and Glutamate-Rich Protein Is Female Salivary Gland-Specific and ...

MOLECULAR BIOLOGY/GENOMICS

A Glycine- and Glutamate-Rich Protein Is Female Salivary Gland-Specific and Abundant in the Malaria Vector Anopheles dirus B (Diptera: Culicidae) NARISSARA JARIYAPAN,1, 2 WEJ CHOOCHOTE,1 ATCHARIYA JITPAKDI,1 THASANEEYA HARNNOI,1 PADET SIRIYASATEIN,3 MARK C. WILKINSON,4AND PAUL A. BATES5, 6

J. Med. Entomol. 43(5): 867Ð874 (2006)

ABSTRACT Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may inßuence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton & Harrison (⫽An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of ⬇4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis conÞrmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcriptionpolymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-speciÞc. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding. KEY WORDS Anopheles, salivary gland, GE-rich, mosquito, malaria

Malaria control strategies are facing increasing difÞculties because of the acquired resistance of Plasmodium parasites to antimalarial drugs and the growing insecticide resistance in vector populations as well as the lack of an effective malaria vaccine. Therefore, there is an urgent need for novel malaria control strategies (Beerntsen et al. 2000). Rational approaches to new strategies are anticipated to originate from studies of insect physiology, immunology, biochemistry, and molecular biology. These approaches will beneÞt from a detailed understanding of the interactions that occur between parasites and insects in all of these disciplines (Hurd 1994). Salivary glands and the saliva of mosquito vectors have attracted considerable attention because of their role in blood feeding (Ribeiro and Francischetti 1 Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. 2 Corresponding author, e-mail: [email protected]. 3 Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. 4 School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom. 5 Molecular and Biochemical Parasitology Group, Liverpool School of Tropical Medicine, University of Liverpool, Liverpool, United Kingdom (e-mail: [email protected]).

2003), transmission of diseases (James 2003, Rodriguez and Hernańdez-Hernańdez 2004) and allergic responses in humans and animals (Peng and Simons 2004). Recently, proteomic approaches have generated comprehensive lists of the individual gene products in the salivary glands of various mosquitoes, including Aedes aegypti (L.) (Valenzuela et al. 2002), Anopheles gambiae Giles (Francischetti et al. 2002), Anopheles stephensi Liston (Valenzuela et al. 2003), Anopheles darlingi Root (Calvo et al. 2004), and Culex pipiens quinquefasciatus Say (Ribeiro et al. 2004). These studies have revealed an amazing diversity of salivary gland proteins. Examples include the apyrases, maltase-like proteins, thrombin inhibitors, mucins, cecropins, the D7 family, and the allergen/ antigen-5-related family. One of the most abundant classes of mRNAs expressed in the salivary glands of these Þve mosquito species includes those encoding products related to the 30-kDa allergen of Ae. aegypti. The biological function of this protein remains unknown, but it is highly immunogenic (Simons and Peng 2001) and assumed to assist in blood feeding. The cDNA sequence of the gene was Þrst reported by Xu et al. (1998), and conceptual translation revealed a long N-terminal sequence of low amino acid complex-

0022-2585/06/0867Ð0874$04.00/0 䉷 2006 Entomological Society of America

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ity, with many glycine (G), glutamic acid (E), and aspartic acid (D) residues; thus, some of these proteins have also been called GE-rich proteins (Valenzuela et al. 2003, Calvo et al. 2004). The members of the GErich/30-kDa allergen family in the Þve mosquito species listed above have apparent molecular masses ranging from 30 to 35 kDa when separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Anopheles dirus B Peyton & Harrison (Diptera: Culicidae), which is one of the malaria vectors in Southeast Asia, has been investigated with respect to genetic studies of the Anopheles species complex (Baimai et al. 1984; Green et al. 1992; Walton et al. 1999, 2000a,b, 2001) and malaria transmission (Klein et al. 1991, Frances et al. 1996, Coleman et al. 2001). Sallum et al. (2005) proposed to rename An. dirus B as An. cracens. So far, little is known regarding the molecules expressed in the salivary glands of this mosquito species. We began our analysis by isolating cDNAs from an An. dirus B female salivary gland cDNA library, and in parallel identifying the most abundant salivary proteins in the females by SDS-polyacrylamide electrophoresis (PAGE), 2D gel electrophoresis (2DPAGE), and peptide sequencing. Characterization of mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) also was performed. Here, we describe the cDNA encoding one of the major saliva proteins, GE-rich, and its mRNA expression pattern in An. dirus B mosquitoes. Materials and Methods Mosquitoes. An. dirus B mosquitoes (originally from the Armed Forces Research Institute of Medical Sciences laboratory, Bangkok, Thailand) were reared in an insectary of the Department of Parasitology, Faculty of Medicine, Chiang Mai University at 26 Ð28⬚C, 70 Ð 80% RH, and a photoperiod of 12:12 (L:D) h. Groups of mosquitoes were reared simultaneously from the same cohort of eggs. Mosquitoes aged between 3 and 7 d after emergence were used. The mosquitoes were given continuous access to cotton wool saturated with 10% sucrose solution. Salivary Gland Dissection. Salivary glands of the adult mosquitoes were dissected as described previously (Suwan et al. 2002). For 2D-PAGE, 10 female salivary glands of An. dirus B were dissected in sterile water and immediately placed in a rehydration solution [8 M urea, 50 mM dithiothreitol, 4% (wt:vol) CHAPS, 0.2% (vol:vol) 3/10 Bio-lyte Ampholyte, and 0.002% (wt:vol) bromphenol blue]. The 2D samples were kept at ⫺80⬚C until use. SDS-PAGE and 2D Electrophoresis. SDS-PAGE, N-terminal, and internal-peptide sequencing were carried out as described previously (Suwan et al. 2002). 2D-PAGE was performed using the IPGphor system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) for the Þrst dimension isoelectrofocusing (IEF) gel. A broad pH range (3Ð10) was used in the IEF gel. After loading the 2D samples, IPG strips (isoelectric point [pI] 3Ð10, 7 cm, GE Healthcare) were rehydrated for 12 h at 20⬚C and run

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according to the manufacturerÕs protocol. The strips were then equilibrated in SDS equilibration buffer [6 M urea, 2% (wt:vol) SDS, 50 mM Tris, pH 8.8, and 20% (vol:vol) glycerol] for 15 min and loaded onto 12% SDS-PAGE gels for the second dimension. N-Terminal and Internal Peptide Sequencing. A sample of 50 female salivary glands was separated on a 12% SDS-polyacrylamide gel. After transfer to polyvinylidene dißuoride membranes, N-terminal sequences of individual protein bands were determined by Edman degradation on an Applied Biosystems model 471A Protein Sequenator (Applied Biosystems, Warrington, United Kingdom). For internal peptide sequencing, in-gel digestion was then carried out using an adaptation of the method of Rosenfeld et al. (1992). Gel slices from SDS-PAGE were washed with 50% (vol:vol) acetonitrile, 0.2 M ammonium bicarbonate, pH 8.9, and then freeze-dried. The slices were reswollen in 0.2 M ammonium bicarbonate, pH 7.8, 0.02% (vol:vol) Tween 20, and 2 M urea containing trypsin, and incubated at 37⬚C overnight. Excess buffer was then removed to a second tube, and peptides were extracted from the gel slices twice with 60% (vol:vol) acetonitrile and 0.1% (vol:vol) trißuoroacetic acid. The latter and the excess buffer were pooled, concentrated by centrifugal evaporation, and applied to a reverse phase (C18) high-performance liquid chromatography column to separate the peptides. Suitable peptides were then subjected to sequencing by Edman degradation using an Applied Biosystems model 471A Protein Sequenator (Applied Biosystems). Isolation of mRNA and Construction of cDNA Library. Approximately 2 to 3 ␮g of poly(A)⫹ RNA was isolated from 150 pairs of An. dirus B female salivary glands by using a Micro FastTrack2.0 kit (Invitrogen, Carlsbad, CA) and used as a template for doublestranded (ds)cDNA synthesis using cDNA synthesis kit (GE Healthcare). A Zero Background/Kan Cloning kit (Invitrogen) was used to construct the female salivary gland cDNA library. EcoRI/NotI adaptors were added to the blunt-ended dscDNA before ligating into pZEro-2 vector (Invitrogen), and then 2 ␮l of ligation mixture (from a total volume of 10 ␮l) was transformed into One Shot TOP 10 Competent cells (Invitrogen). Kanamycin (Invitrogen) was used for colony selection. All procedures were performed following manufacturersÕ instructions. A pool of the bacterial colonies was mixed with glycerol, and this cDNA library was stored at ⫺20⬚C. Sequence Analysis and Comparison. Sequence analyses and comparisons were performed using the BLAST program (http://www.ncbi.nlm.nih.gov/). Signal peptides were predicted by submission of the sequences to the PSORT (http://psort.nibb.ac.jp/). Sequence alignments and analyses were performed using CLUSTALW and EMBOSS programs available at the European Bioinformatics Institute (http:// www.ebi.ac.uk). Domain analysis was performed using GlobPlot (http://globplot.embl.de). Isolation of Genomic DNA. Approximately 200 ␮g of genomic DNA (gDNA) was isolated from 200 mg of

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JARIYAPAN ET AL.: GE-RICH SALIVA PROTEIN OF An. dirus B

fourth instars using QIAGEN tips 500 for genomic DNA isolation (QIAGEN, Crawley, United Kingdom). Isolation procedures followed the manufacturerÕs instructions. RT-PCR Analysis. Poly(A)⫹ RNA samples of An. dirus B were isolated from 50 female salivary glands, 20 female heads with salivary glands removed, 20 female thoraxes and abdomens with salivary glands removed, 50 male salivary glands, 20 male heads with salivary glands removed, and 20 male thoraxes and abdomens with salivary glands removed, by using the Micro FastTrack 2.0 kit (Invitrogen). First strand cDNA of each sample was synthesized using ⬇1 ␮g of the poly(A)⫹ RNA as template (First Strand cDNA Synthesis kit for RT-PCR, Invitrogen). Primers were designed for detection of the An. dirus B GE-rich mRNA: Andi054F (5⬘-GATTCACATGATGGAGCGG-3⬘) and Andi054R (5⬘-GTGCATCACGTTTCTCAACG-3⬘). Positive control primers used in a PCR reaction to check the efÞciency of poly(A)⫹ RNA isolation were designed using the sequence of a ribosomal L12 protein cDNA clone (accession no. DQ018867) obtained from the An. dirus B salivary gland library: Andi008F (5⬘-CTTCCCTGATCGTGAAAG-3⬘) and Andi008R (5⬘-TGGTACAGCCAACGCTCT-3⬘). Possible genomic DNA contamination of RNA was checked by using gDNA isolated from mosquito larvae as template for comparable PCR reactions, because the gDNA products were determined to be of different sizes than those obtained with cDNA as template. Previously synthesized dscDNA with adaptors was used as a positive control. PCR reactions were set up using 5 ␮l of the Þrst strand cDNA mixture of each sample or 1 ␮l of the gDNA or the 1 ␮l of the cDNA as template for each pair of primers. The reactions were conducted in a PTC-100TM thermal cycle (MJ Research, Watertown, MA). The PCR proÞle consisted of an initial denaturation for 5 min at 94⬚C followed by 35 cycles of a denaturation at 94⬚C for 1 min, annealing for 1 min at 55⬚C, and extension for 2 min at 72⬚C. An additional cycle included an extension for 10 min at 72⬚C. AmpliÞed PCR products were veriÞed by electrophoresis in a 1.0% agarose gel. Results Female salivary gland extracts of An. dirus B were separated by SDS-PAGE and stained with Coomassie Blue or silver (Fig. 1). Several major and several minor protein bands were observed, including a protein with an apparent molecular mass of 35 kDa that was negatively stained with silver. In Coomassie Blue stained gels, this 35-kDa protein was detected as an abundant protein. Several attempts were made to sequence the protein by automated Edman degradation, but no sequence was obtained due to possible N-terminal blocking. Therefore, internal peptide sequencing was performed. Salivary gland proteins of An. dirus B females were separated on 2D gels (Fig. 1). A protein with an estimated molecular mass of 35 kDa and a pI of ⬇4 was identiÞed as a potential GE-rich protein, because it correlated with the 35-kDa protein band on

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Fig. 1. Female salivary gland proteins of An. dirus B. Salivary gland proteins of An. dirus B were separated by 12% SDS-PAGE or 2D gel electrophoresis. Lane 1, silver-stained gel of two salivary glands. Lane 2, Coomassie Blue-stained gel of 10 salivary glands. Panel pl, Coomassie Blue-stained 2D gel of 10 salivary glands. Arrows indicate the 35-kDa GE-rich protein band or spot. Molecular mass markers in kilodaltons are as indicated on the left.

SDS-PAGE, was acidic, and was detected as an abundant protein on the 2D gel. This spot was excised, trypsin-digested, and the N-terminal sequences of three internal peptides were obtained: QVHDQL, DGYLK, and SFVVAR. These showed high similarity to sequences present in the conserved C-terminal domain of other GE-rich proteins. They were subsequently found to be identical to peptides in the deduced amino acid sequence of a full-length An. dirus B salivary gland cDNA clone encoding a GE-rich protein (see below; Fig. 2). Thus, the abundant 35-kDa An. dirus B salivary gland protein is also a member of the GE-rich protein family. To further characterize the GE-rich, we randomly picked colonies from the An. dirus B cDNA library and sequenced their inserts. As expected, cDNA sequences encoding proteins with homology to GE-rich proteins were readily obtained from the cDNA library. A cDNA clone named Andi054 was obtained from An. dirus B (Fig. 2). Inspection of the sequence and BLAST analysis indicated that Andi054 was a fulllength cDNA clone with signiÞcant similarity to Anopheles GE-rich proteins. Andi054 consists of 863 nucleotides and encodes a protein of 256 amino acids. A putative signal peptide cleavage site was found between position 19 and 20 (VTA-RP), yielding a predicted minimum mass of the mature protein of 25.4 kDa with a pI ⫽ 3.90. A region rich in glycine (19%), glutamic acid (27%), and aspartic acid (17%) was present from amino acids 21Ð141. In the Andi054 protein sequence, two potential N-linked glycosylation sites were found at Asn177 and Asn223. Hydropathy analysis did not show any potential hydrophobic transmembrane sequences, or sequence motif for attaching a GPI-anchor. These data together with the presence of a signal peptide indicates that Andi054 is a secreted

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Fig. 2. Nucleotide sequence and deduced amino acid sequence of the cDNA clone Andi054. The stop codon is indicated by an asterisk. The predicted signal peptide, two potential N-linked glycosylation sites, and the polyadenylation addition site are underlined. The three peptides obtained by protein sequencing are boxed. Sequence was deposited under accession no. AAP68774.

salivary protein. BLAST analysis produced the closest match to An. gambiae (EAL39244) with 79% identity and An. stephensi (BAC78821) with 74% identity, both GE-rich salivary proteins. The three internal peptide sequences obtained by protein sequencing mapped onto the translated sequence. Overall, these data demonstrate that Andi054 encodes an Anopheles dirus B GE-rich protein. Although it is probable that the 35kDa protein is encoded by Andi054 (given the peptide sequence data), this may not be the case, because there is evidence that in other mosquito species multiple GE-rich proteins exist. The difference in molecular mass predicted from the gene sequence (25.4 kDa) and electrophoresis (35 kDa) could be due to glycosylation or other posttranslational modiÞcations. To determine the key properties of the GE-rich proteins of hematophagous mosquitoes, compositional analysis (Table 1) and sequence alignment (Fig. 3) was performed on 13 related GE-rich/30-kDa allergen proteins from Þve Aedes, four Anopheles, and one Culex species. After removal of their putative secretion peptides the resulting proteins (excluding any possible posttranslational modiÞcation) have predicted molecular masses ranging from 19.2 to 26.5 kDa and show pI values ranging from 3.69 to 4.44, i.e., all are highly acidic proteins (Table 1). Alignment revealed two discrete domains in the protein sequences. The N-terminal region begins at the Þrst amino acid of the predicted mature protein and ends

before a conserved TY doublet. This region shows limited conservation of sequence and overall length (67Ð137 amino acids) but has very similar amino acid content. The N-terminal region contains a very high percentage of glutamic acid and/or aspartic acid residues (27.7Ð 47.1%), making this part of the protein very acidic (pI ⫽ 3.03Ð3.83) and highly negatively charged (Table 1). Many but not all of the proteins also contain a high percentage of glycine residues. The C-terminal domains are more conserved in terms of length (109 Ð117 residues) and sequence, containing 10 identical residues and 17 well conserved residues that form a clear signature for inclusion in this group of salivary proteins (Fig. 3). Four conserved cysteine residues that could form intramolecular disulÞde bridges were found in the 13 sequences. The existence of the two regions was also supported by GlobPlot software analysis, which predicted a disordered N-terminal region and a potential globular Cterminal domain. Thus, although the N-terminal region may not form a distinct domain, it clearly has a structure and important function. Sex and tissue speciÞcity of GE-rich mRNA expression was investigated in female and male mosquitoes by RT-PCR (Fig. 4). In female An. dirus B transcription (541-bp product) was speciÞc to salivary glands (Fig. 4, lanes 2Ð 4), indicating that protein expression was conÞned to female salivary glands. Control ribosomal protein L12 products (195 bp) were detected in each mRNA preparation (Fig. 4B), indicating that the

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Table 1. Properties of mosquito GE-rich/30-kDa allergen salivary gland proteins deduced from translated sequences of Anopheles, Aedes, and Culex species

Species and gene Ae. albopictus 30k-1 Ae. albopictus 30k-2 Ae. aegypti allergen-like Ae. albopictus 30k-5 Ae. albopictus 30k-4 Ae. albopictus 30k-3 Ae. aegypti allergen Aed a3 An. dirus B GE-richf An. gambiae ENSANGP00000028522 An. stephensi GE-rich An. stephensi GE-rich precursor An. darlingi GE-rich Cx. p. quinquefasciatus salivary protein

N-terminal region

NCBI accession no.

MMa kDa

SPb

MMc kDa

pld

AAV90691 AAV90692 AAL76031 AAV90695 AAV90694 AAV90693 AAB58417 AAP68774 EAL39244 BAC78821 AAO74840 Calvo, E., personal communication AAR18452

22.8 23 23.6 21.4 27.9 28.3 27.1 27.4 26.9 28.5 28.5 27.4

19 19 19 20 19 19 19 19 19 19 19 19

20.8 20.9 21.4 19.2 25.7 26.2 25.0 25.4 24.9 26.5 26.5 22.6

24.4

19

22.3

e

AA

%G

%E

%D

% (D⫹E)

pI

4.11 4.07 4.29 4.19 3.75 3.74 3.94 3.90 3.83 3.72 3.69 4.44

77 77 82 67 132 137 119 122 118 135 135 100

9.1 6.5 11.0 9.0 25.8 25.5 19.3 18.9 17.8 22.2 22.2 22.0

19.5 20.8 19.5 23.9 24.2 24.1 28.6 27.0 22.9 30.4 30.4 13.0

16.9 16.9 18.3 9.0 18.9 18.2 18.5 17.2 16.9 9.6 9.6 22.0

36.4 37.7 37.8 32.9 43.1 42.3 47.1 44.2 39.8 40.0 40.0 35.0

3.64 3.62 3.82 3.46 3.06 3.05 3.28 3.40 3.15 3.03 3.03 3.41

4.25

94

8.5

16.0

11.7

27.7

3.83

NCBI, National Center for Biotechnology Information (Bethesda, MD). a Molecular mass (MM) of the putative translated protein. b Size in amino acids of the secretory signal peptide predicted by PSORTII. c MM of the mature protein without the signal peptide. d Isoelectric point of the mature protein. e Number of amino acids in the N-terminal domain, deÞned as the sequence from the predicted N-terminal amino acid of the mature protein to the conserved TY doublet (see Fig. 3). f An. dirus B data from this study.

quality of mRNAs was acceptable in all samples. Also, the PCR products of gDNA (lane 8) were larger than other products for both primer pairs, indicating that no gDNA contaminated in the poly(A)⫹ RNA samples. The differences in size between the PCR products of cDNA and gDNA were assumed to be due to the presence of at least one intron in the gDNA sequences. Discussion Mosquito salivary glands have been studied because of their roles in blood feeding, pathogen transmission, and involvement in allergic responses in the vertebrate hosts (Ribeiro and Francischetti 2003, James 2003, Rodriguez and Hernańdez-Hernańdez 2004, Peng and Simons 2004). Recently, genomic and proteomic approaches have led to not only the discovery of several new members of gene/protein families but also novel gene/protein families. Among others, members of the GE-rich/30-kDa allergen family have been reported in Ae. aegypti (Valenzuela et al. 2002), An. gambiae (Francischetti et al. 2002), An. stephensi (Valenzuela et al. 2003), An. darlingi (Calvo et al. 2004), and Cx. pipiens quinquefasciatus (Ribeiro et al. 2004). Each protein has a long region of low amino acid complexity, with a high content of glycine and/or glutamic acid and/or aspartic acid residues. However, the biological function of these proteins remains unclear. In this report, we describe the gene for a GE-rich protein in An. dirus B for the Þrst time, and examined the expression of this gene. The analyses performed in this study demonstrated that the GE-rich is among the most abundant female saliva-speciÞc proteins in An. dirus B. The results also provide further evidence

that the GE-rich/30 kDa salivary proteins are highly expressed in all blood-feeding mosquitoes, and that expression of these secretory proteins is both femalespeciÞc and salivary gland-speciÞc. The speciÞc function of the GE-rich proteins in blood feeding remains to be determined, but the current report and analysis provides several important clues. The high quantity of the protein expressed suggests some kind of binding, neutralizing, or lubricating function, rather than a catalytic function associated with an enzyme. All the proteins in the GE-rich family are highly acidic, resulting from their high content of glutamic acid and/or aspartic acid residues. However, these residues are not randomly distributed within each protein, rather they are concentrated in an Nterminal region of variable length. Thus the resulting N-terminal region is very negatively charged, suggesting that it would readily interact with any blood/ salivary proteins that were positively charged or contained externally exposed local regions of positive charge. It is also possible that the GE-rich proteins interact with or bind to other salivary proteins. However, interestingly, almost all both plasma proteins and most mosquito salivary proteins are themselves negatively charged overall (http://www.expasy.org/ch2d/; Valenzuela et al. 2003). Therefore, another possibility is that the GE-rich proteins serve as a kind of lubricant, facilitating the ßow of other salivary components with more direct pharmacological properties. This would also be consistent with the abundance of the GE-rich proteins. To elucidate their function(s) structural analysis of the GE-rich proteins is required. In addition, expression of large quantities of recombinant GE-rich proteins may yield enough material for bioassays. If facilitation of blood feeding is the core function of

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Fig. 3. Multiple sequence alignment of mosquito GE-rich/30-kDa allergen salivary gland proteins deduced from translated sequences of Anopheles, Aedes, and Culex species. The two vertical arrows mark the boundaries between the 19Ð20 amino acid signal peptides, and the N-terminal and C-terminal regions, respectively. Identical residues are indicated by a Þlled circle (F), and well conserved residues (10Ð12/13 identical) are indicated with an open circle (E). Abbreviated gene names are used; full names and accession numbers are provided in Table 1.

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Fig. 4. RT-PCR analysis of GE-rich mRNA expression. (A) Analysis of An. dirus B with primers speciÞc for GE-rich mRNA (Andi054F/Andi054R). (B) Analysis of An. dirus B with primers speciÞc for ribosomal protein L12 mRNA (Andi008F/Andi008R). RNA samples were lane 1, negative control, no mRNA; lane 2, female salivary glands; lane 3, female heads with salivary glands removed; lane 4, female thoraxes and abdomens with salivary glands removed; lane 5, male salivary glands; lane 6, male heads with salivary glands removed; lane 7, male thoraxes and abdomens with salivary glands removed; and lane 8, An. dirus B gDNA. Arrows indicate size of expected RT-PCR products.

these proteins, the recombinant proteins could be added to blood and tested by nondenaturing gel or other chromatography protocols to see whether there is binding of these proteins to any blood proteins that are positively charged. Alternatively, speciÞc GErich gene silencing by RNA interference, driven by the infection of mosquitoes with Sindbis virus expressing double-stranded GE-rich RNA (Kuwabara and Coulson 2000; Adelman et al. 2001), could be attempted to evaluate mosquito feeding efÞciency or its impact on parasite transmission. These proteins may be exploited by malaria parasites to enhance their transmission in some way, as has been suggested for other salivary gland proteins (Montero-Solis et al. 2004). Adsorption to the sporozoite surface might assist transmission if the GE-rich proteins function as salivary lubricants. Because mosquito control through environmental perturbation and insecticide application has been limited by environmental and human health concerns and the development of insecticide-resistant mosquitoes (Hemingway and Ranson 2000), novel malaria control strategies are needed (Beerntsen et al. 2000). A strategy of applying molecular genetic techniques to control the mosquito vectors has been proposed (Curtis and Graves 1988). Recently, success in achieving stable transformation of An. stephensi (Catteruccia et al. 2000) has raised hopes for the production of mosquito strains that are unable to transmit malaria parasites. Engineering such a parasite-resistant mosquito population will depend not only on identiÞcation of a suitable anti-pathogen effector molecule but also on linking it to a suitable promoter. An ideal promoter for this purpose is a promoter that is expressed continuously at the developmental site of the pathogen and with a minimal Þtness cost to the mosquito (Beerntsen et al. 2000). A few salivary gland promoters have been tested, mainly in the mosquito Ae. aegypti. The promoter

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regions of the Ae. aegypti maltase-like l (Mall) and apyrase (Apy) genes were shown to direct tissuespeciÞc expression of the luciferase (luc) reporter gene in the salivary glands of transgenic Ae. aegypti. But both promoters yielded only very low expression levels (Coates et al. 1999). Recently, short promoter fragments located upstream of the An. gambiae female salivary gland-speciÞc genes apyrase (AgApy) and D7related 4 (D7r4) were analyzed for their transactivation properties. The AgApy promoter directed speciÞc expression of the LacZ reporter gene in the salivary glands of transgenic An. stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-speciÞc expression in Drosophila melanogaster (Meigen) but only low transcription levels in transgenic An. stephensi (Lombardo et al. 2005). Therefore, investigation and identiÞcation of other salivary gland-speciÞc promoters is still desirable. In this study, the An. dirus B GE-rich is identiÞed as a highly expressed female saliva-speciÞc protein. The promoter driving the expression of this protein may have the potential of being a female, salivary glandspeciÞc promoter for the expression of antiparasite genes within transgenic mosquitoes. Acknowledgments We are grateful to Eric Calvo of Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing the An. darlingi GE-rich sequence. We also thank Anuluck Junkum for technical assistance. We thank the Faculty of Medicine Endowment Fund for Þnancial support of this research project and publication costs.

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