, and H). Cellularviability of control tissues at �20 8C was below 40% for alltreatments (Fig. 1C, F, and I). Similar to tissues held at �10 8C,viability of midgut and Malpighian tubule tissues was reduced ifthe media was supplemented with HgCl2 before exposure to�20 8C (Fig. 1F and I). Fat body viability was very low at �20 8C, andnot significantly different among the three treatments (Fig. 1C;ANOVA, P > 0.05). These results suggest that aquaporins arecritical for B. antarctica to retain cellular viability during freezing.

3.2. Dehydration and overhydration tolerance

When midgut tissues were held in Coast’s solution (control),water content remained stable throughout the course of theexperiments (Fig. 2A and B). Tissues moved to dehydratingconditions (4� Coast’s solution) experienced a rapid drop inwater content that was significantly different from controls after2 min and remained so for 10 min (Fig. 2A; ANOVA, P < 0.05).Blocking aquaporin channels reduced tissue desiccation underdehydrating conditions, but water levels were still lower thancontrols (Fig. 2A). Addition of b-mercaptoethanol restoredfunction to the water channel as evidenced by tissue dehydrationwhen tissues were exposed to dehydrating conditions (Fig. 2A).

When midgut samples were moved to hydrating conditions (1/4 Coast’s solution), water content within samples increased by 30%within 2 min (Fig. 2B), reaching a higher level than observed incontrols (a condition we refer to as overhydration). Under thesehydrating conditions blocking aquaporins with HgCl2 reduced the

rapid influx of water into midgut tissues (Fig. 2B, ANOVA, P < 0.05),while this protective effect was eliminated by the addition of b-mercaptoethanol to the solution (Fig. 2B). These results stronglysuggest a functional role for aquaporins in regulating tissue watercontent in B. antarctica.

3.3. Localization of aquaporins in larval tissues

To confirm that midge tissues had no preexisting immuno-reactions to either normal rabbit serum or secondary antibodies,negative immuno-reactivity was verified in tissue sections usingrabbit preimmuno-serum and staining procedures that omittedprimary antibodies (Fig. 3). No positive reactions were detected infat body, salivary glands, midgut, hindgut or Malpighian tubuleswith the preimmuno-serum (Fig. 3A–D). Similarly, tissue sectionsof fat body, midgut, Malpighian tubules, and trachea showednegative immuno-reactions when primary anti-aquaporin anti-bodies were omitted in the staining process (Fig. 3E–G). In contrast,actin, a positive protein control for many tissues, was immuno-detected in the fat body, midgut, hindgut, muscles, and trachea, butwas not observed in salivary glands or Malpighian tubules (Fig. 4and Table 1).

While AQP2- (Fig. 5) and AQP3-like activity (Fig. 6) were bothfound in fat body, foregut, hindgut, trachea, and integumentmuscles, only AQP2-like activity was detected in the midgut (Table1). AQP4-like activity was not detected in any tissue tested (brain,VNC, FB, MG, and MT) (Fig. 6I–K). DRIP-like activity was localized inthe same tissues as AQP2, with the addition of salivary glands(Fig. 7). No immuno-reactivity from any of these aquaporins wasdetected in Malpighian tubules (Table 1).

Fig. 4. Immuno-detection of actin in larval fat body (A), salivary gland (B), midgut (C), hindgut (D and E), Malpighian tubules (F and G), and integumental muscles and trachea

(H). (+) positive and (�) negative. Blue spots are nuclei stained with DAPI, while red spots indicate immunological reactivity. These color codes are also used for Figs. 5–8. (For

interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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3.4. Localization of aquaporins in adult tissues

Since both AQP2- and DRIP-like activity was present in mostlarval tissues, we also tested for these in adult tissues (Fig. 8 andTable 1). As in the larvae, AQP2-like activity was detected in theadult midgut and hindgut but not in Malpighian tubules (Fig. 8A–C). DRIP-like activity was immunologically positive in themouthparts and hindgut but not in the foregut and midgut(Fig. 8D–F).

3.5. Immuno-blotting of aquaporin proteins

To compare overall changes in aquaporins induced bydehydration and rehydration we used immuno-blotting analysisagainst specific anti-aquaporin antibodies. These data showeddifferent banding patterns between larvae and adults (Fig. 9 andTable 2). Originally actin was chosen with the assumption that itmay be an appropriate control protein. However, the 42 kDa

actin levels changed with dehydration and freezing treatmentsin both larvae and adults, a result consistent with dramaticchanges in actin and other cytoskeletal proteins in response todehydration (Li et al., 2009). In larvae, AQP2 generated fourdistinct protein bands at 31, 30, 28, and 26 kDa (Fig. 9A). The 30-kDa band was more abundant following dehydration, rehydra-tion and freezing. In adults, only three bands were noted and the30-kDa band was missing. Dehydration increased abundance ofthe 26 kDa protein and decreased the 31 kDa protein (Fig. 9B).Two AQP3 bands (26 and 25 kDa) were present in both lifestages. While both bands were abolished by all treatments inlarvae (Fig. 9A), dehydration resulted in elevated levels in adults(Fig. 9B). DRIP yielded a 28 kDa protein band that was moreabundant following dehydration in both larvae (Fig. 9A) andadults (Fig. 9B). Again, no AQP4 was detected in either stage(Fig. 9A and B), further supporting the aforementioned immuno-cytochemical data showing an absence of this aquaporin in alltissues examined (Fig. 6).

Table 1Distribution of actin and aquaporins in larval (L) and adult (A) tissues detected with fluorescence immuno-cytochemistry using specific antibodies. Tissue abbreviations: FB,

fat body; FG, foregut; HG, hindgut; IN, integument; MG, midgut; MT, Malpighian tubules; MU, muscles; SG, salivary glands; TC, trachea; VNC, ventral nerve cord. Protein

abbreviations: AQPs, aquaporins; DRIP, a Drosophila equivalent of AQP. (+) positive; (�) negative; blank, not detected.

Tissue Protein localization

Actin AQP2 AQP3 AQP4 DRIP

L L A L L L A

Brain � �VNC � �FB + + + � +

SG � � � +

Mouth +

FG + + + �MG + + + � � + �HG + + + + + +

MT � � � � � �TC + + + +

MU + + +

IN + +

Fig. 5. Immuno-detection of AQP2 in larval fat body (A), salivary gland (B), foregut (C), midgut (D), hindgut and Malpighian tubules (E), Malpighian tubules (F), tracheae (G),

integument (H), and integumental muscles (I). (+) positive and (�) negative.

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4. Discussion

Survival of desiccation stress and cycles of freezing and thawingdepend significantly on appropriate rates of water flux betweencells and extracellular compartments. Both desiccation and

freezing result in cellular dehydration; during extracellular iceformation only water joins the ice lattice, creating a hypertonicmilieu that osmotically withdraws water from cells (Zachariassen,1991). Furthermore, insects must maintain an appropriate balancebetween water ingestion and excretion. Evidence is accumulating

Fig. 6. Immuno-localizing AQP3 (A–H) and AQP4 (I–K) in larval tissues. (A) Fat body; (B) salivary gland; (C) foregut; (D) midgut; (E) hindgut and Malpighian tubules; (F)

hindgut; (G) Malpighian tubules; (H) tracheae and integument; (I) brain and ventral nerve cord; (J) fat body; and (K) foregut, midgut and Malpighian tubules. (+) positive and

(�) negative.

Fig. 7. Immuno-localization of DRIP in larval tissues. (A) Brain and fat body; (B) brain, thoracic ganglion and muscles; (C) salivary gland; (D) foregut; (E) midgut; (F) hindgut

and Malpighian tubules; (G) Malpighian tubules; and (H) trachea and muscles. (+) positive and (�) negative.

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that aquaporins play primary roles in water homeostasis as eight ofthe 11 known insect AQPs function directly or indirectly inexcretory systems (Spring et al., 2009). Data from our studyprovide further evidence that AQPs are involved in cellular

responses to desiccation and freezing in the Antarctic midge, B.

antarctica.

4.1. Role of aquaporins in cellular tolerance of desiccation and freezing

Larvae of B. antarctica are exposed to multiple periods offreezing, dehydration and overhydration throughout their lifetime(Hayward et al., 2007; Benoit et al., 2007, 2009; Lopez-Martinezet al., 2009). Physiological results from our study show that (1)AQPs are likely involved in the cellular response to dehydrationand overhydration and (2) freeze tolerance depends on functionalAQPs. The fact that both freezing and water stress are influenced byAQPS is not surprising, since freezing and dehydration stress bothlead to cellular dehydration (Ruelland et al., 2009). This commonfeature between cold and dehydration frequently yields crosstolerance between these two stresses as documented in manyinsects, including B. antarctica (Hayward et al., 2007; Benoit et al.,2009). This cross tolerance is likely due to a combination ofcryoprotectants, which double as protectants against dehydration,and stress proteins that accumulate after exposure to cold anddehydration (Danks, 2000; Benoit et al., 2010; Benoit and Lopez-Martinez, in press).

Studies on AQPs in Chilo suppressalis and Eurosta solidaginis

demonstrated that these channels are critical for cold and freezingtolerance (Izumi et al., 2006; Philip et al., 2008; Philip and Lee,

Fig. 8. Immuno-localizing AQP2 (A–C) and DRIP (D–F) in adult tissues. (A) Midgut and Malpighian tubules; (B) hindgut; (C) Malpighian tubule; (D) mouth and foregut; (E)

mouth, foregut, and midgut; and (F) hindgut. (+) positive and (�) negative.

Fig. 9. Immunoblotting identification of actin (42 kDa), AQP2-like protein (26, 28 or

30, and 31 kDa), AQP3-like protein (25 and 26 kDa), AQP4-like protein (30 kDa), and

DRIP-like protein (28 kDa) in the soluble proteins of control (C), dehydrated (DH),

rehydrated (RH) and frozen (FR) larvae (A) and adults (B). Fifty micrograms of proteins

were loaded onto each lane. No AQP4-like protein was detected in either larvae or

adults.

Table 2Dehydration increased the abundance of actin and aquaporins in the soluble protein fraction of larvae and adults as detected by western blotting. Designations of signs: (+)

positive; (�) negative; up-headed arrow, increased abundance; down-headed arrow, decreased abundance; up-and down-arrows, increase of some proteins and decrease of

others; Blank, not detected; C, control; DH, dehydrated; RH, rehydrated after dehydration; and FR, frozen.

Protein Larva Adult

C DH RH FR C DH RH

Actin + " " " + " +

AQP2 + " " " + "# "AQP3 + � � � + " "#AQP4 � � � � � � �DRIP + " " + " "

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2010). We obtained similar results with B. antarctica larvae byshowing reduced cellular survival during freezing when AQPchannels were blocked (Fig. 1). This reduced survival after AQPblockage is likely due to restriction of water and solute flux thatoccurs during freezing or thawing. During freezing, water is drawnfrom cells as ice forms in the hemolymph and thawing results inthe return of water into cells, however, it is difficult to determinewhether AQPs are critical for freezing, thawing or both. Retentionof cellular water during freezing may lead to lethal intracellular iceformation. As the larvae thaw, the inability to rehydrate the cellcould have negative and potentially lethal impacts. Similarly,facilitation of glycerol movement by GLPs may be critical for cellsduring cycles of freezing and thawing.

The physiological role of AQPs has been most thoroughlystudied in relation to periods of water stress in insects (Campbellet al., 2008; Tomkowiak and Pienkowska, 2010). One key exampleis the African sleeping midge, Polypedilum vanderplanki, a midgemore tolerant of dehydration than B. antarctica and capable ofundergoing anhydrobiosis (Crowe et al., 1992; Watanabe, 2006).This African midge increases expression of AQPs during dehydra-tion, likely as a mechanism to control water loss from the cells(Kikawada et al., 2008). Studies on fluid-feeding insects (i.e. bloodand sap), demonstrate that AQPs are critical for removing excesswater in the food (Le Caherec et al., 1996; Spring et al., 2009; Benoitand Denlinger, 2010). Although few studies have examinedrehydration in insects (Campbell et al., 2008; Tomkowiak andPienkowska, 2010), it is important to note that rehydration is notsimply the reverse of dehydration; instead, this stress yields adistinctly different response from that of dehydration (Haywardet al., 2004; Lopez-Martinez et al., 2009; Benoit et al., 2010).Overhydration has an adverse efffect on B. antarctica larvae (Lopez-Martinez et al., 2009), and based on our results, this couldpotentially be due to aquaporins allowing water movement intothe cells when the hemolymph is hypotonic. Our results suggestthat AQPs play a role in both dehydration and overhydrationtolerance in larvae of B. antarctica.

4.2. Distribution of aquaporins among tissues

Although the study of insect AQPs is relatively limitedcompared to mammals, the evidence to date suggests that AQPsare widely distributed and affect cellular functions in every tissue.The number and type of AQPs within a particular membraneappear to be tailored to that tissue’s requirements for transport ofwater and glycerol (Spring et al., 2009). AQPcic (originally P25) isfound solely in the filter chamber of the xylem-sucking leafhopperCicadella viridis (Le Caherec et al., 1996, 1997). AQPs are knownfrom the larval midgut and hindgut of the silkworm B. mori and theoriental fruit moth G. molesta (Kataoka et al., 2009a,b). AeaAQP islocated in distal cells of tracheoles attached to the Malpighiantubules of the yellow fever mosquito Aedes aegypti (Pietrantonioet al., 2000; Duchesne et al., 2003). DRIP (Drosophila integralprotein) is found in Malpighian tubules of both embryonic andadult D. melanogaster (Kaufmann et al., 2005). DRIP shares 44%amino acid sequence identity with human AQP4 and is 65%identical to mosquito AeaAQP (Kaufmann et al., 2005). Immuno-localization using an antibody against DRIP has demonstrated thepresence of DRIP-like protein in Malpighian tubule cells of thecricket Acheta domesticus (Spring et al., 2007), suggesting thataquaporin proteins retain conserved elements shared by differentorganisms.

Our previous studies showed that mammalian AQP-like waterchannel proteins are expressed in freeze-tolerant gall fly larvae(Philip et al., 2008; Philip and Lee, 2010). In the present study, wedemonstrated tissue specific immuno-reactivity of antibodiesagainst mammalian AQP2 and AQP3, and Drosophila DRIP activity

in larvae and adults of B. antarctica (Table 1). However, unlike inlarvae of the gall fly, mammalian AQP4 was not detected in anytissue of the midge, suggesting variation of immuno-reactivitybetween insect species. The presence of AQP2-, AQP3-, and DRIP-like activity in larval fat bodies, foregut, midgut (except for AQP3),hindgut, trachea, and muscle shown in our study indicates thatAQPs are histotypically expressed to control the water content ofeach tissue type. Some of the AQPs are widely distributed, yet thereis evidence suggesting some level of tissue specificity in thisspecies.

4.3. Dehydration and rehydration alter aquaporin levels

In the present study, we demonstrated that a 30-kDa band ofAQP2-like protein was elevated in response to dehydration,rehydration and freezing in larvae (Fig. 9A), whereas a 26-kDaband was increased only by dehydration in adults (Fig. 9B).Desiccation also increased levels of adult AQP3-like protein (anaquaglyceroporin), and larval and adult DRIP-like protein.

Recently, two aquaporins from the sleeping insect P. vander-

planki were reported to be responsible for regulating the transitionof larvae into an anhydrobiotic state (Kikawada et al., 2008).PvAQP1 is proposed as a ‘‘desiccation-tolerance’’ aquaporinwhereas PvAQP2 is considered a ‘‘desiccation-avoidance’’ AQP.PvAQP1 not only facilitates rapid dehydration to protect cellsagainst osmotic damage but also facilitates water uptake duringrehydration. Desiccation activates the phosphorylation cascadeinvolving protein kinase A or kinase C, a mechanism that regulatesAQP intracellular trafficking from storage vesicles to the apicalplasma membrane, as demonstrated in human AQP2 (Fushimiet al., 1997; van Balkom et al., 2002). By altering AQP isoforms,expression levels, intracellular trafficking, tissue distribution, andregulating channel activities, homeostasis of water content ismaintained in response to both internal and external environmen-tal changes (Kikawada et al., 2008).

5. Conclusion

On the Antarctic Peninsula B. antarctica may experience hydricand osmotic stress at any time of the year due to rain or flooding byfreshwater as ice melts and by inundation from seawater duringstorms as well as freezing temperatures and desiccating conditions(Lee and Denlinger, 2006; Elnitsky et al., 2009). Thus far, we haveidentified a number of physiological and molecular mechanismsthat not only protect this species from a specific stress, but alsoconfer cross-tolerance to other stresses. Larvae tolerate severedehydration that enhances their tolerance of desiccation and cold(Baust and Lee, 1987; Hayward et al., 2007; Benoit et al., 2007).Among the stress responses we have identified, thus far, are thecontinuous elevation of heat shock proteins (Rinehart et al., 2006),and the accumulation of low molecular mass solutes (i.e. glycerol,glucose, and trehalose) (Elnitsky et al., 2009; Benoit et al., 2009).Here we provide evidence that aquaporins play an integral role inpromoting desiccation and cold tolerance in this unique insect, aswell. An accompanying paper (Goto et al., in press), providessequence information, expression patterns and a functional assayfor one of the aquaporins we have recently obtained from B.

antarctica.

Acknowledgements

This research was supported by National Science Foundationgrants, OPP-0337656 and ANT-0837559 to REL and OPP-0413786and ANT-0837613 to DLD. This work was also supported in part byNational Institutes of Health grant DK79307 (the University ofPittsburg George O’Brien Kidney Research Core Center) to JLB.

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