Chymotrypsin genes in the malaria mosquitoes Anopheles aquasalis and Anopheles darlingi

Insect Biochemistry and Molecular Biology 33 (2003) 307–315 www.elsevier.com/locate/ibmb

Chymotrypsin genes in the malaria mosquitoes Anopheles aquasalis and Anopheles darlingi R.W. de Almeida a,∗, F.J. Tovar a, I.I. Ferreira b, O. Leoncini a a

Instituto de Biologia, Departamento de Gene´tica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Instituto Oswaldo Cruz, Departamento de Bioquı´mica e Biologia Molecular, Rio de Janeiro, Brazil Received 30 July 2002; received in revised form 15 October 2002; accepted 15 November 2002

Abstract Four closely related chymotrypsin genes were identified in Anopheles aquasalis and Anopheles darlingi (Anachy1, Anachy2, Andchy1 and Andchy2). The deduced amino-acid sequences were compared to other chymotrypsin sequences. These sequences were used to infer phylogenetic relationships among the different species. Genomic cloning revealed that, in contrast to An. aquasalis and A. gambiae, the chymotrypsin genomic locus in An. darlingi had a short intergenic region that accompanied the inverted position of the genes, suggesting inversion mechanisms in this species related to transposable elements. Alignments of the sequences upstream of the transcription start sites of Anachy1, Anachy2, Andchy1 and Andchy2 revealed areas with high similarity containing palindromic sequences. Northern analysis from An. aquasalis indicated that the transcription of chy 1 and 2 are induced by blood feeding.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Anopheles aquasalis; Anopheles darlingi; Chymotrypsin; Serine protease; Mosquito

1. Introduction Malaria is a serious public health problem in Brazil, mainly in the Amazon region. Approximately 19 million people, or 12.3% of the Brazilian population, live in malaria high risk areas. An increase in the number of malaria cases concentrated in Legal Amazonia has occurred in the last 17 years, from 94.9% of the total registered cases in Brazil in 1980 to 99.4% in 1996. In 1996, 444 049 cases of the disease were reported in the country (Pan American Health Organization, 1996). Anopheles (Nyssorhynchus) darlingi is broadly distributed in the neotropics, ranging from southern Mexico to northern Argentina and from the Andes mountains east to the Atlantic coast of South America (Forattini, 1962). This species is the principal vector of human malaria in Brazil (Rachou, 1958; Lourenc¸o-de-Oliveira et al., 1989) and contributes to malaria endemicity throughout its distribution. Anopheles (Nyssorhynchus) aquasalis is also an important vector of malaria that

∗

Corresponding author. Tel.: +1-55-2125626383. E-mail address: [email protected] (R.W. de Almeida).

breeds in brackish coastal marsh habitats. This species cannot develop in undiluted sea water but finds suitable habitats in landlocked coastal mashes and swamps (Beaty and Marquardt, 1996). One of the main reasons that malaria is a central health problem in tropical countries is due to the failure to control anopheline vectors effectively. Widely used residual insecticides and antiparasitic drugs have been inadequate solutions to the problem of vector-borne disease control. There is growing interest for genetic strategies to manipulate the genomes of these insect vectors. The practical aim of these new strategies is either to eliminate the insect pest or to render it inefficient as a vector. The increase in knowledge of basic mosquito biology resulting from these studies will inevitably stimulate novel approaches to the control of mosquitoborne diseases. The effect of mosquito proteases on the development of the malaria parasite Plasmodium suggest that blood digestion and parasite development in the mosquito midgut are intimately connected. The digestion of blood, the kinetics of protease expression and the complexity of digestive enzymes present in the midgut have been studied in great detail (Clements, 1992). Among the dif-

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ferent proteases, trypsin is the most prevalent. These enzymes are responsible for the bulk of anopheline endoproteolytic activity, which reaches a peak one day after a blood meal (Billingsley and Hecker, 1991). Two An. gambiae trypsin genes (Antry1 and Antryp2) were induced by a blood meal (Mu¨ ller et al., 1993). Antryp1 mRNA accumulation is considerably greater than Antryp2 mRNA. These genes are members of a cluster of seven trypsin genes (Mu¨ ller et al., 1995). In Aedes aegypti there are two groups of trypsin forms produced by the female mosquito midgut folowing the blood meal: early and late trypsin. Early forms appear in the midgut within 2 h of the blood meal, and disappears by 8 h after the blood meal. Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene (Barillas-Mury et al., 1995). The late trypsin protein is produced in large amounts, begins to appear 8–10 h after the blood meal, and accounts for most of the endoproteolytic activity present in the midgut during blood meal digestion. Noriega et al. (1999) and Noriega and Wells (1999) showed that feeding per se or filling of the midgut is not sufficient to stimulate early trypsin translation but, several proteins of variable molecular weight and different amino acid sequences are able to induce early trypsin synthesis. Chymotrypsins are also known to play a role in insect digestion (Terra and Ferreira, 1994). The regulation of chymotrypsin synthesis in Aedes aegypti midgut is different from that of other adult female-specific endoproteases. Like early trypsin (Noriega et al., 1996), the chymotrypsin gene is first transcribed at the beginning of adult life; its mRNA reaches its maximal level before feeding, and the protein product is not translated until the ingestion of a meal. Unlike early trypsin, the chymotrypsin mRNA does not decrease after feeding and the chymotrypsin protein is produced continuously during meal digestion. The fact that protein expression and enzymatic activity in crude extracts increase after the ingestion of a protein meal, suggests an involvement in protein digestion (Jiang et al., 1997). Chymotrypsin genes have been identified in A. gambiae. Trypsin activation studies suggest that in A. gambiae both trypsins and chymotrypsins are components of a zymogen cascade. Vizioli et al. (2001) reported the cDNA and genomic cloning and the expression analysis of two closely related chymotrypsin genes, Anchym1 and Anchym2, which are clustered in tandem within 6 kb. Nothing is known about how Brazilian anopheline mosquitoes regulate genes encoding digestive enzymes after a blood meal. For this reason we present here the results of genomic cloning and sequencing of two closely related chymotrypsins from An. aquasalis (Anachy1 and Anachy2) and An. darlingi (Andchy1 and Andchy2). We also describe the expression analysis from An. aquasalis

and a comparative analysis of regulatory regions that flank the chymotrypsin coding region.

2. Materials and methods 2.1. Insects An. aquasalis (kindly provided by Dr. Bento, Biology Institute of the Brazilian Army, Rio de Janeiro, Brazil) were reared at 27 °C and 80% relative humidity under a 12L:12D photoperiod regime. Adults were supplied with a cotton wool pad soaked in a 10% sucrose solution until 12–16 h before the experimental feeding. For purification of total RNA used in Northern bloting, 2–4-dayold females were allowed to feed with human blood. In this paper, we will refer to the sucrose-fed females as unfed. After feeding, the insects were maintained at room temperature for various times. 2.2. Chymotrypsin (CY) and trypsin (TY) probes The probes were kindly provided by Dr. H.-M. Mu¨ ller. The chymotrypsin PCR fragment (CY) was amplified with the degenerated oligonucleotides pTry2, 5⬘-TTCgARATIgAYgTI(AT)(gC)IgARgCICCITAYCA -3⬘, and pTry3, 5⬘-CACCAgCACCAgYggICCICCI (gC)(TA)RTC(TA)CCYTgRCA-3⬘ (Vizioli et al., 2001). The trypsin PCR fragment (TY) was amplified with the primers pTry1, designed according to the sequence of the first nine amino acids (VVGGFEIDV) of the aminoterminal peptide sequence of the A. quadrimaculatus trypsin (Graf et al., 1991) and pTry3 (Mu¨ ller et al., 1993). These probes were obtained from cDNA derived from blood-fed A. gambiae females. 2.3. EMBL3A libraries Genomic An. aquasalis and An. darlingi libraries were constructed in lEMBL3Acos after Sau3A partial digestion and fractionation of the DNA (Sambrook et al., 1989). Approximately 5×105 plaques were plated from an amplified mosquito genomic library, using as host cells Escherichia coli strain LE392 (Stratagene, La Jolla, CA, USA). The plaques were screened with the radioactive chymotrypsin (CY) and trypsin (TY) probes described above. 2.4. Mapping and subcloning of the genomic DNA clones The positive clones were purified and phage DNA was obtained performing a large-scale preparation of bacteriophage l (Sambrook et al., 1989). The genomic DNA clones were digested with different restriction enzymes and the digestion products subjected to Southern analysis

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(Sambrook et al., 1989), using the labeled CY and TY as probes. The 2.0 and 1.0 Kb BamHI digested fragments from An. aquasalis DNA clone and the 3.5 and 1.5 Kb EcoRI digested fragments from An. darlingi DNA clone were isolated using gene clean II kit (Bio 101) and subcloned into pUC9, pGEM-2, pALTER and pGEM-2 vectors, respectively (Stratagene). Nucleotide sequences were determined from both strands by the dideoxy chain termination method, using an automatic DNA sequencer (model 373A; Applied Biosystems, Foster City, CA, USA). Sequence analyses were performed with the GCG software package at the Instituto Oswaldo Cruz, Rio de Janeiro, Brazil. Database searches were conducted using the Blastp program (NCBI, NIH). Additional deduced amino acid sequences were obtained from the GenEMBL database using Stringsearch software. All invertebrate chymotrypsins were subject to multiple sequence alignments using PileUp program (Feng and Doolittle, 1987). The An. aquasalis and An. darlingi chymotrypsin sequences have been deposited in GenBank with accession numbers AF051778, AF051779, AF051780 and AF051781. 2.5. Oligonucleotides Two primers, pAq-2 (5’-AAgCTCgTCCTggATgATCA-3’) and pAq-1 (5’-ACgTTCCCggTACgCATCAC-3’) were endlabelled using g-32P ATP (4500 Ci/mM) and T4 polynucleotide kinase kit (Gibco BRL). pAq-2 and pAq-1 were designed to the highly conserved regions in Anachy2 and Anachy1, respectively. 2.6. RNA extraction and Northern blots from An. aquasalis Total RNA from larvae, adult males, unfed adult female, and adult females fed human blood and killed at different intervals was extracted using QuickPrepTM Total RNA Extraction Kit (Amersham Pharmacia Biotech Inc.). Northern analysis was carried out using an amount of total RNA equivalent to two mosquitoes per lane on denaturing 1.5% agarose/formaldehyde gels containing 2.2 M formaldehyde in 0.1 M MOPS pH 7.0, 40 mM sodium acetate, 5 mM ethylenediamine tetraacetic acid (EDTA) 0.5 M. An RNA ladder (Gibco/BRL) was loaded into the outside lane of the gel and cut from the gel after electrophoresis and stained with ethidium bromide (0.5 µg/ml in 0.1 M ammonium acetate) for 30– 45 min. The gel was run at 4 V/cm. Capillary transfer of RNA to MSi-NITROPURE membranes was performed in 20× sodium chloride/sodium citrate (SSC) overnight. RNA was cross-linked to the membrane using a UV crosslinker (Stratalinker, Stratagene). In this experiment, we used the oligonucleotides pAq-1 and pAq-2 as probes as they have sequences specific for Anachy1 and Anachy2, respectively. Prehybridization was performed

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in a buffer containing 100 µg sperm salmon DNA, 50% formamide, 20 mM phosphate buffer, 0.5% sodium dodecyl sulphate (SDS), 1× denhardt s solution, 5× SSC. The membranes were washed in 2× SSC, 0.5% SDS at room temperature, followed by a more stringent wash in 0.1× SSC, 0.1% SDS at 42 °C. Membranes were exposed to X-ray film with an intensifying screen at ⫺80 °C (Sambrook et al., 1989). Total RNA was stained on the membrane after hybridization (Herrin and Schmidt, 1988). 2.7. DNA extraction and Southern blots Total genomic DNA was extracted from individual mosquitoes placed in 1.5 ml tubes with 100 µl of homogenization buffer (50 mM Tris pH 7.5, 0.3 M NaCl, 25% sucrose, 0.5 M EDTA). After homogenization, 100 µl of lysis buffer (10% SDS, 2.4 M Tris pH 8.5, 25% sucrose, 0.5 M EDTA) was added and the samples were incubated at 65 °C for 30 min. While tubes were still warm, 30 µl 8 M potassium acetate was added and mixed by tapping. The samples were incubated on ice for 45 min to precipitate SDS and centrifuged at 14 000 g for 5 min in a microcentrifuge. The supernatant was transfered to fresh 1.5 ml tubes. The nucleic acids were precipited adding 100% ethanol (EtOH) and ressuspended in 100 µl of TE (0.05 M Tris-HCl, EDTA pH 8.0). Genomic DNA was digested with restriction enzymes and fractionated by eletrophoresis on 0.7% agarose gels. Prior to transfer to MSi-NITROPURE membranes, the DNA was nicked by incubating the gel in 0.25 M HCl for 15 min and then denatured with 1.5 M NaCl/0.5 M NaOH for 60 min at room temperature. DNA probes for library screening and Southern analysis were labeled by the random primer method (Gibco BRL) using [α-32P] dATP (3000 Ci/mmol). Subsequent procedures were the same as for Northern blots. 2.8. PCR amplification of An. darlingi chymotrypsin intergenic sequence Two primers, sense pAd-1 from Andchy1 (5⬘CTCgTCCCggACgATCACTA-3⬘) and antisense pAd-2 from Andchy2 (5⬘-gACCCCAgACATCTAgCATA-3⬘) were used to amplify the 2 kb intergenic region from An. darlingi to confirm the genomic organization of the chymotrypsin locus. PCR reaction was performed in a volume of 50 µl containing 200 µM of each desoxynucleotide, 50 pmol of each primer, 25 pg of genomic λEMBL3A clone CHY1, 4 µl of buffer A and 6 µl of buffer B (60 mM Tris-SO4 pH 9.1, 18 mM (NH4)2SO4, with MgSO4 between 1–2 mM) and 2 µl of eLONGase enzyme (Life Technologies). PCR was done following the cycling conditions: one step of 30 s at 94 °C and three steps of 30 s at 94 °C, 30 s at 55 °C, 5 min at 68 °C (35 cycles).

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3. Results 3.1. Organization of the An. aquasalis chymotrypsin genomic clone The genomic lEMBL3A clone CHY11 carrying a 11 kb insert was isolated and characterized from An. aquasalis. Two fragments of 1.0 and 2.0 kb were recognized by a mixed PCR product as a probe (Ty/Cy) in Southern blots prepared with BamHI-digested CHY11 DNA (data not shown). These two internal BamHI fragments of CHY11 were isolated, subcloned, sequenced and contained most of the Anachy2 and Anachy1 coding sequences, respectively. Fragments of corresponding sizes were detected in genomic Southern blot analysis (Fig. 1). Sequence analysis of genomic subclones containing Anachy2 and Anachy1, indicated that the coding sequences of each gene was interrupted by two introns (Fig. 2). Hybridization experiments carried out on the genomic lEMBL3A clone CHY11 (not shown) and on An. aquasalis DNA digested with a set of restriction enzymes revealed several overlapping DNA fragments. Two of them, CHY8 (SalI digested fragment of 8 kb) and CHY6 (SalI/EcoRI digested fragment of 6 kb), con-

Fig. 1. Southern blot performed on genomic DNA digested with BamHI (B), EcoRI (E), HindIII (H), SalI (S), BamHI and EcoRI (B/E), BamHI and SalI (B/S), EcoRI and SalI (E/S). The DNA was separeted on a 0.7% agarose gel and hybridized with 32P-labelled Anachy1 EcoRI/BamHI fragment. The selected marker positions indicated are derived from lDNA digested with HindIII.

Fig. 2. Restriction map of the 11 kb genomic clone containing Anachy2 and Anachy1. The relative position of the four overlapping DNA fragments CHY1, CHY2, CHY8 and CHY6 used for restriction mapping and subcloning are shown above the restriction map, plasmid vector borders are shown dashed. The relative position and transcriptional direction of the chymotrypsin genes are indicated by arrows. S, SalI; E, EcoRI; B, BamHI. Coding regions, interrupted by two introns each, are shown as black boxes.

nected the two BamHI digested fragments of 1.0 and 2.0 kb. 3.2. Organization of the An. darlingi chymotrypsin genomic clone The genomic lEMBL3A clone CHY18 carrying a 18 kb insert was isolated and characterized from An. darlingi. Two EcoRI digested fragments of 3.5 and 1.5 kb were recognized in a Southern blot analysis (not shown) using Anachy1 and Anachy2 derived probes. These two internal EcoRI fragments of CHY18 were isolated, subcloned, sequenced and contained the Andchy1 and Andchy2 coding sequences, respectively. Sequence analysis of genomic subclones containing Andchy2 and Andchy1, indicated that the coding sequences of each gene were interrupted by two introns (Fig. 3). Hybridization experiments carried out on the genomic clones digested with a set of restriction enzymes (not shown), revealed several overlapping DNA fragments. Three of them, CHY12.1 (SalI/BamHI digested fragment of 12.1 kb), CHY2 (BamHI digested fragment of 2 kb) and CHY3.8 (SalI/BamHI digested fragment of 3.8 kb) contained both chymotrypsin genes and connected the two EcoRI digested fragments of 3.5 and 1.5 kb. The genomic arrangement from An. darlingi was confirmed by ampli-

Fig. 3. Restriction map of the 18 kb genomic clone containing Andchy1 and Andchy2 from An. darlingi. The relative position of the 5 overlapping subclones CHY1.5, CHY3.5, CHY2, CHY3.8 and CHY 12.1 used for restriction mapping and subcloning are shown above the restriction map, plasmid vector borders are shown dashed. The relative position and transcriptional direction of the chymotrypsin genes are indicated by arrows. S, SalI; E, EcoRI; B, BamHI. Coding regions, interrupted by two introns each, are shown as black boxes.

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fication with eLONGase (BRL), and DNA sequencing (data not shown). 3.3. Comparison and analysis of the deduced protein sequence of the putative serine protease A multiple sequence alignment of the amino acid sequence of the putative An. aquasalis and An. darlingi serine proteases with the sequences of other chymotrypsins is shown in Fig. 4A. The Brazilian mosquito species sequences (Anachy1, Anachy2, Andchy1 and Andchy2)

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have the typical catalytic triad including the conserved histidine, aspartic acid and serine residues. Three conserved cysteine bridges, common to all known invertebrate chymotrypsinogens, are found also in analogous positions in the Brazilian anophelines sequences. The putative activation peptides endind with an arginine residue just prior to the conserved N-terminal valine residue indicates zymogen activation by tryptic cleavage. Sequence comparison of the predicted amino acid sequences of Anachy1, Anachy2, Andchy1 and Andchy2 revealed a higher degree of identity between different

Fig. 4. (A) Comparison of deduced amino acid sequences from: Anachy1, chymotrypsin 1, An. aquasalis (AF051778); Anachy2, chymotrypsin 2, An. aquasalis (AF051779); Andchy1, chymotrypsin 1, An. aquasalis (AF051780); Andchy2, chymotrypsin 2, An. aquasalis (AF051781); Anchym1, chymotrypsin 1, A. gambiae (Z18887); Anchym2, chymotrypsin 2, A. gambiae (Z18888); Aecy1, chymotrypsin 1, Aedes aegypti (AY008348); Aecy2, chymotrypsin 2, Aedes aegypti (AY008348); Anstcy, chymotrypsin from A. stephensi (personal communication); Ctenocy, chymotrypsin from Ctenocephalides felis (AF053903); Rhyzocy, chymotrypsin from Rhyzopherta dominica (AF127088; Culexcy, chymotrypsin from Culex pipiens pallens (AF468495). The residues of the catalytic triad are marked with 왓. Three pairs of Cys conserved in both vertebrate and invertebrate chymotrypsins are labelled 1–3. The predicted signal peptide cleavage site is indicated by a star. (B) Neighbour-joining tree of eight species using P-distance. Bootstrap values (500 replicates) indicated above internodal branches are shown for branches with support ⬎50%. Mature peptide regions were used to minimize the noise. Sequences were aligned using CLUSTALW programme (Lombard et al., 2002). The data matrix was subjected to neighbour joining analysis using MEGA2 programme (Kumar et al., 2001).

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species (Anachy1 and Andchy1, 90.3%; Anachy2 and Andchy2, 84.3%) than that observed among sequences of the same species (Anachy1 and Anachy2, 68.2%; Andchy1 and Andchy2 69.8%). Anachy1, Anachy2, Andchy1 and Andchy2 form a separate group that ranges between 60.1 and 72.8% in identity with the A. gambiae chymotrypsins.

detect any similar sequences, especially in Drosophila melanogaster and An. gambiae genomes.

3.4. Blood meal-induced expression of chymotrypsin genes Anachy1 and Anachy2

Most studies regarding brazilian anophelines have investigated the genetic structure of natural populations of An. darlingi in Brazil, determining the amount of intra and interpopulational mtDNA sequence divergence and correlating these findings with morphological data. The objective was to compare this information with results from previous genetic and behavioral studies to obtain a clear understanding of the taxonomic status of An. darlingi in Brazil (Freitas-Sibajev et al., 1995). We identified four serine protease genes, Anachy1 and Anachy2 (in An. aquasalis) and Andchy1 and Andchy2 (in An. darlingi). The high similarity to other chymotrypsins of related organisms and the perfect conservation of amino acid residues that are critical for function argue that these genes encode chymotrypsins. Genomic cloning and sequencing revealed that these genes contain two short introns located in conserved positions and clustered in tandem. The pattern of the expression of chymotrypsin genes in An. aquasalis (Anachy1 and Anachy2) indicated that both genes are induced by blood feeding and are only detectable after 24 h post feeding. By 44 h, there were no detectable levels of Anachy1 or Anachy2, indicating that the transcripts were eliminated at the conclusion of digestion. Vizioli et al. (2001) showed in A. gambiae that Anchym1 and Anchym2 are blood meal induced, reaching a peak at 30 h after blood meal. This is the first study with Brazilian malaria vector serine proteases. One of our major objectives was to initiate a functional characterization of the pattern of expression of serine proteases in a Brazilian anopheline. The identification of inducible chymotrypsin genes, Anachy1 and Anachy2, represents a crucial step towards understanding the blood meal digestion process in Brazilian anophelines. Moreover, the remarkable divergence among Brazilian anopheline chymotrypsin promoters and other inducible promoters make the regulatory regions of Anachy1, Anachy2, Andchy1 and Andchy2 good candidates to design experiments to test the expression of a transgenic protein that hinders the development of pathogenic agents within the gut. Alignments of upstream regions of the four sequences revealed areas with high similarity (Fig. 6). This segment is interesting in Anachy1 and Andchy1 because it contains palindromic sequences. Similar sequences were previously found in an An. gambiae trypsin gene cluster, where the mapping of that site revealed high specificity of a GATA transcriptor factor (Giannoni et al., 2001).

Chymotrypsin gene expression was analysed by Northern blot analysis using total RNA extracted from female An. aquasalis mosquitoes killed at several time points after blood feeding. The analysis was performed for each chymotrypsin gene, using highly specific oligonucleotides pAq1 and pAq2, which hybridize to Anachy1 and Anachy2, respectively. As controls, larvae, adult males and unfed adult females were also analysed. No chymotrypsin RNA was detected in larvae, adult males and unfed adult females. In adult females, chymotrypsin RNA could only be detected approximately 24 h after the blood meal. The chymotrypsin transcript was not detected 44 h post blood meal (Fig. 5). Alignments of upstream regions of the four sequences, ranging from the ATG to nucleotide –89 (in the case of Andchy1), ⫺130 (Anachy1), ⫺138 (Andchy2) and –139 (Anachy2), revealed areas with high similarity (Fig. 6). The first segment with extensive similarities encompassed the entire 5⬘-untranslated region of all the genes (23/30 identities between Anachy1 and Andchy1; 15/20 identities between Anachy2 and Andchy2). Database searches using the fragment from upstream regions of Anachy1, Andchy1, Anachy2 and Andchy2 failed to

Fig. 5. Induction of Anachy1 and Anachy2 mRNA in An. aquasalis following blood meal. Total RNA of An.aquasalis larvae (L), male (M), unfed female mosquitoes (N), female mosquitoes killed immediatelly after blood meal (0) and female mosquitoes killed at 7, 24 and 44 h after blood meal was hybridized with specific oligonucleotides for Anachy2 (A) and Anachy1 (B). RNA size is indicated.

4. Discussion

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Fig. 6. Comparison of the sequences upstream of transcrition start sites (arrowheads) of Anachy1, Andchy1, Anachy2 and Andchy2. The sequences were aligned using the BESTFIT programme of the GCG package. Bold characters indicate upstream conserved segments. AUG translation start codons are underlined, horizontal arrows indicate palindromic sequences and the TATA boxes are doubled-underlined. Italic characters indicate transcribed sequences.

It is interesting to note that the order of the genomic organization of the two genes is Anachy2-Anachy1 in An. aquasalis, whereas it is Andchy1-Andchy2 in An. darlingi. In contrast to An. aquasalis and A. gambiae, the chymotrypsin genomic locus in An. darlingi had a short intergenic region (approximately 2 kb) that accompanied the inverted position of the genes. This short intergenic region was confirmed in An. darlingi by amplification with eLONGase, BRL, and DNA sequencing (data not shown). These data suggest a genomic rearrangement in which the order of the genes was changed while retaining the head-to-tail orientation (and identical transcription direction), characteristic of most genes duplicated by unequal crossover. High frequencies of inversion heterozygotes were observed in An. darlingi from Manaus and the surrounding municipalities (Kreutzer et al., 1972). Little is known about the molecular mechanisms underlying the generation of these inversions. In situ hybridization studies detected the transposon hobo around the breakpoints of four endemic inversions of D. melanogaster (Lyttle and Haymer, 1992). Cloning of inversion breakpoints in An. gambiae detected a transposable element (TE) at the inversion junction (Mathiopoulos et al., 1998). Chymotrypsin loci from An. aquasalis and An. darlingi has not

been sequenced. However, analysing the chymotrypsin locus from A. gambiae, we found a short segment of Pegasus, a small terminal inverted repeat transposable element first reported in the white gene of Anopheles gambiae (Besansky et al., 1996), located into the intergenic region between Anchym1 and Anchym2 and not reported by Vizioli et al., 2001 (Fig. 7). If present in the genomic locus of chymotrypsin in Brazilian anophelines, Pegasus may be associated with inversion mechanisms in this species. Apart from the effects on the biology of their hosts, TEs are of interest for their potential as vehicles for introducing exogenous DNA into the germline or as means of driving genes of interest into natural populations. Phylogenetic analysis among several chymotrypsin amino acid sequences could help in the investigation regarding the event of chymotrypsin gene duplication and the species divergence. The fact that chymotrypsin 1 is less related to chymotrypsin 2 of the same species than to chymotrypsin 1 of the other species suggests that the gene duplication preceded the divergence of An. aquasalis and An. darlingi (Anachy1 is the ortholog of Andchy1 and Anachy2 is the ortholog of Andchy2). On the other hand, the An. stephensi and An. gambiae divergence occurred after the event of gene duplication.

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Fig. 7. Comparison of nucleotide sequences from Pegasus (A) and intergenic region of chymotrypsin genomic locus from Anopheles gambiae (b).

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