Two laboratory colonies were used in this work, one at Dr. Ribeiro's laboratory at the National Institutes of Health (NIH), and the other in Dr. Wikel's laboratory at the University of Connecticut Health Center (UCHC). Both mosquito colonies were the Liverpool/blackeye strain of Æ. ægypti. Insectary rooms were kept at 26°C ± 0.5°C (NIH) or 27°C ± 0.5°C (UCHC), with a relative humidity of 70% to 75% and a 16 h:8 h light:dark photoperiod. Adult female mosquitoes used in the experiments were 0–7 days old, took no blood meals, and were maintained on a diet of 10% Karo syrup solution (NIH) or raisins (UCHC).
At the NIH, SG from adult female mosquitoes were dissected and transferred to 20μl Hepes saline (HS; NaCl 0.15 M, 10 mM Hepes, pH7.0) in 1.5 ml polypropylene vials in groups of 20 pairs of glands in 20 μl of HS or as individual glands in 10μl of HS. SG were kept at -75°C until needed. At UCHC, adult female mosquitoes were primed for blood feeding before dissecting out their SG by placing a human hand close to the mosquito cage for 3–5 min (without letting the mosquitoes probe). SG were dissected and placed into a solution of 75% RNA-Later (Ambion) 25% 1 × PBS (RNAse free) and stored in 100% RNA-Later at -20°C for isolating polyA+ RNA.
Æ. ægypti SG mRNA was isolated from 80SG pairs (NIH) or 110 pairs (UCHC) from adult females at days 1 and 2(NIH) or 1–4 (UCHC) after emergence using the Micro-FastTrack mRNA isolation kit (Invitrogen) (NIH) or the Oligotex™ direct mRNA isolation kit (Qiagen) (UCHC). The PCR-based cDNA library was made following the instructions for the SMART (switching mechanism at 5' end of RNA transcript) cDNA library construction kit (Clontech). This kit provided a method for producing high-quality, full-length cDNA libraries from nanogram quantities of polyA+ or total RNA. It utilizes a specially designed oligonucleotide named SMARTIV™ in the first-strand synthesis to generate high yields of full-length, double-stranded cDNA. Æ. ægypti SG polyA+ RNA (300 ng) was used for reverse transcription to cDNA using PowerScript reverse transcriptase (Clontech), the SMARTIV oligonucleotide, and the CDS III/3' primer (Clontech). The reaction was carried out at 42°C for 1 h. Second-strand synthesis was performed by a long-distance PCR-based protocol using the 5' PCR primer and the CDS III/3' primer as sense and antisense primers, respectively. These two primers also create Sfi1A and Brestriction enzyme sites at the end of nascent cDNA. Advantage™ Taq polymerase mix (Clontech) was used to carry out the long-distance PCR reaction on a Perkin Elmer GeneAmp® PCR system9700 (Perkin Elmer Corp.). The PCR conditions were: 95°C for 20s; 24 cycles of 95°C for 5s, 68°C for 6 min. A small portion of the cDNA was analyzed on a 1.1% agarose/EtBr (0.1 μg/ml) gel to check for the quality and range of the cDNA synthesized. Double-stranded cDNA was immediately treated with proteinaseK (0.8 μg/ml) at 45°C for 20 min, extracted with phenol:chloroform:iso-amyl alcohol mixture, and precipitated using sodium acetate (200 mM), glycogen (0.12 μg/ml), and 95% ethanol. The clean double-stranded cDNA was then digested with SfiI restriction enzyme at 50°C for 2 h followed by size fractionation on a ChromaSpin-400 drip column (Clontech). The profiles of the fractions were checked on a 1.1% agarose/EtBr (0.1 μg/ml), and fractions containing cDNA of more than 400 bp were pooled and concentrated by precipitation. The cDNA were then ligated into a λ TriplEx2 vector (Clontech), and the resulting ligation mixture was packaged using GigaPack® IIIPlus packaging extract (Stratagene) according to the manufacturer's instructions. The packaged library was plated by infecting log-phase XL1-Blue Escherichia coli cells (Clontech). The percentage of recombinant clones was determined by performing a blue-white selection screening on LB/MgSO4 plates containing X-gal/IPTG. Recombinants were also determined by PCR, using vector primers (5' λ TriplEx2 and 3' λ TriplEx2 sequencing primers) flanking the inserted cDNA and visualizing the products on a 1.1% agarose/EtBr gel.
Sequencing of the Æ. ægypti cDNA Library
The Æ. ægypti SG cDNA library was plated on LB/MgSO4 plates containing X-gal/IPTG, to an average of 250 plaques per 150 mm Petri plate. Recombinant (white) plaques were randomly picked up and transferred to 96-well MICROTEST™ U-bottom plates (BD BioSciences) containing 100 μls of SM buffer (0.1 M NaCl, 0.01 M MgSO4, 0.035 M Tris-HCl [pH7.5], 0.01% gelatin) per well. The plates were covered and placed on a gyrating shaker for 30 min at room temperature. The phage suspension was either immediately used for PCR or stored at 4°C for future use.
To amplify the cDNA using a PCR reaction, 4 μl of the phage sample was used as a template. The primers were sequences from the λ TriplEx2 vector and named pTEx2 5 seq (5' -TCCGAGATCTGGACGAGC-3' ) and pTEx2 3LD (5' -atacgactcactatagggcgaa ttggc-3' ), positioned at the 5' end and the 3' end of the cDNA insert, respectively. The reaction was carried out in 96-well flexible PCR plates (Fisher Scientific) using TaKaRa EX Taq polymerase (TAKARA; Mirus Bio), on a Perkin Elmer GeneAmp® PCR system9700 (Perkin Elmer Corp.). The PCR conditions were: 1 hold of 95°C for 3 min, 25 cycles of 95°C for 1 min, 61°C for 30s, 72°C for 2 min. The amplified products were analyzed on a 1.5% agarose/EtBr gel. cDNA library clones (1100 clones) were PCR amplified, and those showing a single band were selected for sequencing. Approximately 200–250 ng of each PCR product was transferred to Thermo-Fast 96-well PCR paltes (ABgene Corp.) and frozen at -20°C. Sequencing of the Wikel's laboratory library was performed by Agencourt Bioscience Corp., and a total of 1,017 cDNA library clones was sequenced. The library constructed in Ribeiro's lab was sequenced locally using an 8 capillary CEQ 2000 DNA sequencing instrument (Beckman Coulter, Inc) to provide 2,759 sequences.
2D gel electrophoresis was performed using ZOOM IPGRunner System (Invitrogen) under manufacturer's recommended running conditions. Briefly, approximately 50 μg of sample proteins (approximately 15 pairs of SG) were solubilized with 155 μl rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT, 0.5% carrier ampholytes, pH3-10). The samples were absorbed by rehydration ZOOM strips (7 cm; pH3-10NL) overnight at room temperature and then focused under manufacturer's recommended conditions. The focused IPG strips were reduced/alkylated/equilibrated with reducing and then alkylation reagents dissolved in the sample buffer. The strips were then applied onto NuPAGE 4–12% Bis-Tris ZOOM gels (Invitrogen). The gels were run under MOPS buffer and stained with SeeBlue staining solution (Bio-Rad). A total of 75 spots were selected for tryptic digestion, based on their staining intensity. The gel picture and 23 protein bands matched to Æ. ægypti proteins are shown in Figure .
Protein identification by mass spectrometry
Protein identification of 2Dgel-separated proteins was performed on reduced and alkylated trypsin-digested samples prepared by standard mass spectrometry protocols. Tryptic digests were analyzed by coupling the Nanomate (Advion BioSciences) – an automated chip-based nano-electrospray interface source – to a quadrupole time-of-flight mass spectrometer, QStarXL MS/MS System (Applied Biosystems/Sciex). Computer-controlled, data-dependent automated switching to MS/MS provided peptide sequence information. AnalystQS software (Applied Biosystems/Sciex) was used for data acquisition. Data processing and databank searching were performed with Mascot software (Matrix Science). The NR protein database from the NCBI, National Library of Medicine, NIH, was used for the search analysis, as was a protein database generated during the course of this work.
Bioinformatic tools and procedures
EST were trimmed of primer and vector sequences, clusterized, and compared with other databases as described before [102
]. The BLAST tool [162
], CAP3 assembler [163
], ClustalW [164
], and Treeview software [165
] were used to compare, assemble, and align sequences and to visualize alignments. Phylogenetic analysis and statistical neighbor-joining bootstrap tests of the phylogenies were also done with the Mega3 package [166
]. For functional annotation of the transcripts we used the tool blastx [107
] to compare the nt sequences with the NCBI NR protein database of the NCBI and to the Gene Ontology (GO) database [167
]. The tool rpsblast [107
] was used to search for conserved protein domains in the Pfam [168
], Smart [169
], Kog [170
], and conserved domains (CDD) databases [171
]. We have also compared the transcripts with other subsets of mitochondrial and rRNA nt sequences downloaded from NCBI and to several organism proteomes downloaded from NCBI (yeast), Flybase (D. melanogaster
), or ENSEMBL (An. gambiæ
). Segments of the three-frame translations of the EST (as the libraries were unidirectional, we did not use six-frame translations) starting with a methionine in the first 100 predicted aa – or the predicted protein translation, in the case of complete coding sequences – were submitted to the SignalP server [172
] to help identify translation products that could be secreted. O-glycosylation sites on the proteins were predicted with the program NetOGlyc [173
]. Functional annotation of the transcripts was based on all the comparisons above. Following inspection of all results, transcripts were classified as either (S)ecretory, (H)ousekeeping, or of (U)nknown function, with further subdivisions based on function and/or protein families. To map the EST and contigs in the genome, blastn was used [107
]. To speed the program, each genomic fasta file was broken into 30-kb fragments with 5 kb from previous sequence. For visualization of EST on the Æ. ægypti
genome, we used the Artemis tool [174
] after transforming the blastn output to a file compatible to Artemis using a program written in Visual Basic.
To compare the EST frequency in Æ. ægypti
salivary cDNA libraries with EST frequency in other libraries whose mRNA derive from other sources (downloaded from the NCBI EST database DBEST), all available EST from Æ. ægypti
plus the EST set from a EST hemocyte library from DrBruce Christensen's laboratory [175
] plus our own salivary EST set were pooled to obtain a total of 232,921 EST; these were assembled as described above to create a searchable annotated database of 28,458 contigs and singletons, which is available for browsing at Anobase [10
]. The combined EST database thus derives from 29 different EST libraries, 2 of which are from SG of adult female mosquitoes (4,040 from Ribeiro/Wikel laboratories, and 11,585 from Dr. Sergio Verjovski's laboratory); the remainder are from different organs or whole organisms at different developmental stages, or from adult mosquitoes infected or not with different pathogens. Details of these libraries are available at the EST dataset website [10
]. From each of these 28,458 contigs, we determined the EST contribution from each of the 29 libraries to the final assembled contigs, thus obtaining for each contig the total salivary and nonsalivary contribution. A χ2
test was applied to the data set to determine whether a salivary contribution was above or below the null hypothesis of no differential library contribution when the expected EST frequency was above 5, as indicated for the correct use of the test. When the Pvalue was below 0.05, we considered the deviation of equal EST distribution among salivary and remaining libraries as significant.
RT-PCR expression analysis
For RT-PCR analysis, SG were dissected from adult females 1 to 5 days after emergence and stored at -80°C. Total RNA was extracted from female glands, carcasses (i.e. adult females with SG removed), and adult males using the TRIZOL reagent (Invitrogen).
Approximately 50 ng RNAse-free DNase-treated total RNA (Invitrogen) was used for the RT-PCR amplification by the SuperScript one-step RT-PCR system (Invitrogen) according to manufacturer's instructions. Typically, reverse transcription (50°C, 30 min) and heat inactivation of the reverse transcriptase (94°C, 2 min) were followed by 30 PCR cycles: 30s at 94°C, 30s at 55°C, 1 min. at 72°C; 25 cycles were used for the amplification of the ribosomal protein S5 mRNA (rpS5) to keep the reaction below saturation levels and to allow reliable normalization. For some clones (gi|94468620, gi|94468350, gi|94468634, and gi|42632615), 35 cycles of amplification were needed to obtain detectable bands. The oligonucleotide primers used for rpS5 amplification were: rpS5-F, 5' -ATTACATCGCCGTCAAGG AG-3' , and rpS5-R, 5' -TCATC ATCAGCGAGTTGGTC-3'. The sequence of the other oligonucleotide primers is available as Supplemental Material. Amplification reactions were analyzed on 1.2% agarose gels. Each sample was analyzed by RT-PCR two to three times using independent batches of total RNA.