Journal of Insect Physiology 56 (2010) 1599–1610

Cloning and functional expression of Rh50-like glycoprotein, a putative ammoniachannel, in Aedes albopictus mosquitoes

Yu Wu a,b,*, Xiaoying Zheng a,b, Meichun Zhang a,b, Ai He a,b, Zhuoya Li a,b, Ximei Zhan a,b

a Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, Chinab Key Laboratory of Tropical Disease Control, Sun Yat-sen University, Ministry of Education, Guangzhou, Guangdong 510080, China

A R T I C L E I N F O

Article history:

Received 27 April 2010

Received in revised form 23 May 2010

Accepted 24 May 2010

Keywords:

Rh50 glycoprotein

Ammonia channel

Amino acid metabolism

Gonotrophic cycle

A B S T R A C T

Evidence has shown that female mosquitoes can deaminate more than 80% of the ingested bloodmeal

protein amino acids, and thus lead to a massive amount of ammonia production. Ammonia transport is a

critical step for detoxifying ammonia in organisms. Here we characterized a putative ammonia channel

gene, Rhesus (Rh) 50 glycoprotein, from Aedes albopictus (AalRh50) and determined the difference of its

expression profile in different tissues at both message and protein levels as well as its response to a blood

meal. We showed that AalRh50 shares a low identity with E. coli ammonia transporter (EcoAmtB), but

higher identities with human RhBG and Drosophila Rh50 genes. The analysis of ammonia-conductance

sites indicates that AalRh50 has residue substitutions of S237L (equivalent to S219 in AmtB) in the

external vestibule, F127I (equivalent to F107 in AmtB) in the pore entrance, and S281N (equivalent to

S263 in AmtB) in the internal vestibule, which could alter or reduce ammonia-conductance activity. The

results from quantitative real-time-PCR and immunohistochemistry revealed that AalRh50 is expressed

at significantly higher levels in the head, Malpighian tubules, and thorax of the non-blood-fed females,

suggesting that AalRh50 might play roles in maintaining normal neurotransmitter metabolism, acid–

base balance, and flight energy production in different tissues of mosquitoes at the non-blood-fed

condition. A blood meal significantly increases AalRh50 expression in midgut, fat body, and Malpighian

tubules from 3 or 6 to 24 h post feeding, indicating that AalRh50 plays an important role in detoxification

of excess systemic ammonia of female adults during the gonotrophic cycle.

� 2010 Elsevier Ltd. All rights reserved.

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1. Introduction

Metabolism of blood meal-derived amino acids is a process ofnitrogen metabolism in mosquitoes (Briegel, 1985, 1986; Franceand Judson, 1979; Zhou et al., 2004). Evidence showed that femaleAedes aegypti mosquitoes can deaminate more than 80% of theingested bloodmeal protein amino acids and use them formetabolic purposes other than egg protein synthesis (Zhouet al., 2004). In the same mosquito species after a blood meal, of60% excretory nitrogen, about 11% is excreted as ammonium(NH4

+) (Briegel, 1986). On the other hand, in Anopheles stephensi

adult females, the hemolymph ammonia (NH3) concentrationvaries from 1.2 mg/ml at emergence to maximal 2.2 mg/ml aftertwo successive blood meals (Mack et al., 1979), suggesting that thefemale mosquitoes possess a huge capacity to process excess

* Corresponding author at: Department of Parasitology, Zhongshan School of

Medicine, Sun Yat-sen University, #74 Zhongshan Er Rd, Guangzhou, Guangdong

510080, China. Tel.: +86 20 87331638; fax: +86 20 87331638.

E-mail addresses: wuyu@mail.sysu.edu.cn (Y. Wu), zhanximei@yahoo.com.cn

(X. Zhan).

0022-1910/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2010.05.021

ammonia in the body to maintain ammonia homeostasis in thecirculatory system when they face a massive load of exogenousnitrogen.

Ammonium is a key nitrogen source for bacteria, archaea, fungi,and plants, and its transport across cellular membrane can beachieved by ammonium transporter (Amt) and methylaminepermease (MEP) proteins (Zidi-Yahiaoui et al., 2009). Based on thesequence comparisons, it has been proposed that homologues ofthese transporters in animals are Rhesus (Rh) proteins (Huang andPeng, 2005; Marini et al., 2000). In humans, the Rh family includingboth erythroid (RhAG, RhD, and RhCE) and nonerythroid (RhCG,RhBG, and RhGK) also shares conservation with the Amt/MEPfamily throughout their sequence (Marini et al., 1997). Amt is an 11transmembrane helix protein with a periplasmic N-terminus(Khademi et al., 2004; von Wiren and Merrick, 2004; Zheng et al.,2004). Crystal structural analysis reveals that Amt has a recruit-ment vestibule for cations such as NH4

+ or neutral NH3, a site thatcan bind CH3NH3

+ or NH4+ using p-cation interactions (a

noncovalent molecular interaction between the face of anelectron-rich p system, e.g. benzene and ethylene, with anadjacent cation), and a hydrophobic channel that incorporatesNH3 using weak interactions with C–H hydrogen bond donors

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–16101600

(Khademi et al., 2004). In animals, ammonium transport acrossmembranes is important for maintaining ammonium homeostasis(Weiner and Verlander, 2003) and is specifically implicated in themaintenance of pH balance in the kidney (Nakhoul et al., 2005;Wall, 2003; Weiner, 2004).

In Manduca sexta larva, an Rh-like ammonia transporter wasfound with high mRNA expression levels in the hindgut,Malpighian tublules and ganglia and has been confirmed to haveammonia transport properties in this insect species (Weihrauch,2006). However, no reports have been found in mosquitoes. In thepresent study, we sequenced an Aedes albopictus Rh50 glycoproteingene (AalRh50). AalRh50 shares 42% identity with human RhBGand has a putative ammonia-conductance structure that is similarto both Escherichia coli ammonium transporter B and Human RhBG.We also report the differential tissue expressions of AalRh50message and protein as well as its response to a blood meal. Resultsfrom these studies may give new insights into several importantaspects of physiological processes in A. albopictus and couldprovide novel targets for mosquito vector control.

2. Materials and methods

2.1. Mosquitoes

A. albopictus (Guangzhou strain) larvae were fed on a mixture ofBrewer’s yeast, lactalbumin hydrolysate and finely ground ratchow (1:1:1). Adult mosquitoes were routinely maintained at27 8C, 80% relative humidity with a photoperiod of 14:10 h (L:D) on10% sucrose solution ad libitum.

2.2. RNA isolation and cDNA preparation for cloning AalRh50 gene

Eight 3- to 5-day-old A. albopictus females were homogenized in1 ml of TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and total RNAwas extracted by following the manufacturer’s instructions. Afterthe final isopropanol precipitation, the RNA pellet was re-suspendedin 30 ml of RNase-free H2O, and 5 ml was used for spectroscopicquantitation at OD260 nm. All RNA samples were treated with DNaseI to remove any residual genomic DNA, and cDNA was synthesizedfrom 1 mg of total RNA using reverse transcriptase and oligo-dT withPrimeScript First cDNA Synthesis Kit (Takara, Tokyo, Japan)according to the manufacturer’s instructions.

2.3. Molecular cloning and bioinformatics of AalRh50 gene

A multiple amino acid sequence alignment of Rh-like glycopro-teins from A. aegypti (GenBank accession numbers: AY926463 andAY926464), Anopheles gambiae (XP_001663123), Drosophila mela-

nogaster (XP_001661801), C. p. quinquefasciatus (XP_001662114),and Homo sapiens (RhBG, NP_065140) was performed by usinghttp://www.ebi.ac.uk/clustalw/. Based on the highly conservedamino acid regions, two pairs of degenerate primers were designedas follows: (1) outer forward primer: 50- GCGCG GAATTC TTY GGNGCN TAY TT-30, the corresponding amino acid sequence: FGAYF;outer reverse primer: 50-GCGCGC AAGCTT CAT NCC RTG NAG RTT-30,the corresponding amino acid sequence: NLHGM; (2) inner forwardprimer: 50- GCGCG GAATTC GCN ATG ATH GGN AC-30, thecorresponding amino acid sequence: AMIGT; inner reverse primer:50- GCGCGC AAGCTT NAC NCC RCA NGT RTC-30, the correspondingamino acid sequence: DTCGV. Added additional sequence at the 50-end of each primer (i.e. a regular restriction enzyme site and a fewmore GC) can increase the PCR efficiencies by increasing primerlength and CG content and hence annealing temperature.

The first PCR was performed using the outer forward andreverse primers. The product of the first PCR amplification wasused for a nested PCR with the inner forward and reverse primers.

PCR conditions were: one cycle at 94 8C for 3 min, 20 cycles at 94 8Cfor 30 s, 50 8C for 30 s, 72 8C for 2 min, and final extension at 72 8Cfor 10 min. The nested PCR was performed under the sameconditions as the first PCR except for 25 cycles at 55 8C of annealingtemperature for 30 s. The predicted inner PCR fragment of Rh50gene is about 379 bp. The inner PCR product was ligated withpMD18-T vector (Takara) and transformed into E. coli DH5a.Positive colonies were selected and sequenced as usual. PlasmidDNA was extracted using a Qiagen Plasmid Mini-prep kit (Qiagen,CA, U.S.A.). Sequence encoding mosquito Rh50 protein wasdetermined by homology searches in the NCBI databases usingthe BLAST program. The obtained putative A. albopictus Rh50 genewas named as AalRh50.

Rapid amplification of cDNA ends (RACE) PCR was used toobtain overlapping cDNA fragments of the putative AalRh50 genewith pairs of outer and inner gene-specific primers (GSPs) designedfrom the blasting result. For 30-RACE, forward outer GSP1 wasdesigned as 50-GCGGATCCGCGGAACAGGAACGAGCAAT-30 andforward inner GSP3 as 50-GCAACCGTTACCACCTTTGT-30. For 50-RACE, reverse outer GSP2 was designed as 50-GCAAGCTTCCAT-GATTGCACCGAATGGA-30 and reverse inner GSP4 as 50-CAAATG-GATCCAACGGCTAC-30. 50 and 30-RACE were conducted using 50-and 30-full RACE Core Set Kits (Takara). The forward GSP1 and GSP3

and reverse primer 30-RACE T17 Adaptor Primer provided by the kitwere used for the outer and inner 30-RACE PCR, respectively. ThePCR reactions were conducted using Takara LA Taq DNA polymer-ase (Takara) at: 94 8C, 3 min, 1 cycle; 94 8C, 30 s, 53 8C (outer PCR)or 55 8C (inner PCR), 30 s and 72 8C, 2 min, 20 cycles for outer PCRand 25 cycles for inner PCR; 72 8C, 10 min, 1 cycle. This proceduregave an about 800-bp product containing a portion of the 30-UTR.The reverse GSP2 and GSP4 and forward 50-RACE outer and innerprimers provided by the kit were used for the outer and inner 50-RACE PCR, respectively. The PCR reactions were conducted usingTakara LA Taq DNA Polymerase (Takara) at: 94 8C, 3 min, 1 cycle;94 8C, 30 s, 55 8C, 30 s and 72 8C, 2 min, 20 cycles for outer PCR and25 cycles for inner PCR; 72 8C, 10 min, 1 cycle. This procedure gavean about 1000-bp product containing a portion of the 50-UTR. AllPCR products were cloned into the pMD18-T Vector (Takara) andsequenced as mentioned above. The overlapping cDNAs betweenthree products from degenerate PCR, 50-RACE and 30-RACE werealigned to determine the complete AalRh50 transcription se-quence. ClustalW2 (www.ebi.ac.uk/Tools/clustalw2/index.html)(Larkin et al., 2007) was used to align the deduced amino acidsequences and determine sequence identity. The ammonia-conductance sites were determined from alignments comparingAmtB with mosquito AalRh50 and conserved ammonia-conduc-tance residues were identified manually.

2.4. Quantitative real-time polymerizing chain reaction (QRT-PCR)

Total RNA was extracted from adult female tissues (head, thorax,fat body, midgut, Malpighian tubules, and ovary) at non-blood-fed(named as ‘‘unfed’’ throughout the manuscript), 3, 6, 12, 18, 24, 36,48, 72, 96, and 120 h post-blood meal (PBM) using TRIzol Reagent(Invitrogen) as described above. All RNA samples were treated withDNase I to remove any residual genomic DNA, and cDNA wassynthesized from 250 ng of total RNA using reverse transcriptaseand oligo-dT, as described above. QRT-PCR was performed usingSYBR Premix Ex Taq (Takara) by MJ Research Opticon (MJ ResearchOpticon, CA, USA). We cloned and sequenced ribosomal protein S7(RPS7) gene from A. albopictus (data not shown). The RPS7 transcriptlevels were used as an internal control for normalization of mRNAyields in all samples. The primers for the QRT-PCR reactions ofAalRh50 were designed as: AalRh50-QFP: 50- GCGGAACAGGAAC-GAGCAAT-30 and AalRh50-QRP: 50-CCATGATTGCACCGAATGGA-30

and RPS7 primers as: 50-ATGGTTTTCGGATCAAAGGT-30 and

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–1610 1601

50-CGACCTTGTGTTCAATGGTG-30. Before real-time RT-PCR, thetargeted, specific PCR product was cloned and sequenced todetermine that the product sequence represented that of thedesired message. Real-time RT-PCR reactions were conducted usingMJ Research Opticon with the buffers provided at: 95 8C, 3 min, 1cycle; 95 8C, 10 s, 60 8C, 30 s and 72 8C, 30 s, 45 cycles; with a meltcurve over a temperature range starting at 55 8C and ending at 95 8C.RT-PCR reactions were analyzed by Opticon Monitor SoftwareVersion 1. PCR product quality was monitored using post-PCR meltcurve analysis. Standard curves were obtained using a two-foldserial dilution of pooled cDNA from all the stages. The quantities ofthe mRNA expression relative to RPS7 were obtained. Both PCRefficiency and R2 (correlation coefficient) values are taken intoaccount prior to estimating the relative quantities. Mean andstandard errors for each time point were obtained from the averagesof three independent sample sets (Wu et al., 2006).

2.5. Preparation, purification and confirmation of antibody against

AalRh50 glycoprotein in mosquitoes

A 15-mer peptide (RQLEKDEHHKDDAY) from AalRh50 aminoacid sequence was determined as antigen using an online antigendesign tool EMBOSS (http://bioweb.pasteur.fr/seqanal/EMBOSS)for producing monospecific polyclonal antibody against AalRh50.The peptide was synthesized by Aviva Systems Biology Corpora-tion (Aviva Systems Biology Corporation, Beijing, China) andconjugated with KLH vector and then immunized New Zealandrabbits. The initial injection was carried out with 300 mg of thesynthesized antigen dissolved in phosphate buffered saline (PBS)(0.5 ml) and Freund’s complete adjuvant (0.5 ml) followed by twobooster injections with a 200 mg of antigen each. One week afterthe last injection, blood was collected and allowed to stand at roomtemperature for 1 h, then centrifuged to separate out the serum.The antiserum was purified with Protein A treatment and peptideaffinity purification.

Immunoprecipitation of AalRh50 protein from mosquito bodyextracts with anti-AalRh50 serum was performed using the ProteinA Agarose Beads (Invitrogen). About 20 mg of mosquito tissues washomogenized in pre-cold RIPA buffer (1 ml/100 mg tissue) andcentrifuged at 4 8C, 14,000 � g for 15 min. About 15 ml of thesupernatant (tissue lysate) was aliquoted for SDS-PAGE. Of theremaining supernatant, 100 ml was mixed with 4 ml of anti-AalRh50 serum (20 mg/ml), and incubated at room temperature for1 h. The Protein A Agarose was washed twice with PBS andcentrifuged at 5000 rpm for 30 s and then adjusted to 50% withPBS. About 100 ml of 50% Protein A Agarose in PBS was added intothe antigen–antibody complex followed by an hour of incubationwith gentle shaking at room temperature and centrifuged at5000 rpm for 30 s. About 15 ml of the supernatant that containsproteins not to bind anti-AalRh50 antibody (unbound proteins)was collected for SDS-PAGE. The pellet (antigen–antibody com-plex) was washed 5 times with pre-cold PBS and centrifuged at5000 rpm for 30 s. The washed pellet was suspended with 100 mlof 0.1 M glycine (pH 2.8) followed by centrifuge. Of the collectedsupernatant, about 15 ml (Elution 1) was used for SDS-PAGE andthe remaining pellet was re-suspended with another 100 ml of0.1 M glycine (pH2.8) and centrifuged. Of the resulted supernatant,15 ml (Elution 2) was taken for SDS-PAGE. All protein samples werestabilized with 1/10 V of 1 M Tris–HCl pH8.0. All stabilized proteinsamples were resolved by 10% SDS-PAGE at 80 V for 40 min. Theseparated proteins were transferred onto a PVDF membrane(Millipore, Eschborn, Germany) and membranes were blockedwith 5% skim milk (Difco Lab., Detroit, MI) in 50 mM Tris/HCl (pH7.6), 0.15 M NaCl (TBS). The membranes were incubated with anti-AalRh50 (1:1000) at 4 8C overnight in TBS containing 0.1% Tween-20. Alkaline phosphatase-conjugated goat anti-rabbit IgG was used

as the second antibody at a 1:10,000 dilution in the same buffer.The membranes were then washed twice with TBS containing 0.1%Tween-20 and the protein visualized with BCIP and NBT (Promega,Madison, WI). Molecular markers (Bio Rad, Hercules, CA) wereresolved under identical conditions. Membranes were exposed to aKodak Biomax Light Film (Kodak, New Haven, CT) in a KodakBioMax Cassette with an intensifying screen mounted inside(Kodak) at�80 8C overnight and developed with a Mini-Med/90 X-ray film processor (AFP Imaging, Elmsford, NY).

2.6. Immunohistochemistry (IHC) with anti-AalRh50 antibody

2.6.1. Tissue preparation for immunohistochemical localization of

AalRh50

Three-day-old non-blood-fed females and blood-fed females at3 days post feeding were anesthetized with cold and fixed with 4%paraformaldehyde (PLP) at 4 8C overnight. The fixative mosquitoeswere embedded in polyester wax (polyethylene glycol 400distearate, Polysciences, Warrington, PA), and 4-mm-thick sectionswere cut and mounted on gelatin-coated glass slides.

2.6.2. Immunohistochemistry

Immunolocalization of AalRh50 was accomplished by usingimmunoperoxidase procedures and a commercially available kit(ChemMateTM EnVision +/HRP/DAB Rb&Mo, DAKO, Denmark). Thesections were dewaxed in xylene and rehydrated, rinsed in PBS.Endogenous peroxidase activity was blocked by incubating thesections in 3% H2O2 for 10 min followed by washing in PBS (pH 7.4)for 5 min. The sections were placed in a microwaveable vesselcontaining appropriate amount of antigen retrieval buffer (1 mMEDTA, pH8.0) and boiled for 5 min at full power and then heated for20 min at medium power in a microwave (850 W) followed byincubation at 40 8C for 30 min. After antigen retrieval, the sectionswere rinsed with PBS (pH 7.4) for 3 times followed by blockingwith normal goat serum, and then incubated at 27 8C for 1 h withthe anti-AalRh50 antibody (5 mg/ml in PBS, 50 ml per slide). Fornegative control, the anti-AalRh50 antibody was replaced byantibody diluent PBS buffer. The sections were washed in PBS andincubated for 1 h with ChemMate EnVision + /HRP (50 ml/slide)(Dako) and again washed with PBS. The sections were treated for5 min with DAB working solution, rinsed with distilled H2O. Thesections were counterstained with hematoxylin, then dehydratedwith xylene, mounted with Permount (Fisher Scientific, Fair Lawn,NJ), and observed by light microscopy.

2.7. Inductive expression of Rh50-like glycoprotein among different

tissues with a blood meal

About 150 3-day-old females were allowed to feed on citratedporcine blood at 37 8C for 1 h administered by membrane feeding(Kogan, 1990). Fifteen engorged females were collected at 3, 6, 12,18, 24, 36, 48, 72, 96, and 120 h post-blood meal (PBM),respectively. The unfed cohorts were used as negative control.Fat body (FB), midgut (MG), ovary (OV), Malpighian tubules (MT),head (HD) and thorax (TX) tissues were dissected into Buffer RLT(Qiagen) containing b-ME. Total RNA was extracted as described inSection 2.2. QRT-PCR was performed as described in Section 2.4.

2.8. Data analysis

Data were obtained from three separate experimental samples.Treatment differences were determined by one-way analysis ofvariance with the Tukey’s multiple comparisons test or one-tailedunpaired t-test for comparison of selected data sets (Graph PadSoftware, Inc., San Diego, CA). The significance level, a, was set as0.05.

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–16101602

3. Results

3.1. Characterization of AalRh50 gene

The cDNA sequence for AalRh50 is 1389 bp with a 50-UTR of127 bp and a 30-UTR of 201 bp (GenBank accession number[(Fig._1)TD$FIG]

Fig. 1. The cDNA and deduced amino acid sequence of A. albopictus Rh50 glycoprotein (A

shown with lowercase letters. The bold ATG = translational start codon, TAG = translat

GU013469) (Fig. 1). The ORF encodes a 462 residue polypeptidewith a predicted mass �50 kDa and a theoretical isoelectric point(pI) of 5.37 (Fig. 1).

Blast analysis (Table 1) shows the deduced AalRh50 amino acidsequence has 95% identity with an A. aegypti Rh50-GP1 (AaeRh50-GP1) sequence from the NCBI protein database. Similar Rh50 genes

alRh50) (GenBank accession number GU013469). 50-UTR and 30-UTR sequences are

ional stop codon, and aataaa = polyadenylation signal sequence.

Table 1Comparison of the AalRh50 sequence with others in the NCBI database.

Sequence Identity (%)

AaeRh50-1 AaeRh50-2 CpqRh50 AgaRh50 DmeRh50 HsaRhBG EcoAmtB AfuAmt1

AalRh50 95 85 82 78 61 42 16 16

AaeRh50-1 86 83 78 61 42 17 11

AaeRh50-2 79 73 58 41 16 13

CpqRh50 78 61 42 17 12

AgaRh50 59 40 18 12

DmeRh50 43 15 14

HsaRhBG 15 15

EcoAmtB 16

AfuAmt1

Abbreviations and GenBank accession numbers are: our new Aedes albopictus (Aal) Rhesus 50 (Rh50) (AalRh50, GU013469); Aedes aegypti Rh50 glycoprotein 1 (AaeRh50-1,

AY926463) and Rh50 glycoprotein 2 (AaeRh50-2, AY926464); Culex pipiens quinquefasciatus Rh50 (CpqRh50, XP_001662114); Anopheles gambiae Rh50 (AgaRh50,

XP_001663123); Drosophila melanogaster Rh50 (DmeRh50, XP_001661801); Homo sapiens RhBG (HsaRhBG, NP_065140); Archaeglobus fulgidus ammonium transporter 1

(AfuAmt1, NP_069810); and Escherichia coli ammonium transporter B (EcoAmtB, 1U77A) (Khademi et al., 2004).

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–1610 1603

in the NCBI database also include A. aegypti Rh50-GP2 (AaeRh50-GP2, 85%), Culex pipiens quinquefasciatus Rh (CpqRh, 83%), A.

gambiae (AgaRh, 79%) and D. melanogaster (DmeRh, 59%, Table 1).AalRh50 also shares 47% identity with Homo sapiens Rh (HsaRhBG)(Table 1).

Comparative alignments of AalRh50 with those of other speciesare shown in Fig. 2. The residues for the ammonium conductance inmosquito AalRh50 are shown in Table 2. AalRh50 monomerconsists of 11 transmembrane (TM) a-helices with the N-terminuslocated on the periplasmic side of the membrane and the C-terminus on the cytoplasmic side (data not shown). A criticalaspartic acid (D160 in AmtB), potentially involved in a NH4

+

deprotonation mechanism (Zidi-Yahiaoui et al., 2009; Khademiet al., 2004), was shown to be conserved in AalRh50 (D174,Table 2). Another residue (S219 in the external vestibule of AmtB)responsible for NH4

+ deprotonation is replaced by leucine (L) inAalRh50 (L237, Table 2). Two critical residues located in the porecenter (H168 and H318 in AmtB) are highly conserved in AalRh50(H182 and H341, Table 2). In the part of the channel that separatesthe extracellular vestibule and the pore, the ‘‘twin-Phe’’ (F107 andF215 in AmtB) are present in AmtB and human RhBG, but one ofthem (F107) is replaced by isoleucine (I) in insect Rh glycoproteins(I127 in AalRh50, Table 2). Similar to the extracellular vestibule, oftwo residues S263 and D310, playing a critical role in NH3

reprotonation (Zidi-Yahiaoui et al., 2009; Khademi et al., 2004), theformer (S263 in AmtB) is replaced by N in AalRh50 (N281, Table 2),whereas D310 conserved in all Rh glycoproteins (D333 in AalRh50,Table 2).

3.2. Tissue-specific expression and the effects of blood meal on the

expression of AalRh50 glycoprotein message

We determined the differences in message expression of AalRh50in mosquitoes at different tissues. The expression is significantlygreater in head, Malpighian tubules, and thorax of non-blood-fedfemales three days old, compared to other tissues (P < 0.05, Fig. 3).AalRh50 message is up-regulated over time in midgut, fat body andMalpighian tubules between 3 or 6 and 24 h PBM, and then declinesto levels of equal-aged, unfed females by 36 h post-blood meal(PBM) (Fig. 4C, D and F). AalRh50 message is unresponsive to bloodfeeding in head, thorax, and ovary although it can be expressed at acertain level in these tissues (Fig. 4A, B and E).

3.3. Immunohistochemical localization of AalRh50 glycoprotein in

mosquitoes

We carried out immunohistochemical analysis to determine thetissue localization of the AalRh50 glycoprotein in mosquitoes.

Antibody against the peptides that are specific to mosquitoAalRh50 protein was raised in rabbits. The specificity of theantiserum was assessed by immunoprecipitation (IP) and Westernblot analysis. The antiserum showed a specific signal with theappropriate protein with the predicted molecular weight (Fig. 5).There were no any other non-specific bands after the IP (Fig. 5).

The immunohistochemical staining revealed that AalRh50protein was widely localized in the head, thorax, fat body, andMalpighian tubules of non-blood-fed females (Fig. 6), but not inunfed midgut and ovaries (Fig. 6). Particularly, the AalRh50immunoreactive signals were significantly detected at the apicalmembrane of epithelial cells of Malpighian tubules in both blood-fed and non-blood-fed female mosquitoes (Fig. 6K and L).

4. Discussion

Rh glycoproteins are members of the ammonium transporter(Amt)/methylamine permease (Mep)/Rh family facilitating move-ment of NH3 across plasma membranes (Zidi-Yahiaoui et al., 2009).Although Rh proteins have been extensively studied in bacteria,plants, and mammals, the expression and importance of Rhglycoproteins in mosquitoes remain unknown. We were interestedin the potential role of Rh glycoprotein in mosquito physiology andin response to blood meal ingestion and digestion. We report thesequence of Rh glycoprotein from A. albopictus, message expres-sion in response to a blood meal, as well as its immunohistochemi-cal localization.

Our homology analysis shows that AalRh50 has a putativeammonia channel structure that is similar to both E. coli AmtB andhuman RhBG with residue substitutions of S237L (equivalent toS219 in AmtB) in the external vestibule, F127I (equivalent to F107in AmtB) in the pore entrance, and S281N (equivalent to S263 inAmtB) in the internal vestibule (Table 2). This suggests thatAalRh50 could have an altered or reduced ammonia-conductanceactivity. Evidence demonstrates that human RhBG protein-mediated methylammonium uptake in oocytes has a property ofsaturation with apparent Km values in the low millimolar rangeand is effectively inhibited by ammonium at similar concentra-tions (Ludewig, 2004). A lower ammonium ion affinity of thehuman RhBG protein is functionally plausible as it mediatesammonia conduction typically at millimolar concentration inhumans rather than micromolar ammonium concentration inbacteria (Winkler, 2006). Similar situation may also existin mosquitoes. Hence the putative mechanism of AalRh50ammonia-conductance in mosquitoes might be expected to besimilar to that of human RhBG. Clearly, more rigorous biochemicalstudies of mosquito Rh50 glycoprotein functions involved inammonia metabolism in mosquitoes are needed.

[(Fig._2)TD$FIG]

Fig. 2. Comparative alignments of the deduced amino acid sequence of A. albopictus Rh50 glycoprotein with those of other species. GenBank accession numbers: A. albopictus

Rh50 (AalRh50, GU013469); A. aegypti Rh50 glycoprotein 1 (AaeRh50-1, AY926463) and Rh50 glycoprotein 2 (AaeRh50-2, AY926464); C. pipiens quinquefasciatus Rh50

(CpqRh50, XP_001662114); A. gambiae Rh50 (AgaRh50, XP_001663123); D. melanogaster Rh50 (DmeRh50, XP_001661801); H. sapiens RhBG (HsaRhBG, NP_065140); and E.

coli ammonium transporter B (EcoAmtB, 1U77A) (Khademi et al., 2004). Conserved ammonia-conducting residues are highlighted in grey. The symbols ‘‘*’’ = identical,

‘‘:’’ = conservative residue substitution, and ‘‘.’’ = partial conservation of the residue.

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–16101604

In non-blood-fed female mosquitoes, the greatest expression ofAalRh50 mRNA was observed in head (Figs. 3 and 6). It has beenknown that brain is one of the major organs where ammoniaproduction is localized in vertebrates (Planelles, 2007). In thecentral nervous system of vertebrates, the ammonia production is

consequent to the metabolism of neurotransmitters (Planelles,2007). High expression of AalRh50 mRNA in mosquito headsuggests that there may be a highly active and constitutiveneurotransmitter metabolism occurred in mosquito head at thenon-blood-fed condition. A line of evidence may support this

[()TD$FIG]

Fig. 2. (Continued ).

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–1610 1605

suggestion. For instance, lamina glia in Drosophila or Musca cantake up GABA (Kretzschmar and Pflugfelder, 2002), and inEuropean corn borer the putative neurotransmitters acetylcholineand dopamine were preferentially found in glial cells of larval brain(Houk and Beck, 1977). Particularly, in locust, the changes in levelsof neurotransmitters such as glutamate, GABA, dopamine, seroto-nin, acetylcholine, tyramine, and citrulline are widespread in thecentral nervous system and reflect the time course of behavioraland physiological processes (Rogers et al., 2004). Of neurotrans-mitters, glutamate can be directly used for synthesizing glutamineby reaction with ammonia, which is an important pathway fordetoxification of ammonia in the brain (Nissim, 1999). Obviously, aroutine high neurotransmitter metabolism requires a highconstitutive expression of ammonia channel to timely removethe produced ammonia in the brain. In addition, glutamate can also

Table 2Comparison of the residues of the ammonium/ammonia-conductance sites for

various Rh glycoproteins.

Sequence Residues

External

vestibule

Pore

entrance

Pore center Internal

vestibule

EcoAmtB S219 D160 F107 F215 H168 H318 D310 S263

AalRh50 L237 D174 I127 F233 H182 H341 D333 N281

AaeRh50-1 L237 D174 I127 F233 H182 H341 D333 N281

AaeRh50-2 L237 D174 I127 F233 H182 H341 D333 N281

CpqRh50 L239 D176 I129 F235 H184 H344 D336 N283

AgaRh50 L238 D175 I128 F234 H183 H342 D334 N282

DmeRh50 L234 D171 I124 F230 H179 H338 D330 N278

HsaRhBG L210 D148 F101 F206 H156 H315 D307 N255

Abbreviations and GenBank accession numbers are: our new Aedes albopictus (Aal)

Rhesus 50 (Rh50) (AalRh50, GU013469); Aedes aegypti Rh50 glycoprotein 1

(AaeRh50-1, AY926463) and Rh50 glycoprotein 2 (AaeRh50-2, AY926464); Culex

pipiens quinquefasciatus Rh50 (CpqRh50, XP_001662114); Anopheles gambiae Rh50

(AgaRh50, XP_001663123); Drosophila melanogaster Rh50 (DmeRh50,

XP_001661801); Homo sapiens RhBG (HsaRhBG, NP_065140); and E. coli ammoni-

um transporter B (EcoAmtB, 1U77A) (Khademi et al., 2004).

be metabolized via the oxidative deamination through glutamatedehydrogenase (GDH) pathway forming ammonia and a-ketoglu-tarate (aKG). The aKG so formed is oxidized in the tricarboxylicacid cycle (TCA) to supply energy and lactate in astrocytes(Schousboe et al., 1997). Thus, AalRh50 may play important roles inmaintaining normal neurotransmitter metabolism as well as inlinking ammonia detoxification and energy metabolism in brain.Further studies in this area in mosquitoes are needed. In mammals,the sites for rapid accumulation and detoxification of ammonia inbrain are astrocytes (a kind of glial cells, also called as microglia)(Brookes, 2000; Planelles, 2007). Referring the information fromvertebrates, there may be two pathways for ammonia productionin the mosquito head. The most important pathway is the oxidative[(Fig._3)TD$FIG]

Fig. 3. Differential expression of AalRh50 gene in different tissues of non-blood-fed

A. albopictus females. Three-day-old unfed females were dissected and different

tissue mRNA levels were evaluated using real-time RT-PCR with S7 ribosomal RNA

as an internal control. Data represent the mean � SEM of three independent

experiments. One-way ANOVA with Tukey’s multiple comparison test was used for

statistical analysis of data. Bars with different letters are significantly different

(P < 0.05). HD = head; TX = thorax; FB = fat body; MG = midgut; MT = Malpighian

tubules; and OV = ovary.

[(Fig._4)TD$FIG]

Fig. 4. Effects of blood feeding on AalRh50 transcripts in mosquitoes. Female mosquitoes were dissected at unfed, 3, 6, 12, 18, 24, 36, 48, 72, 96, and 120 h PBM. AalRh50 mRNA

levels in head, thorax, fat body, midgut, ovary, and Malpighian tubules were evaluated using real-time RT-PCR with S7 ribosomal RNA as an internal control. (A) AalRh50

mRNA in head (HD) is not induced by a blood meal, compared to that of unfed females (P > 0.05). (B) AalRh50 mRNA in thorax (TX) is not induced by a blood meal compared to

that of unfed females (P > 0.05). (C) AalRh50 mRNA in fat body (FB) is up-regulated at 3, 6, 12, 18, and 24 h PBM, compared to that of unfed females (P < 0.05). (D) AalRh50

mRNA in midgut (MG) is up-regulated at 3, 6, 12, 18, and 24 h PBM, compared to that of unfed females (P < 0.05). (E) AalRh50 mRNA in ovary (OV) is not induced by a blood

meal, compared to that of unfed females (P > 0.05). (F) AalRh50 mRNA in Malpighian tubules (MT) is up-regulated at 6, 12, 18, and 24 h PBM, compared to that of unfed

females (P < 0.05). Data represent the mean � SEM of three independent experiments. One-way ANOVA with Tukey’s Multiple Comparison Test was used for statistical analysis of

data. The symbols ‘‘*’’ represents P < 0.05, ‘‘**’’ means P < 0.001.

[(Fig._5)TD$FIG]

Fig. 5. Characterization of specificity of anti-AalRh50 serum. Immunoprecipitation

of AalRh50 was done with rabbit specific anti-AalRh50 serum. All proteins are

resolved by 12% SDS-PAGE. Western blots were done as described in the methods by

the same antiserum. Lane 1: mosquito whole body tissue lysate (15 ml, 66.7 mg

protein/ml); lane 2: unbound proteins (without anti-AalRh50) (15 ml, 66.7 mg

protein/ml); lane 3: Elution 1 (anti-AalRh50 bound protein) (15 ml, 0.4 mg protein/

ml); and lane 4: Elution 2 (anti-AalRh50 bound protein) (15 ml, 0.5 mg protein/ml).

Detailed procedures are referred to Section 2.5. RH = AalRh50.

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–16101606

deamination of glutamate by the glutamate dehydrogenase(Planelles, 2007). A second pathway for ammonia production inthe brain may be involved in diffusion of ammonia fromhemolymph to the central nervous system by crossing theblood–brain barrier in insects, which may be driven by theammonium chemical gradient and by the pH gradient, as proposedin human (Planelles, 2007). However, our data revealed thatAalRh50 mRNA expression in mosquito head is not induced by ablood meal (Fig. 4). Thus, this second pathway for the ammoniaproduction in the head might not exist in mosquitoes. Alterna-tively, the AalRh50 gene localized in head may not have a putativebloodmeal-inducible cis-element. Such an alternative explanationmay also be suitable for the similar observation obtained in thoraxand ovary.

The second greatest expression of AalRh50 mRNA was observedin Malpighian tubules in non-blood-fed female mosquitoes (Figs. 3and 6). Malpighian tubules consist of epithelium and are a distallyblind-ended complex that inserts the gut region between themidgut and hindgut (Bradley, 1985; Garrett et al., 1988). The apical

[(Fig._6)TD$FIG]

Fig. 6. Immunolocalization of AalRh50 in mosquito tissues. Hematoxylin and eosin (HE) staining of formalin-fixed, paraffin-embedded sections of mosquito tissues (A and B)

and IHC analysis of mosquito tissues with rabbit anti-AalRh50 serum (C–L) were performed. Immunoreactivity was visualized after incubation with peroxidase-conjugated

anti-rabbit IgG secondary antibody and development with the peroxidase substrate DAB. Counterstaining was performed with hematoxylin. The positive color is brown.

Panel A shows HE staining of tissues from the non-blood-fed 3 days old adult female tissues (Magnification�100). Panel B shows HE staining of tissues from the adult female

at 3 day PBM, mature eggs are shown (�40). Panels C, E, G, and J are negative control for IHC assay of non-blood-fed adult female A. albopictus with different magnifications (C

and E:�40; G and J:�100). Panels D, F, H, I, K, and L show the positive immunostaining results with different magnifications (D and F:�40; H and I:�100; K and L:�200) in

non-blood-fed females and blood-fed females at 3 days PBM (L only). Light microscopic immunohistochemistry revealed that AalRh50 is mainly located in HD, TX, FB, and MT,

but not detectable in the unfed MG and OV at current conditions. HD = head, TX = thorax, FB = fat body, MG = midgut, OV = ovary, MT = Malpighian tubules.

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–1610 1607

polarized location of AalRh50 protein (Fig. 6) is similar to that ofRhcg responsible for ammonium secretion in mouse kidney(Weiner, 2006). In the kidney, NH4

+ secretion is a main componentof net acid excretion (Nakhoul and Hamm, 2004). Therefore, renalammonia metabolism and transport are critically important for theregulation of systemic acid–base homeostasis (Knepper et al.,1989; Weiner and Verlander, 2003; Weiner, 2004). Clearly, highmessage expression of AalRh50 in Malpighian tubules of mosqui-toes at the non-blood-fed condition might be essential to maintaina routine systemic acid-base homeostasis by ammonia channel-mediated ammonia excretion. Like in brain, the major pathway forammonia production in Malpighian tubules would be thedeamination of glutamine that is taken up from hemolymph. Inaddition, the glutamine may also be formed in Malpighian tubulesthemselves. Evidence showed that locust Malpighian tubule wallhomogenate is able to convert glutamic acid into glutamine (Kilbyand Neville, 1957), which is subsequently metabolized intoglutamate or a-ketoglutarate and ammonia (Planelles, 2007). In

M. sexta, an Rh-like ammonia transporter was found with highexpression levels in Malpighian tubules (Weihrauch, 2006), whichis consistent with our result. Inducement of AalRh50 mRNAexpression by a blood meal reflects an increased need of ammoniadetoxification or excretion after a blood meal. The inducementmechanism of AalRh50 in Malpighian tubules by a blood meal inmosquitoes is unknown. In addition, the formation and secretion ofuric acid is one of the major pathways for detoxification ofammonia in insects, but whether the AalRh50 is involved in thispathway in mosquitoes is unclear.

Unfed mosquito thorax shows a medium level of AalRh50mRNA expression, compared to high levels in both head andMalpighian tubules as well as low levels in unfed midgut, fat body,and ovary (Fig. 4). This reflects a medium ammonia production inthorax. Thorax is rich in flight muscles. In Colorado beetle, flightmuscles exhibit high activities of both glutamic-pyruvate trans-aminase and glutamate dehydrogenase (Khan and de Kort, 1978),which leads to the accumulation of ammonia in flight muscles

[()TD$FIG]

Fig. 6. (Continued ).

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–16101608

(Bursell et al., 1974; Bursell and Slack, 1976). Of all amino acids,proline is a dominant one that plays an important role in theoxidative metabolism of the flight muscles (thorax) of the Coloradobeetle (de Kort et al., 1973). During flight, the proline concentrationin the flight muscles of Leptinotarsa decemlineata decreases sharply(Weeda and de Kort, 1979). Similar results are also seen in A.

aegypti mosquitoes, in which, whether sugar-fed or blood-fed,flight behavior leads to a significant decrease in proline and asignificant increase in glutamine concentration in thorax, and thelevels of the enzymes involved in proline oxidation were higher inthorax than in fat body and midgut (Scaraffia and Wells, 2003). Theincreased glutamine concentration in thorax of mosquitoes duringthe flight may be the major pathway for removing ammonia that isproduced by proline deamination. Thus, the medium expression ofAalRh50 message in unfed thorax is a constitutive requirement formeeting the need of both energy production and ammoniametabolism during the flight of females who are seeking a bloodsource. Another possible source for ammonia production in thoraxis deamination via purine nucleotide cycle by AMP deaminase(Lowenstein, 1972), the activity of which in vertebrates is adeterminant of adenylate energy charge and energy metabolism inskeletal muscles (Flanagan et al., 1986; Morisaki et al., 1992). Thisis based on the analysis of bioinformatics on A. aegypti genome inwhich a homologue of human AMP deaminase (AAEL015410) is

found (data not shown), although the tissue location of this gene inmosquitoes is yet unknown. Like in head, a blood meal cannotinduce AalRh50 expression in thorax of mosquitoes.

Gut is also one of the major organs to produce ammonia invertebrates (Planelles, 2007). However, our results revealed thatAalRh50 expression at both transcriptional and translational levelsin unfed midgut is very low. This is probably because adult femalesonly take sugar meals before a blood meal so that it is not necessaryto have a high constitutive expression without any substrates. Incontrast, after a blood meal the AalRh50 message was significantlyup-regulated in midgut, suggesting occurrence of oxidativedeamination of a transient bulk of amino acids derived from theblood meal and subsequent ammonia transport in midgut. A line ofevidence may support this point. For instance, the midgut of A.

aegypti mosquitoes responds efficiently to high levels of ammoniain vitro (Scaraffia et al., 2010). Further studies demonstrated thatglutamine synthetase 1 (GS1) and GDH in midgut of the samespecies are up-regulated by blood feeding (Scaraffia et al., 2010)and several key enzymes related to ammonium/ammonia metab-olism also showed the activities in homogenates of midgut in A.

aegypti (Scaraffia et al., 2005). Therefore, the highly inducementexpression of AalRh50 by a blood meal in midgut might be a criticalstep for dietary amino acid metabolism at the entry of meal aminoacids and its metabolites into mosquitoes. The property of AalRh50

Y. Wu et al. / Journal of Insect Physiology 56 (2010) 1599–1610 1609

expression with low level at the non-blood-fed condition and highlevel induced by a blood meal in midgut that is the entry of dietaryamino acids and its metabolites for mosquitoes may allow themidgut AalRh50 to be an ideal candidate target to develop aneffective agent for the control of both mosquito population andmosquito-borne diseases.

Like in midgut, the expression of AalRh50 message in unfed fatbody is very low, but can be significantly up-regulated by a bloodmeal. This reflects an increased requirement of detoxification ofexcess systemic ammonia in mosquitoes after a blood meal. Themajor source of ammonia production in fat body after a bloodmeal might be the deamination of meal amino acids in midgut. Atleast, this is the case in vertebrates as the vertebrate liver candetoxify the gut ammonium production as a result of proteindigestion and deamination processes by hepatic ureagenesis(Planelles, 2007). Another major source of ammonia productionin fat body after a blood meal would be oxidative deamination ofamino acids in fat body itself. The mRNA expression of GDH in A.

aegypti fat body can be up-regulated by a blood meal from 3 to24 h PBM (Scaraffia et al., 2005). The expression pattern of GDHin A. aegypti appears to exactly match that of AalRh50 in A.

albopictus (Fig. 4). Thus, AalRh50 may play an important role inmeal amino acid metabolism and ammonia detoxification in fatbody after a blood meal. The detoxification of ammoniaproduction in fat body may include two major pathways:ureagenesis and conversion into glutamine by GS (Scaraffiaet al., 2010).

In summary, we have sequenced AalRh50 gene from A.

albopictus. The predicted ammonia-conductance sites of AalRh50as well as its tissue-specific expression and bloodmeal-responsesuggest that this protein likely serves a critical role in mosquitoneurotransmitter metabolism, flight, and acid–base homeostasisat the non-blood-fed condition, as well as in detoxification ofexcess systemic ammonia of female adults during the gono-trophic cycle. Further studies are needed to determine AalRh50function by using RNAi technique and to decipher its molecularmechanisms of regulation at both transcriptional and transla-tional levels.

Acknowledgement

This work was supported by fund from the National NaturalScience Foundation of China (no. 30600514) to YW.

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