Intraspecific chromosomal polymorphism in the Anopheles gambiae complex as a factor affecting malaria transmission in the Kisumu area of Kenya.

The paracentric inversion polymorphisms of Anopheles gambiae and An. arabiensis populations in the Kisumu area of western Kenya were studied in relation to parameters of Plasmodium falciparum transmission. Anopheles gambiae (n = 1,387) was polymorphic for inversions b on chromosomal arm 2R and a on arm 2L, with frequencies of the inverted arrangements of 17% and 43%, respectively. Anopheles arabiensis (n = 484) was polymorphic for inversion b on chromosomal arm 2R and a on 3R, with frequencies of the inverted arrangements of 58% and 5%, respectively. Observed karyotypic frequencies did not deviate from Hardy-Weinberg equilibrium, indicating a condition of panmixia (i.e., random mating) for both species. The overall degree of intraspecific polymorphism was low, confirming findings from other zones of East Africa. No significant differences in inversion frequencies of either An. gambiae or An. arabiensis were observed, either between collecting sites or between similar sampling periods of consecutive years. At the same time, a stable, significant two-fold difference in Plasmodium infection rates was detected among An. gambiae carriers of different inversion karyotypes on chromosome 2. A significant non-uniform distribution of human- and bovid-fed specimens was also detected among the carriers of different 2Rb inversion karyotypes in indoor resting An. arabiensis. Relationships among inversion karyotypes of the two major malaria vectors in the An. gambiae complex and key factors affecting malaria transmission intensity emphasize that intraspecific variation could contribute significantly to the diversity and stability of malaria vectorial systems in Africa.     

A physical map for an Asian malaria mosquito, Anopheles stephensi

Physical mapping is a useful approach for studying genome organization and evolution as well as for genome sequence assembly. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to develop high-resolution physical maps. We report a 0.6-Mb-resolution physical map consisting of 422 DNA markers hybridized to 379 chromosomal sites of the Anopheles stephensi polytene chromosomes. This makes An. stephensi second only to Anopheles gambiae in density of a physical map among malaria mosquitoes. Three hundred sixty-three (363) probes hybridized to single chromosomal sites, whereas 59 clones yielded multiple signals. This physical map provided a suitable basis for comparative genomics, which was used for determining inversion breakpoints, duplications, and origin of novel genes across species.     

Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae

Eight novel families of miniature inverted repeat transposable elements (MITEs) were discovered in the African malaria mosquito, Anopheles gambiae, by using new software designed to rapidly identify MITE-like sequences based on their structural characteristics. Divergent subfamilies have been found in two families. Past mobility was demonstrated by evidence of MITE insertions that resulted in the duplication of specific TA, TAA, or 8-bp targets. Some of these MITEs share the same target duplications and similar terminal sequences with MITEs and other DNA transposons in human and other organisms. MITEs in A. gambiae range from 40 to 1340 copies per genome, much less abundant than MITEs in the yellow fever mosquito, Aedes aegypti. Statistical analyses suggest that most A. gambiae MITEs are in highly AT-rich regions, many of which are closely associated with each other. The analyses of these novel MITEs underscored interesting questions regarding their diversity, origin, evolution, and relationships to the host genomes. The discovery of diverse families of MITEs in A. gambiae has important practical implications in light of current efforts to control malaria by replacing vector mosquitoes with genetically modified refractory mosquitoes. Finally, the systematic approach to rapidly identify novel MITEs should have broad applications for the analysis of the ever-growing sequence databases of a wide range of organisms.     

Molecular karyotyping of the 2La inversion in Anopheles gambiae

The African malaria vector Anopheles gambiae is polymorphic for alternative arrangements on the left arm of chromosome 2 (2La and 2L+(a)) that are non-randomly distributed with respect to degree of aridity. Detailed studies on the ecological role of inversion 2La have been hindered by the technical demands of traditional karyotype analysis and by sex- and stage-specific limitations on the availability of polytene chromosomes favorable for analysis. Recent molecular characterization of both inversion breakpoints presented the opportunity to develop a polymerase chain reaction (PCR)-based method for karyotype analysis. Here we report the development of this molecular diagnostic assay and the results of extensive field validation. When tested on 765 An. gambiae specimens sampled across Africa, the molecular approach compared favorably with traditional cytologic methods, correctly scoring > 94% of these specimens. By providing ready access to the 2La karyotype, this tool lays groundwork for future studies of the ecological genomics of this medically important species.     

Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication.

The genome of herpes simplex virus 1 or 2 consists of two components, L and S, which invert relative to each other during infection. As a result, viral DNA consists of four equimolar populations of molecules differing solely in the relative orientations of the L and S components. Previous studies have shown that the a sequences, located in the same orientation at the genomic termini and in inverted orientation at the L-S junction, play a key role in the inversion of L and S components. In this report we describe a virus-dependent system designed to allow identification of the viral genes capable of acting in trans to invert DNA flanked by inverted copies of a sequences. In this system, cells are converted to the thymidine kinase-positive phenotype with a chimeric plasmid carrying the thymidine kinase gene flanked by inverted copies of the a sequence and linked to an origin of viral DNA replication derived from the S component. The DNA introduced into the cells is retained and propagated in its original sequence arrangement as head-to-tail concatemers. Infection of these cells with herpes simplex virus 1 or 2 results in as much as 100-fold amplification of the plasmid sequences and inversion of the DNA flanked by copies of the a sequence. In infected cells, the amplified resident DNA accumulates in head-to-tail concatemers and no rearrangement other than the inversions could be detected. These results suggest that the a sequence-dependent inversions required trans-acting viral gene products.     

A test of the chromosomal theory of ecotypic speciation in Anopheles gambiae

The role of chromosomal inversions in speciation has long been of interest to evolutionists. Recent quantitative modeling has stimulated reconsideration of previous conceptual models for chromosomal speciation. Anopheles gambiae, the most important vector of human malaria, carries abundant chromosomal inversion polymorphism nonrandomly associated with ecotypes that mate assortatively. Here, we consider the potential role of paracentric inversions in promoting speciation in A. gambiae via "ecotypification," a term that refers to differentiation arising from local adaptation. In particular, we focus on the Bamako form, an ecotype characterized by low inversion polymorphism and fixation of an inversion, 2Rj, that is very rare or absent in all other forms of A. gambiae. The Bamako form has a restricted distribution by the upper Niger River and its tributaries that is associated with a distinctive type of larval habitat, laterite rock pools, hypothesized to be its optimal breeding site. We first present computer simulations to investigate whether the population dynamics of A. gambiae are consistent with chromosomal speciation by ecotypification. The models are parameterized using field observations on the various forms of A. gambiae that exist in Mali, West Africa. We then report on the distribution of larvae of this species collected from rock pools and more characteristic breeding sites nearby. Both the simulations and field observations support the thesis that speciation by ecotypification is occurring, or has occurred, prompting consideration of Bamako as an independent species.     

Distribution and chromosomal characterization of the Anopheles gambiae complex in Angola

Mosquitoes of the Anopheles gambiae complex (N = 1,336) were sampled (2001-2005) across Angola to identify taxa, study inversion polymorphisms, and detect the circumsporozoite protein of Plasmodium falciparum. Anopheles gambiae s.s. was found in all sites; it was characterized as M-form in localities of the tropical dry and semi-desertic belts, whereas the S-form was predominant in comparatively more humid and less anthropized sites. Both forms were characterized by low degrees of chromosomal polymorphism based solely on the 2La inversion, a pattern usually associated with An. gambiae populations from forested, humid, and derived savanna areas. Unexpectedly, this pattern was also observed in M-form populations collected in dry/pre-desertic areas, where this form largely predominates over An. arabiensis, which was also detected in central/inland sites. Anopheles melas was found in northern coastal sites. Three of 534 An. gambiae s.s. were positive for P. falciparum CS-protein, whereas none of the 105 An. melas were positive.     

Low-resolution genome map of the malaria mosquito Anopheles gambiae.

We have microdissected divisions of the Anopheles gambiae polytene chromosomes, digested the DNAs with a restriction enzyme, and PCR-amplified the DNA fragments to generate a set of pooled probes, each corresponding to approximately 2% of the mosquito genome. These divisional probes were shown to have high complexity. Except for those derived from near the centromeres, they hybridize specifically with their chromosomal sites of origin. Thus, they can be used to map cloned DNAs by a dot blot procedure, which is much more convenient than in situ hybridization to polytene chromosomes. We discuss additional potential uses of these probes, such as easier isolation of molecular markers and genes, including those that cross-hybridize with clones available from other insects. It is expected that the probes will substantially accelerate molecular genetic analysis of this most important malaria vector.     

When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F(ST), R(ST), (delta mu)(2), and D. Further, we have introduced new parameters, D' and D(K), which have well defined statistical significance tests and complement the standard parameters to advantage. D' is a modification of D, whereas D(K) is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.     

Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principal malaria vectors.

The six Afrotropical species of mosquitoes comprising the Anopheles gambiae complex include the most efficient vectors of malaria in the world as well as a nonvector species. The accepted interpretation of evolutionary relationships among these species is based on chromosomal inversions and suggests that the two principal vectors, A. gambiae and Anopheles arabiensis, are on distant branches of the phylogenetic tree. However, DNA sequence data indicate that these two species are sister taxa and suggest gene flow between them. These results have important implications for malaria control strategies involving the replacement of vector with nonvector populations.     

Birth and death of genes linked to chromosomal inversion

The birth and death of genes is central to adaptive evolution, yet the underlying genome dynamics remain elusive. The availability of closely related complete genome sequences helps to follow changes in gene contents and clarify their relationship to overall genome organization. Helicobacter pylori, bacteria in our stomach, are known for their extreme genome plasticity through mutation and recombination and will make a good target for such an analysis. In comparing their complete genome sequences, we found that gain and loss of genes (loci) for outer membrane proteins, which mediate host interaction, occurred at breakpoints of chromosomal inversions. Sequence comparison there revealed a unique mechanism of DNA duplication: DNA duplication associated with inversion. In this process, a DNA segment at one chromosomal locus is copied and inserted, in an inverted orientation, into a distant locus on the same chromosome, while the entire region between these two loci is also inverted. Recognition of this and three more inversion modes, which occur through reciprocal recombination between long or short sequence similarity or adjacent to a mobile element, allowed reconstruction of synteny evolution through inversion events in this species. These results will guide the interpretation of extensive DNA sequencing results for understanding long- and short-term genome evolution in various organisms and in cancer cells.     

Distribution and chromosomal polymorphism of the malaria vector Anopheles dirus species D

Preliminary results indicate that species D of the Anopheles dirus complex is widespread west of the Thai-Burma border in Burma and Bangladesh. A chromosomal study of An. dirus species D in these areas has revealed that this malaria vector is highly polymorphic for chromosomal rearrangements in salivary gland polytene chromosomes. The data from the limited number of wild-caught samples suggest that different geographically isolated populations may occur with respect to the frequency of inversions 2La, 3Ra and 3La. The distribution of chromosomal polymorphisms may be associated with the geography and epidemiology of human malaria in this region.     

Long-range mapping of the Philadelphia chromosome by pulsed-field gel electrophoresis

The Philadelphia chromosome (Ph1) of chronic myelogenous leukemia (CML) contains sequences from chromosome 9, including the ABL protooncogene, that have been translocated to the breakpoint cluster region (bcr) of chromosome 22, giving rise to a bcr-ABL fusion gene, whose product has been implicated in the genesis of CML. Although chromosome 22 translocation breakpoints in CML virtually always occur within the 5.8- kilobase (kb) bcr, chromosome 9 breakpoints have been identified within the known limits of ABL in only a few instances. For a better understanding of the variability of the breakpoints on chromosome 9, we studied the CML cell line BV173. Using pulsed-field gel electrophoresis (PFGE), large-scale maps of the t(9;22) junctions were constructed. The chromosome 9 breakpoint was shown to have occurred within an ABL intron, 160 kb upstream of the v-abl homologous sequences, but still 35 kb downstream of the 5'-most ABL exon. bcr-ABL and ABL-bcr fusion genes were demonstrated on the Ph1 and the 9q+ chromosomes, respectively; both of these genes are expressed. These results suggest that the 9;22 translocation breakpoints in CML consistently occur within the limits of the large ABL gene. RNA splicing, sometimes of very large regions, appears to compensate for the variability in breakpoint location. These studies show that PFGE is a powerful new tool for the analysis of chromosomal translocations in human malignancies.     

Differences in the organization and methylation patterns of integrated avian sarcoma proviral DNA sequences in nonpermissive and permissive mammalian cells

Four types of avian sarcoma virus (ASV)-transformed mammalian cells were analyzed for the presence of ASV-specific sequences in their genome DNA. A great variability in the number of proviral copies and their structure within the DNA of these lines was observed. In all cells tested gag and src sequences were present in a flexible arrangement. The greatest variation was detected within proviral sequences corresponding to pol and env regions. The number of integrated ASV proviral copies do not correlate with the capability of these cells to produce viral particles. Virus-producing (K2S and K12) and virogenic (XC) cells contain in their chromosomal DNA at least one complete proviral genome, whereas proviral sequences in helper-dependent nonvirogenic cells are substantially changed. Provirus expression level does not correlate with the number of integrated virus copies. In the non-virus-producing cells the proviral sequences are hypermethylated.     

Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation

Attempts to reconstruct the phylogenetic history of the Anopheles gambiae cryptic species complex have yielded strongly conflicting results. In particular, An. gambiae, the primary African malaria vector, is variously placed as a sister taxon to either Anopheles arabiensis or Anopheles merus. The recent divergence times for members of this complex complicate phylogenetic analysis, making it difficult to unambiguously implicate interspecific gene flow, versus retained ancestral polymorphism, as the source of conflict. Using sequences at four unlinked loci, which were determined from multiple specimens within each of five species in the complex, we found contrasting patterns of sequence divergence between the X chromosome and the autosomes. The isolation model of speciation assumes a lack of gene flow between species since their separation. This model could not be rejected for An. gambiae and An. arabiensis, although the data fit the model poorly. On the other hand, evidence from gene trees supports genetic introgression of chromosome 2 inversions between An. gambiae and An. arabiensis, and also points to more broad scale genetic exchange of autosomal sequences between this species pair. That such exchange has been relatively recent is suggested not only by the lack of fixed differences at three autosomal loci but also by the sharing of full haplotypes at two of the three loci, which is in contrast to several fixed differences and considerably deeper divergence on the X. The proposed acquisition by An. gambiae of sequences from the more arid-adapted An. arabiensis may have contributed to the spread and ecological dominance of this malaria vector.     

A new marker, black, a useful recombination suppressor, In(2)2, and a balanced lethal for chromosome 2 of the mosquito Anopheles gambiae.

A new marker for the second chromosome of Anopheles gambiae, black, was isolated from progeny of 60Co-irradiated mosquitoes. The black mutation increases melanization of larval setae and portions of the cuticle that are heavily sclerotized such as the saddle and head capsule. Adults have a sooty color that almost completely eliminates white banding on wings, tarsi, and palps. Fertility and general vigor of black individuals is reduced relative to wild-type; however, this does not prevent routine use for genetic crossing. The black marker was mapped to an interval on chromosome 2 between collarless and Dieldrin resistance 22 centiMorgans (cM) from collarless and 39 cM from Dieldrin resistance. We also isolated from 60Co-irradiated mosquitoes a pericentric inversion, In(2)2, that was marked with dominant alleles of the independently assorting genes collarless and Dieldrin resistance. This inversion is in coupling with the pericentric inversion 2Rd and covers approximately two-thirds of chromosome 2 from divisions 9 to 22. While inbreeding In(2)2 heterozygotes, we isolated a stock in which the inversion was in repulsion to a chromosome marked with c b DlS and an unidentified recessive lethal. This arrangement produced a useful and stable chromosome 2 balancer system that has remained intact for 26 generations without selection. These genetic tools will reduce the effort requires to isolate, among other things, the genetic factors affecting malaria parasite interactions with the mosquito host.     

Silencing of a gene adjacent to the breakpoint of a widespread Drosophila inversion by a transposon-induced antisense RNA

Adaptive changes in nature occur by a variety of mechanisms, and Drosophila chromosomal inversions was one of the first studied examples. However, the precise genetic causes of the adaptive value of inversions remain uncertain. Here we investigate the impact of the widespread inversion 2j of Drosophila buzzatii on the expression of the CG13617 gene, whose coding region is located only 12 bp away from the inversion proximal breakpoint. This gene is transcribed into a 2.3-kb mRNA present in all D. buzzatii developmental stages. More importantly, the expression level of CG13617 is reduced 5-fold in embryos of lines homozygous for the 2j inversion compared with lines without the inversion. An antisense RNA that originates in the Foldback-like transposon Kepler inserted at the breakpoint junction in all of the 2j lines and that forms duplexes with the CG13617 mRNA in 2j embryos is most likely responsible for the near silencing of the gene. Few examples of RNA interference caused by transposable elements (TEs) have been previously described, but this mechanism might be prevalent in many organisms and illustrates the potential of TEs as a major source of genetic variation. In addition, because chromosomal rearrangements are usually induced by TEs, position effects might be more common than previously recognized and contribute significantly to the evolutionary success of inversions.