Journal of Heredity 2004:95(2):103-113
© 2004 The American Genetic Association
Comparative Genome Analysis of the Yellow Fever Mosquito Aedes aegypti with Drosophila melanogaster and the Malaria Vector Mosquito Anopheles gambiae
From the Center for Tropical Disease Research and Training, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 (Severson, deBruyn, Lovin, and Morlais); and from the Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523 (Brown and Knudson). This work was supported by the grants AI33127 and AI34337 from the National Institutes of Health.
Address correspondence to D. W. Severson at the above address, or e-mail: David.W.Severson.1{at}nd.edu.
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An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.
The genomics era offers tremendous opportunities to improve our understanding of gene structure, function, and location, and to allow rapid dissections of the interactions among genes that produce a variety of complex phenotypes. As the availability of genome information for individual species grows, comparative genomics also will provide valuable insights into chromosomal and organismal evolution (Andersson et al. 1996; Nadeau and Sankoff 1998). Of particular interest are comparative relationships among orthologous genes and their associated regulatory elements across diverse taxa. Synteny refers to genes that reside on the same chromosome. Conserved synteny indicates that homologous genes are syntenic between species, regardless of gene order, while conserved linkage indicates conservation of both synteny and linear orders of genes between species (Ehrlich et al. 1997). The more closely related two species are, the greater the expected size of chromosome segments containing conserved linkages. For example, exploitation of conserved linkages between human and mouse is proving invaluable for understanding genetic disorders in humans via their mouse orthologs (Boyd et al. 2000; McPeek 2000; Nadeau 1989).
Several members of the mosquito family, Culicidae, have been or likely will be targeted for complete genome analyses, because of their importance as disease vectors. These include Anopheles gambiae, Aedes aegypti, and Culex pipiens, the primary vectors for malaria, yellow fever and dengue, and lymphatic filariasis transmission to humans, respectively. Indeed, the complete genome sequence was recently reported for A. gambiae (Holt et al. 2002), and an A. aegypti genome project has been initiated (Knudson et al. 2002). The Culicidae is divided into three subfamilies, Anophelinae, Culicinae, and Toxorhynchitinae, with all disease vectors found within the Anophelinae and Culicinae. A. gambiae and A. aegypti are representatives of the Anophelinae and Culicinae, respectively. Because the Culicidae contains about 3,500 species (Knight and Stone 1977), only a restricted group will become subjects for complete genome analysis. Using a comparative genomics approach, one may be able, however, to compare genome information from these species, with draft-level genome efforts for others, to determine gene locations and functions in other mosquito species of interest, rapidly and economically.
Until recently, only limited evidence for synteny conservation between the higher dipterans, such as Drosophila melanogaster, and the lower dipterans, like mosquitoes, has been reported (Fulton et al. 2001; Matthews and Munstermann 1994; Weller and Foster 1993). The availability of the complete sequence for the euchromatic regions of the D. melanogaster genome (Adams et al. 2000) and the A. gambiae genome (Holt et al. 2002) provides gene-order scaffolds that can be used for comparisons with other insects, particularly other dipterans, including other mosquitoes. The comparative analysis of D. melanogaster and A. gambiae identified considerable genome-wide synteny conservation, but only limited evidence for gene-order conservation (Bolshakov et al. 2002; Zdobnov et al. 2002). In this study, an in silico comparative genomics approach was used to identify orthologs to genetically mapped and sequenced A. aegypti genes (Severson et al. 2002) in the D. melanogaster and A. gambiae genome databases. We identified chromosome segments of long-range, or macrosynteny, conservation between these species, and some evidence for local or microsynteny conservation within chromosome segments, but limited evidence for gene-order conservation.
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Molecular Markers
Marker data and assembly of the A. aegypti composite linkage map are described elsewhere (Severson et al. 2002). Most of these markers are cDNA clones for known genes from A. aegypti or D. melanogaster, or random expressed sequence tags (ESTs) from A. aegypti. Three established criteria (Lalley et al. 1987) were used to identify candidate cDNAs as orthologs between A. aegypti, D. melanogaster, and A. gambiae: (1) orthologous gene designations based on high sequence similarities; (2) use of cloned genes from A. aegypti that were isolated with degenerate primers to D. melanogaster gene sequences or with use of D. melanogaster clones as heterologous low-stringency probes to screen A. aegypti libraries; or by (3) use of D. melanogaster cDNA clones directly as probes to A. aegypti Southern blots under high-stringency hybridizations in restriction fragment length polymorphism (RFLP) mapping efforts.
Orthology and Synteny Determination
All A. aegypti cDNA sequences were initially submitted to the BLASTX program (Altschul et al. 1997) for homology searches against the D. melanogaster database and are accessible in the GenBank database (Severson et al. 2002). Searches were performed with use of the PAM30 substitution matrix, with the gap penalty set to the maximum (10). The PAM30 matrix was selected because it is based on a simple evolutionary model and should identify sequences with a high degree of similarity (Altschul 1991). A. aegypti sequences were arbitrarily considered to be orthologous to D. melanogaster sequences when the expected number (E) was less than e–16. A. aegypti sequences that could unambiguously be assigned to chromosome position in D. melanogaster were then submitted to the TBLASTN program for homology searches against the A. gambiae database, with use of the default parameters. A. aegypti sequences were arbitrarily considered to be orthologous to A. gambiae sequences when the expected number (E) was less than e–9. The D. melanogaster database was screened first because of the extensive gene annotation available compared to A. gambiae. This also allowed for identification of the correct reading frame for the A. aegypti sequence and subsequent amino acid translation for TBLASTN searches. Two A. aegypti cDNA sequences with no orthologs in D. melanogaster were included in the A. gambiae comparison, because they represent the only markers located near the end of the p arm of chromosome 2 in A. aegypti (Severson et al. 2002).
Evidence for the nonrandom distribution of D. melanogaster orthologs across the A. aegypti genome was tested with the binomial test (http://home.clara.net/sisa/binomial.htm). This test is based on the exact probability distribution of a single dichotomy and is particularly applicable to small sample sizes (Sokal and Rohlf 1981). The A. aegypti cDNAs were assessed with regard to their general orthologous chromosome locations in D. melanogaster (e.g., X, 2L, 2R, 3L, 3R, 4), and the observed frequencies were calculated for each location. Centromere positions on the A. aegypti chromosomes were estimated according to integrated genetic and physical map data (Brown et al. 2001), which allowed us to define 5 chromosome regions for comparison with D. melanogaster, including chromosome 1, the p arm and q arm of chromosome 2, and the p arm and q arm of chromosome 3. Note that A. aegypti (and other Culicinae) do not exhibit sex chromosome dimorphism, but instead sex determination is controlled by a single autosomal locus on chromosome 1 (Gilchrist and Haldane 1947), with relatively equal recombination rates in both males and females (Severson et al. 1993). The expected number of orthologs for each A. aegypti chromosome region was calculated with use of the observed frequencies of D. melanogaster orthologs and the total number of genes that mapped to each chromosome region in A. aegypti. We tested the null hypothesis that the genes within the designated D. melanogaster chromosome locations are randomly distributed across the A. aegypti genome.
Microsynteny Determination
An A. aegypti region on chromosome 1 (FLpter 
25%, where FLpter represents the fractional length from the operationally defined p terminus) targeted in an ongoing contig construction effort and, where 134 cosmid or BAC end sequences were available, was compared to the A. gambiae predicted proteins by BLASTX, with use of the same parameters as described above. The A. gambiae predicted proteins that were used and their chromosome location came from the Ensembl build version 10.2.1 of the A. gambiae whole genomic sequence (www.ensembl.org).
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Synteny Conservation Between A. aegypti and D. melanogaster
We performed BLASTX analysis on 121 genetically mapped (Severson et al. 2002) A. aegypti genes to identify those with D. melanogaster orthologs. The mapped genes also included those from the direct screening (as RFLP markers) of three D. melanogaster cDNAs in A. aegypti segregating populations; these were included as identified orthologs. With a highly conservative substitution matrix and BLAST analysis conditions, a total of 87 genes (72%) with D. melanogaster orthologs were identified. A small number of these genes (14) had multiple possible orthologs and represent members of multigene families. For these, individual family members are distributed across different chromosomes or chromosome arms in D. melanogaster and could not unambiguously be evaluated for synteny conservation with A. aegypti. Unambiguous chromosome positions in D. melanogaster could be assigned for 73 genes (Table 1).
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Comparative chromosome positions of the D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosome regions, suggesting that some ancestral chromosome elements have been conserved (Table 2). However, it is also very clear that the two genomes reflect extensive reshuffling within and between chromosome regions. A. aegypti chromosome 1 carries a greater than expected number of orthologs from the D. melanogaster X chromosome (P =.0216) and a slight but nonsignificant excess of orthologs from D. melanogaster 3R (P =.0543). The comparative ortholog positions for chromosomes 2 and 3 in both organisms are consistent with whole-arm translocations during chromosome evolution. With A. aegypti chromosome 2, 2p contains a significant excess of orthologs to D. melanogaster 3L (P =.0021), and 2q contains a significant excess of orthologs to D. melanogaster 2L (P =.00005). Conversely, chromosome 3p contains a significant excess of orthologs to D. melanogaster 3R (P =.0139), and 3q contains a significant excess of orthologs to D. melanogaster 2R (P =.0477). Only four orthologs to the dot chromosome 4 in D. melanogaster were identified, providing little power for statistical comparisons.
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Synteny Conservation Between A. aegypti and A. gambiae
We performed TBLASTN analysis of the A. gambiae genome database with the 73 A. aegypti genes that we were able to unambiguously assign to chromosome positions in D. melanogaster, and with two genes (AEGI8 and LF115) that have no D. melanogaster homolog and represent the only genetic markers for the end of A. aegypti 2p. The 75 A. aegypti genes identified 78 orthologs in A. gambiae (Table 1). We used TBLASTN searches because gene annotation for A. gambiae was based largely on in silico gene-finding programs and preliminary BLASTX searches identified likely errors in annotation of some putative genes, usually use of the wrong reading frame. We also used a less stringent cutoff for the A. gambiae TBLASTN searches than that used for BLASTX D. melanogaster searches, because observed sequence similarities with TBLASTN would be impacted in some genes by the presence of introns and BLASTX matches with the conservative PAM30 matrix will give lower E values than TBLASTN with the default BLOSUM matrix for matches to the same sequence. Three A. aegypti genes (LF90, LF178, and VCP) each had essentially equally high scoring sequence identities to duplicate and physically independent genes in A. gambiae. It seems probable that these genes are also represented as duplications in the A. aegypti genome; our efforts to develop physical contigs of BAC clones around the LF178 locus have confirmed the existence of two gene copies (Brown SE, deBruyn BS, Severson DW and Knudson DL, unpublished data). The physical distance between copies has not been determined (also see results for the microsynteny investigations), but our results from multiple genetic mapping experiments (Severson et al. 2002) indicate very low levels of recombination between them (less than 1 cM).
Comparative chromosome positions of the A. gambiae orthologs indicate unequivocally that the five A. aegypti chromosome regions share extensive homology with the five A. gambiae chromosome arms (Figure 1). The conserved synteny pattern is consistent with whole- or near-whole-arm homologies that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within chromosome segments. A. aegypti chromosome 1 contains all orthologs identified on the A. gambiae X chromosome and about half the orthologs identified on the 2R arm. Most of the orthologs to the A. gambiae X are located toward the telomere end of the p arm of A. aegypti chromosome 1. A. aegypti chromosomes 2 and 3 reflect a reciprocal translocation with A. gambiae chromosomes 2 and 3. A. aegypti 2p and 2q correspond to A. gambiae 2L and 3R, respectively. Conversely, A. aegypti 3p and 3q correspond to segments of A. gambiae 2R and the entire 3L arm, respectively. The A. aegypti chromosomes 1 and 3p conserved syntenies with the A. gambiae X and 2R suggest break and fusion events in their ancestral chromosome lineages. This phenomenon is supported by chromosome locations of the putative duplicate loci, LF90 and LF178. Both map genetically to single loci on A. aegypti chromosome 1, but each has homologues on the A. gambiae X and 2R. As discussed above, we have verified that LF178 represents a duplication in A. aegypti. We note that the third putative duplicate gene, VCP, maps genetically to a single locus on A. aegypti chromosome 2 and to two loci on A. gambiae 2L. Whole-arm or near-whole-arm correspondence between conserved synteny segments was contradicted by only two of the genes we examined, LF101 (Ae:1 versus Ag:3R ortholog positions) and para (Ae:3q versus Ag:2L ortholog positions), which is consistent with transpositions. Although caution is warranted regarding the exact centromere position on A. aegypti chromosome 2, the comparative orientations reflected in gene-order conservation around the centromere region suggest that a pericentric inversion occurred in the A. aegypti and A. gambiae chromosome lineages (also see the discussion section).
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Synteny conservation is also consistently greater in A. aegypti–A. gambiae comparisons than in comparisons of either mosquito species with D. melanogaster (Table 3). In addition, these results indicate that, though extensive chromosome rearrangements are evident among all three species, A. aegypti and A. gambiae have largely maintained whole-arm gene associations, whereas comparisons of either with D. melanogaster indicate considerable interarm exchanges.
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Microsynteny Comparison Between A. aegypti and A. gambiae
We performed BLASTX analysis of the A. gambiae proteins with 134 A. aegypti cosmid or BAC end sequences produced during a contig building effort for a region on chromosome 1. A single cosmid (LSBC29.1) was isolated as containing the LF178 gene sequence and used initially as an anchor point for contig construction. As previously indicated, we have determined that there are two tightly linked copies of this gene in A. aegypti and two copies (located on X and 2R) in A. gambiae. For the current analysis, we included sequences from cosmid and BAC clones that are within that contig or physically map near the LSBC29.1 cosmid (Brown SE and Knudson DL, unpublished data). Fourteen A. aegypti end sequences yielded significant BLASTX results, with four representing unique, unambiguous locations on the A. gambiae genome (Table 4). Comparative genome positions of these sequences indicated that they remain microsyntenic in A. gambiae. The LSBC29.1 clone is about 30 kb and contains one copy of LF178 and a wapI ortholog; if we assume the corresponding LF178 ortholog is on the X in A. gambiae, these genes are about 4 Mb apart. The LSBC29.1 clone overlaps partially with the K6.157E6 clone that also is about 30 kb; LSBC29.1 and the Yippee ortholog are, therefore, less than 60 kb apart in A. aegypti and about 11.1 kb in A. gambiae. Two orthologs within clones that physically map near LSBC29.1 also reflect microsynteny conservation in A. gambiae; the K6.2C1 clone contains a CG3822 ortholog that is about 364.5 kb from the wapI ortholog, and the K20.3A10 clone contains a Aats-ser ortholog that is about 631.4 kb from the LF178 ortholog on the X.
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Discussion |
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We combined genetic linkage data and gene and EST sequence data for the mosquito A. aegypti with the public domain D. melanogaster and A. gambiae genome databases to perform an in silico genome comparison of A. aegypti to the other well-described dipterans. We initially submitted 121 A. aegypti sequences for BLASTX comparison with the D. melanogaster whole-genome database because of the extensive gene annotation available. Significant matches to putative D. melanogaster orthologs were observed with 87 (72%) of these sequences, indicating a high level of gene peptide sequence conservation between the two species. Fourteen of these sequences were excluded from comparative genome analyses because they represented multigene families that could not unambiguously be assigned to specific loci in Drosophila. All 73 genes with clear orthologs in D. melanogaster also had significant TBLASTN matches to A. gambiae orthologs. Because these 73 orthologs are distributed across each of the four D. melanogaster and the three A. gambiae chromosomes, they provided an adequate basis for a comparative genome analysis with A. aegypti.
Extent of Synteny Conservation Between A. aegypti and D. melanogaster
Genome-wide, conserved syntenies between A. aegypti and D. melanogaster are clearly evident yet less extensive than those observed in, for example, mammals (Ehrlich et al. 1997; Wiltshire et al. 1999) or even among more distant vertebrates (Barbazuk et al. 2000). For example, 80% of putative orthologous genes belong to conserved synteny groups in the human and zebrafish genomes, despite a divergence time of ca. 420 million (Mya) ago (Barbazuk et al. 2000). We observed significant departures from an expected random distribution of the individual genes. That is, for each of the A. aegypti chromosomes, we observed a greater than expected number of orthologs to genes from particular chromosome regions in D. melanogaster. A. aegypti chromosome 1 shows clear homology to the D. melanogaster X and some evidence for homology to 3R. In general, chromosomes 2 and 3 for the two species suggest whole-arm translocation events in the ancestral chromosome lineages. The A. aegypti 2p and 2q arms have significant homology with D. melanogaster 3L and 2L, respectively. Conversely, the A. aegypti 3p and 3q arms have significant homology with D. melanogaster 3R and 2R, respectively. We note that D. melanogaster 3R has homology with both A. aegypti chromosome 1 and the 3p arm, suggesting a possible break and fusion event in the ancestral chromosome lineages. Only four A. aegypti homologues to D. melanogaster chromosome 4 (the dot chromosome) were observed; these genes are distributed on chromosomes 1 and 3. Overall, our A. aegypti and D. melanogaster comparisons indicate that some orthologs show conserved syntenies that have retained ancestral evolutionary associations, yet these genome segments reflect the effects of considerable intra- and interchromosomal reorganization.
It seems likely that as more A. aegypti genes become available for comparison the individual A. aegypti chromosomes will at best reflect a mosaic of very short homology segments to D. melanogaster chromosomes or individual chromosome arms, as was observed with D. melanogaster–A. gambiae comparisons (Zdobnov et al. 2002). Further, it seems likely that A. aegypti–D. melanogaster homology segments represent the results of uniform random genome rearrangement events that are independent of functional or mechanistic constraints, given that linkage associations across Drosophila species are apparently not based on functional interactions between chromosomal segments (Hilliker and Trusis-Coulter 1987).
From an evolutionary viewpoint, the observation of limited synteny conservation between A. aegypti and D. melanogaster is not unexpected. That is, the main dipteran lineages were evident in the fossil record during the Upper Triassic (Rohdendorf 1974), and, therefore, the two lineages diverged about 250 million years ago. Further and perhaps most significantly, the two most distant lineages within the genus Drosophila, which diverged 40 to 60 million years ago (Russo et al., 1995), exhibit synteny conservation corresponding to individual chromosome arms but extensive interspecific gene-order reshuffling within arms (Ranz et al. 2001). Indeed, the Drosophila genome shows one of the highest rates of chromosome evolution observed for any eukaryote, and as such only tightly linked genes in D. melanogaster would be expected to reflect linkage conservation in nondrosophilid insects (Ranz et al. 2001).
Extent of Synteny Conservation Between A. aegypti and A. gambiae
Our results demonstrate that the A. aegypti and A. gambiae genomes share extensive syntenic associations that correspond largely with a whole chromosome-arm conservation pattern that is consistent with the occurrence of paracentric inversions in the ancestral chromosome lineages. Whole-arm or near-whole-arm correspondence between conserved syntenies was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. Whole-arm homology is clearly represented by Ae:2p with Ag:2L, Ae:2q with Ag:3R, and Ae:3q with Ag:3L. A likely chromosome arm break and fusion event is suggested by the Ag:2R homology with Ae:3p and Ae:1, and by Ag:X with Ae:1. Further, the A. gambiae gene homologies to the general centromere position on Ae:2 suggest a likely pericentric inversion in the chromosome lineages. This interpretation is consistent with our previous comparative mapping data for several culicine mosquito species (Anderson et al. 2001), and particularly with respect to A. aegypti comparisons with Armigeres subalbatus (Ferdig et al. 1998); the linear order of genes mapped across the centromere region in A. subalbatus chromosome 2 is inverted relative to the that of their orthologs in A. aegypti chromosome 2, whereas linear orders are conserved between the two species for genes distal to the respective centromere regions. Armigeres is considered phylogenetically basal to Aedes, and the Anophelinae are basal to the Culicinae (Ross 1951).
Despite the apparent extensive conservation of large syntenic chromosome segments between A. aegypti and A. gambiae, we observed only limited evidence for linear order conservation within these genome segments. In most instances, orthologs to putative A. gambiae genes physically located within the same scaffold accession are clearly not conserved in the same discrete chromosome segments in A. aegypti. Obviously, our results largely represent a broad-scale, or macrosynteny, survey, and additional efforts are needed to adequately address the issue of fine-scale, or microsynteny, and linear gene orders. Although our data set for microsynteny analysis is limited, we observed that A. aegypti genes identified within or very near a contig construction effort on chromosome 1 are represented by orthologs that generally remain within a relatively small genome region on the X in A. gambiae. The same gene associations were not evident in A. aegypti and D. melanogaster comparisons, suggesting that microsynteny conservation is likely to be greater with A. gambiae. Of note, conservation of gene order recently has not been found evident in genome comparisons of A. gambiae and Anopheles funestus, despite an estimated divergence time between them of only 5 million years (Sharakhov et al. 2002). Gene-order conservation between the Anophelinae and Culicinae is, therefore, unlikely to be extensive even at the micro-scale, given a probable divergence time of at least 95 million years (Krzywinski et al. 2001).
Genome Size, Genetic Length, and Evolution in Mosquitoes
A. aegypti and A. gambiae, as representatives of the Culicinae and Anophelinae, reflect interesting dichotomies in mosquito chromosome evolution. For example, genome size varies up to eightfold among mosquito species with strong delineation between the subfamilies (Knudson et al. 1996; Rai and Black 1999). The Anophelinae have smaller genome sizes; for example, the 278 Mb A. gambiae genome is only about 1.6 times larger than the 170 Mb D. melanogaster genome (Holt et al. 2002). Genome size in the Culicinae is typically much larger, with A. aegypti at 813 Mb (Warren and Crampton 1991) or about 2.9 and 4.8 times the A. gambiae and D. melanogaster genome sizes, respectively. Despite such differences in genome size, the basic chromosome number (2n = 6) has remained constant within the Culicidae (the only known exception is the primitive anopheline Chagasia bathana, with 2n = 8), with the differences being attributed to an increase in repetitive DNA among the Culicinae (Rai and Black 1999).
Although the basic chromosome complement is highly conserved, genome evolution in mosquitoes reflects considerable chromosome reorganization due to inversions and translocations (Matthews and Munsterman 1994). Still, however, whole-arm or near-whole-arm conservation seems to be the rule. Our A. aegypti and A. gambiae comparisons clearly demonstrate that, despite extensive rearrangement largely reflecting numerous inversions, chromosome arm syntenies between the two species remain remarkably intact. However, the Anophelinae appear to have followed the Drosophila paradigm, in that genome evolution in both groups is orders of magnitude higher than that reported for any other eukaryote (Ranz et al. 2001; Sharakhov et al. 2002). In contrast, the Culicinae reflect extensive synteny and evidence for broad-scale linear order conservation (Matthews and Munstermann 1994). Indeed, comparative analyses using cDNA-based genetic markers for A. aegypti (Severson et al. 1993, 2002) indicate that they not only have high sequence identities with other mosquito species (Severson et al. 1994), but also provide direct evidence that large-scale inter- and intrachromosome rearrangements may not disrupt gene colinearity within relatively large conserved chromosome segments across a diverse group of culicines, including Aedes albopictus (Severson et al. 1995), Ochlerotatus triseriatus (formerly Aedes triseriatus) (Anderson et al. 2001), A. subalbatus (Ferdig et al. 1998), C. pipiens (Mori et al. 1999), and Culex tritaeniorhynchus (Mori et al. 2001).
The basis for the extreme differences in evolutionary rates between drosophilids/anophelines and culicines is unknown, though three factors have been suggested for the high rate in drosophilids: the short developmental time, a greater mutation rate, and a less detrimental effect on fertility of inversions (Ranz et al. 2001). Although developmental times are similar among the three groups, the other factors are likely applicable, though the exact mechanisms remain to be determined. Of note, there are interesting sex-specific differences in chiasmata formation between the three groups. Crossing over is suppressed in male but not in female drosophilids, but it occurs in relatively equal frequencies in autosomes of males and females of most mosquito species. However, the Anophelinae are characterized by typical X and Y sex chromosomes that reflect limited crossovers in males, whereas the Culicinae show no sex chromosome dimorphism and in general crossovers occur at about equal frequencies in males and females with the sex-determining chromosome (Clements 1992). Curiously, however, crossing over appears to be much reduced in the Culicinae: despite a considerably larger genome size, the genetic map size for A. aegypti is only 205 cM (Severson et al. 2002), compared to 215 cM for A. gambiae (Zheng et al. 1996) and 290 cM for D. melanogaster (O'Brien 1993).
Conclusions
We have performed a broad-scale, genome-wide analysis of synteny conservation of the yellow fever mosquito, A. aegypti, with D. melanogaster and the primary malaria vector, A. gambiae. An in silico comparative genomics approach allowed us to identify orthologs to genetically mapped A. aegypti genes. Although linkage estimates of gene order in A. aegypti could reflect some positional errors, it seems likely that our overall conclusions for residual whole-arm synteny conservation with D. melanogaster and whole-arm synteny conservation but limited linear order conservation with A. gambiae are appropriate. Indeed, accurate determinations of patterns of chromosome rearrangements in mammals have been produced with quite limited genetic linkage data sets (Ehrlich et al. 1997). Further, our interpretations of whole-arm synteny conservation of A. aegypti with both D. melanogaster and A. gambiae are completely concordant with previously reported comparisons of A. gambiae to D. melanogaster (Bolshakov et al. 2002; Zdobnov et al. 2002). Another potential for error in our comparative analysis is the misclassification of paralogous genes as orthologs. However, given our stringent criteria for sequence comparisons and the exclusion of genes representing obvious gene families, most of the identified orthologous pairs are likely valid.
The D. melanogaster and A. gambiae genome databases, though clearly offering tremendous potential for comparative gene identity and function, seem to offer limited value for comparative positional gene determinations among even closely related dipterans. That is, to provide useful positional information, reasonably sized genome segments must reflect not only synteny conservation, but also conservation of linear gene orders. The apparent rapid rate of chromosome rearrangement observed in Drosophila, combined with the drosophilid-mosquito divergence time, suggests that even genome segments on the order of 50 kb have a less than.5 likelihood of linear gene order conservation (extrapolated from Ranz et al. 2001, figure 3). Further, the pattern of rapid chromosome rearrangement seems to be an even more striking feature of anopheline chromosome evolution (Sharakhov et al. 2002). These results and other factors, including differences in genome organization (Knudson et al. 2002), indicate that it remains paramount that genome sequencing projects be conducted for other dipteran species, including representative culicine mosquito species such as A. aegypti.
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Corresponding Editor: Stephen Schaeffer
Received July 9, 2003
Accepted December 15, 2003
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