Genome-wide QTL mapping of saltwater tolerance in sibling species of Anopheles (malaria vector) mosquitoes.

Although freshwater (FW) is the ancestral habitat for larval mosquitoes, multiple species independently evolved the ability to survive in saltwater (SW). Here, we use quantitative trait locus (QTL) mapping to investigate the genetic architecture of osmoregulation in Anopheles mosquitoes, vectors of human malaria. We analyzed 1134 backcross progeny from a cross between the obligate FW species An. coluzzii, and its closely related euryhaline sibling species An. merus. Tests of 2387 markers with Bayesian interval mapping and machine learning (random forests) yielded six genomic regions associated with SW tolerance. Overlap in QTL regions from both approaches enhances confidence in QTL identification. Evidence exists for synergistic as well as disruptive epistasis among loci. Intriguingly, one QTL region containing ion transporters spans the 2Rop chromosomal inversion that distinguishes these species. Rather than a simple trait controlled by one or a few loci, our data are most consistent with a complex, polygenic mode of inheritance.

The white gene of Ceratitis capitata: a phenotypic marker for germline transformation.

Reliable germline transformation is required for molecular studies and ultimately for genetic control of economically important insects, such as the Mediterranean fruit fly (medfly) Ceratitis capitata. A prerequisite for the establishment and maintenance of transformant lines is selectable or phenotypically dominant markers. To this end, a complementary DNA clone derived from the medfly white gene was isolated, which showed substantial similarity to white genes in Drosophila melanogaster and other Diptera. It is correlated with a spontaneous mutation causing white eyes in the medfly and can be used to restore partial eye color in transgenic Drosophila carrying a null mutation in the endogenous white gene.

A retrotransposable element from the mosquito Anopheles gambiae .

A family of middle repetitive elements from the African malaria vector Anopheles gambiae is described. Approximately 100 copies of the element, designated T1Ag, are dispersed in the genome. Full-length elements are 4.6 kilobase pairs in length, but truncation of the 5' end is common. Nucleotide sequences of one full-length, two 5'-truncated, and two 5' ends of T1Ag elements were determined and aligned to define a consensus sequence. Sequence analysis revealed two long, overlapping open reading frames followed by a polyadenylation signal, AATAAA, and a tail consisting of tandem repetitions of the motif TGAAA. No direct or inverted long terminal repeats (LTRs) were detected. The first open reading frame, 442 amino acids in length, includes a domain resembling that of nucleic acid-binding proteins. The second open reading frame, 975 amino acids long, resembles the reverse transcriptases of a category of retrotransposable elements without LTRs, variously termed class II retrotransposons, class III elements or non-LTR retrotransposons. Similarity at the sequence and structural levels places T1Ag in this category.

Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae.

Two distinct site-specific retrotransposon families, named RT1 and RT2, from the sibling mosquito species Anopheles gambiae and A. arabiensis, respectively, were previously identified. Both were shown to occupy identical nucleotide positions in the 28S rRNA gene and to be flanked by identical 17-bp target site duplications. Full-length representatives of each have been isolated from a single species, A. gambiae, and the nucleotide sequences have been analyzed. Beyond insertion specificity, RT1 and RT2 share several structural and sequence features which show them to be members of the LINE-like, or non-long-terminal-repeat retrotransposon, class of reverse transcriptase-encoding mobile elements. These features include two long overlapping open reading frames (ORFs), poly(A) tails, the absence of long terminal repeats, and heterogeneous 5' truncation of most copies. The first ORF of both elements, particularly ORF1 of RT1, is glutamine rich and contains long tracts of polyglutamine reminiscent of the opa repeat. Near the carboxy ends, three cysteine-histidine motifs occur in ORF1 and one occurs in ORF2. In addition, each ORF2 contains a region of sequence similarity to reverse transcriptases and integrases. Alignments of the protein sequences from RT1 and RT2 reveal 36% identity over the length of ORF1 and 60% identity over the length of ORF2, but the elements cannot be aligned in the 5' and 3' noncoding regions. Unlike that of RT2, the 5' noncoding region of RT1 contains 3.5 copies of a 500-bp subrepeat, followed by a poly(T) tract and two imperfect 55-bp subrepeats, the second spanning the beginning of ORF1. The pattern of distribution of these elements among five siblings species in the A. gambiae complex is nonuniform. RT1 is present in laboratory and wild A. gambiae, A. arabiensis, and A. melas but has not been detected in A. quadriannulatus or A. merus. RT2 has been detected in all available members of the A. gambiae complex except A. merus. Copy number fluctuates, even among the offspring of individual wild female A. gambiae mosquitoes. These findings reflect a complex evolutionary history balancing gain and loss of copies against the coexistence of two elements competing for a conserved target site in the same species for perhaps millions of years.

Patterns of mitochondrial variation within and between African malaria vectors, Anopheles gambiae and An. arabiensis, suggest extensive gene flow.

Anopheles gambiae and An. arabiensis are mosquito species responsible for most malaria transmission in sub-Saharan Africa. They are also closely related sibling species that share chromosomal and molecular polymorphisms as a consequence of incomplete lineage sorting or introgressive hybridization. To help resolve these processes, this study examined the partitioning of mtDNA sequence variation within and between species across Africa, from both population genetic and phylogeographic perspectives. Based on partial gene sequences from the cytochrome b, ND1 and ND5 genes, haplotype diversity was high but sequences were very closely related. Within species, little or no population subdivision was detected, and there was no evidence for isolation by distance. Between species, there were no fixed nucleotide differences, a high proportion of shared polymorphisms, and eight haplotypes in common over distances as great as 6000 km. Only one of 16 shared polymorphisms led to an amino acid difference, and there was no compelling evidence for nonneutral variation. Parsimony networks constructed of haplotypes from both species revealed no correspondence of haplotype with either geography or taxonomy. This trend of low intraspecific genetic divergence is consistent with evidence from allozyme and microsatellite data and is interpreted in terms of both extensive gene flow and recent range expansion from relatively large, stable populations. We argue that retention of ancestral polymorphisms is a plausible but insufficient explanation for low interspecific genetic divergence, and that extensive hybridization is a contributing factor.

An Anopheles gambiae cDNA predicts a protein similar to a yeast Suil translation factor.

The nucleotide (nt) sequence of a cDNA cloned from the mosquito Anopheles gambiae was determined. The amino acid (aa) sequence of the deduced protein was 56% identical (60/108 aa) to the recently discovered translation initiation factor Suil of yeast, suggesting that the two proteins are homologs and have similar functions. Database searches also revealed strong similarity to other sequences, including the deduced gene products of cDNAs from organisms as diverse as nematodes, humans and plants. The functions of these putative proteins are unknown, but their homology to Suil suggests that they represent an important component of the eukaryotic translation initiation complex.

Unintegrated two-long terminal repeat circular human T lymphotropic virus DNA accumulation during chronic HTLV infection.

Accumulation of unintegrated human T lymphotropic virus (HTLV) DNA was analyzed in long-term T cell lines infected with HTLV type I (HTLV-I) or type II (HTLV-II). By using a polymerase chain reaction-based assay, amplified products of expected size were obtained in all of the HTLV-I-infected (n = 7) and HTLV-II-infected (n = 8) cell lines. The signal intensities of the hybridizing band varied greatly among the cell lines and did not correlate with HTLV p24gag antigen production. Further analysis of HTLV-I-infected clones demonstrated considerable variability in the unintegrated DNA accumulation, suggesting that either the epigenetic status of the host cell or some environmental factor determines the occurrence of unintegrated DNA. The presence of lower levels of unintegrated DNA in most of the HTLV-infected, long-term cell lines presumably results in persistent noncytopathic infection.

The Tryptophan oxygenase gene of Anopheles gambiae.

The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized. Unlike Drosophila, where it is X-linked, the A. gambiae gene maps to chromosome 2R, subdivision 12E, by in situ hybridization to the polytene chromosomes. Of the six introns present, four are positioned identically to those of the Drosophila homolog, one is similarly positioned, and one is novel. A 1 955 nt cDNA potentially encodes a 392 amino acid protein of an estimated 45 kDa. Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila, and 53% identity between Anopheles and nematode or mammalian proteins. Northern analysis detected a developmentally regulated transcript about 2 kb in length. Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation, one whose expression, unlike that of white, is not cell autonomous.

The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion.

An Anopheles gambiae cDNA encoding tryptophan oxygenase was placed under the control of the constitutive baculovirus promoter, ie-1. The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color. The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects. The functionality of An. gambiae tryptophan oxygenase in a higher fly fulfils predictions based on its structural conservation throughout millions of years of independent evolution.

A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.

A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.

Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae) DNA.

The renaturation kinetics of nuclear DNA from the G3 colony of Anopheles gambiae Giles was studied to estimate the genome size and to determine the proportion of repeated sequences. An. gambiae has a haploid DNA content of 0.27 picograms or 2.6 x 10(8) basepairs. Analysis of reassociation kinetics indicated that the genome is composed of 61% single-copy and 33% repetitive sequences, with 6% foldback sequences.

Complexities in the analysis of cryptic taxa within the genus Anopheles.

Grassi's discovery one hundred years ago brought to light the puzzle of anophelism without malaria in Europe. With the discovery of the European Anopheles maculipennis complex the puzzle was solved but the 'species problem' has not gone away. Meaningful epidemiologic studies and effective vector control programs depend upon efficient methods for discriminating among the major vectors, lesser vectors and non-vectors of ubiquitous anopheline sibling species complexes. We now have a variety of techniques for identifying cryptic species, ranging from crossing studies through morphological, cytogenetic, allozyme and repetitive DNA-based strategies. However, cytogenetic and molecular data can also be used to infer evolutionary relationships among cryptic taxa. This approach has been crucial to understanding the biology of the vector, and may illuminate the speciation process and the human impact upon this process. Nevertheless, the analysis of cryptic taxa has proven unexpectedly complex. Studies of An. funestus and An. gambiae reveal conflicts among classes of markers and between different genomic locations. The data are consistent with a model of speciation in which gene flow may still occur in parts of the genome, and they suggest that caution should be exercised in the interpretation of results from small numbers of loci, only one type of marker, and markers located in specific genomic regions such as chromosomal inversions.

SNP genotyping defines complex gene-flow boundaries among African malaria vector mosquitoes.

Mosquitoes in the Anopheles gambiae complex show rapid ecological and behavioral diversification, traits that promote malaria transmission and complicate vector control efforts. A high-density, genome-wide mosquito SNP-genotyping array allowed mapping of genomic differentiation between populations and species that exhibit varying levels of reproductive isolation. Regions near centromeres or within polymorphic inversions exhibited the greatest genetic divergence, but divergence was also observed elsewhere in the genomes. Signals of natural selection within populations were overrepresented among genomic regions that are differentiated between populations, implying that differentiation is often driven by population-specific selective events. Complex genomic differentiation among speciating vector mosquito populations implies that tools for genome-wide monitoring of population structure will prove useful for the advancement of malaria eradication.

Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences.

The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Genetic exchange in 2La inversion heterokaryotypes of Anopheles gambiae.

In the malaria vector Anopheles gambiae, alternative arrangements of chromosome 2 (2La and 2L+(a)) vary in relative frequency along clines of aridity, suggesting the action of natural selection on targets within the inversion. Our long term goal of detecting such targets depends in part on the level of genetic exchange between arrangements. Accordingly, we estimated recombination rates on 2L from the backcross progeny of 2La/+(a) heterokaryotypes and as a control, from 2L+(a) homokaryotypes. In homokaryotypes, the recombination rate was uniform at ~2.0 centimorgans per megabase (cM/Mb). In heterokaryotypes, recombination within the rearranged region was reduced to < 0.5 cM/Mb, with slightly higher but nevertheless reduced levels (< 1.0 cM/Mb) flanking the rearrangement. Yet, gene exchange was recorded between nearly all markers, including those very near the distal inversion breakpoint. These results suggest that reduced recombination is a necessary but not sufficient mechanism for genetic isolation between alternative arrangements, and that the targets of natural selection can be identified against the different chromosomal backgrounds.

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus.

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.

SINE insertion polymorphism on the X chromosome differentiates Anopheles gambiae molecular forms.

Polymorphic SINE insertions can be useful markers for assessing population structure and differentiation. Maque is a family of SINE elements which, based on bioinformatic analysis, was suggested to have been active recently in Anopheles gambiae, the major vector of malaria. Here, we report the development of polymorphic Maque insertions as population genetic markers in A. gambiae, and the use of these markers to better characterize divergence on the X chromosome between A. gambiae M and S molecular forms in populations from Burkina Faso and Mali. Our data are consistent with the recent activity of Maque. Phylogenetic analysis suggests that at least two recently active lineages may have a role in mediating genome evolution. We found differences in element insertion frequency and sequence between the M and S populations analysed. Significant differentiation was observed between these two groups across a 6 Mb region at the proximal (centromeric) end of the X chromosome. Locus-specific F(ST) values ranged from 0.14 to 1.00 in this region, yet were not significantly different from zero in more distal locations on the X chromosome; the trend was consistent in populations from both geographical locales suggesting that differentiation is not due to local adaptation. Strong differentiation between M and S at the proximal end of the X chromosome, but not outside this region, suggests the action of selection counteracting limited gene flow between these taxa and supports their characterization as incipient species.

Molecular differentiation between chromosomally defined incipient species of Anopheles funestus.

Anopheles funestus Giles is one of the most important vectors of malaria in sub-Saharan Africa. The population structure of this mosquito in Burkina Faso, West Africa based on chromosomal inversion data led to the description of two chromosomal forms, Kiribina and Folonzo. Because both forms co-occur in the same locales yet differ significantly, both in the frequency of inverted arrangements on chromosome arms 3R and 2R and in vectorial capacity, they were hypothesized to be emerging species with at least partial barriers to gene flow. This hypothesis would be strengthened by molecular evidence of differentiation between Kiribina and Folonzo at loci outside chromosomal inversions. We surveyed molecular variation in sympatric populations of the two forms using sequences from the mitochondrial ND5 gene and genotypes at sixteen microsatellite loci distributed across the genome. Both classes of marker revealed slight but significant differentiation between the two forms (mtDNA F(ST) = 0.023, P < 0.001; microsatellite F(ST) = 0.004, P < 0.001; R(st) = 0.009, P = 0.002). Locus-by-locus analysis of the microsatellite data showed that significant differentiation was not genome-wide, but could be attributed to five loci on chromosome 3R (F(ST) = 0.010, P < 0.001; R(st) = 0.016, P = 0.002). Importantly, three of these loci are outside of, and in linkage equilibrium with, chromosomal inversions, suggesting that differentiation between chromosomal forms extends beyond the inversions themselves. The slight overall degree of differentiation indicated by both marker classes is likely an underestimate because of recent population expansion inferred for both Folonzo and Kiribina. The molecular evidence from this study is consistent with the hypothesis of incipient speciation between Kiribina and Folonzo.

Rangewide population genetic structure of the African malaria vector Anopheles funestus.

Anopheles funestus is a primary vector of malaria in Africa south of the Sahara. We assessed its rangewide population genetic structure based on samples from 11 countries, using 10 physically mapped microsatellite loci, two per autosome arm and the X (N = 548), and 834 bp of the mitochondrial ND5 gene (N = 470). On the basis of microsatellite allele frequencies, we found three subdivisions: eastern (coastal Tanzania, Malawi, Mozambique and Madagascar), western (Burkina Faso, Mali, Nigeria and western Kenya), and central (Gabon, coastal Angola). A. funestus from the southwest of Uganda had affinities to all three subdivisions. Mitochondrial DNA (mtDNA) corroborated this structure, although mtDNA gene trees showed less resolution. The eastern subdivision had significantly lower diversity, similar to the pattern found in the codistributed malaria vector Anopheles gambiae. This suggests that both species have responded to common geographic and/or climatic constraints. The western division showed signatures of population expansion encompassing Kenya west of the Rift Valley through Burkina Faso and Mali. This pattern also bears similarity to A. gambiae, and may reflect a common response to expanding human populations following the development of agriculture. Due to the presumed recent population expansion, the correlation between genetic and geographic distance was weak. Mitochondrial DNA revealed further cryptic subdivision in A. funestus, not detected in the nuclear genome. Mozambique and Madagascar samples contained two mtDNA lineages, designated clade I and clade II, that were separated by two fixed differences and an average of 2% divergence, which implies that they have evolved independently for approximately 1 million years. Clade I was found in all 11 locations, whereas clade II was sampled only on Madagascar and Mozambique. We suggest that the latter clade may represent mtDNA capture by A. funestus, resulting from historical gene flow either among previously isolated and divergent populations or with a related species.