Cuticular differences associated with aridity acclimation in African malaria vectors carrying alternative arrangements of inversion 2La.

BACKGROUND: Principal malaria vectors in Africa, An. gambiae and An. coluzzii, share an inversion polymorphism on the left arm of chromosome 2 (2La/2L+a) that is distributed non-randomly in the environment. Genomic sequencing studies support the role of strong natural selection in maintaining steep clines in 2La inversion frequency along environmental gradients of aridity, and physiological studies have directly implicated 2La in heat and desiccation tolerance, but the precise genetic basis and the underlying behavioral and physiological mechanisms remain unknown. As the insect cuticle is the primary barrier to water loss, differences in cuticle thickness and/or epicuticular waterproofing associated with alternative 2La arrangements might help explain differences in desiccation resistance. METHODS: To test that hypothesis, two subcolonies of both An. gambiae and An. coluzzii were established that were fixed for alternative 2La arrangements (2La or 2L+a) on an otherwise homosequential and shared genetic background. Adult mosquitoes reared under controlled environmental conditions (benign or arid) for eight days post-eclosion were collected and analyzed. Measurements of cuticle thickness were made based on scanning electron microscopy, and cuticular hydrocarbon (CHC) composition was evaluated by gas chromatography-mass spectrometry. RESULTS: After removing the allometric effects of body weight, differences in mean cuticle thickness were found between alternative 2La karyotypes, but not between alternative environments. Moreover, the thicker cuticle of the An. coluzzii 2La karyotype was contrary to the known higher rate of water loss of this karyotype relative to 2L+a. On the other hand, quantitative differences in individual CHCs and overall CHC profiles between alternative karyotypes and environmental conditions were consistent with expectation based on previous physiological studies. CONCLUSIONS: Our results suggest that alternative arrangements of the 2La inversion are associated with differences in cuticle thickness and CHC composition, but that only CHC composition appears to be relevant for desiccation resistance. Differences in the CHC composition were consistent with previous findings of a lower rate of water loss for the 2L+a karyotype at eight days post-eclosion, suggesting that CHC composition is an important strategy for maintaining water balance in this genetic background, but not for 2La. Despite a higher rate of water loss at eight days, higher body water content of the 2La karyotype confers a level of desiccation resistance equivalent to that of the 2L+a karyotype.

Patterns of genomic differentiation between ecologically differentiated M and S forms of Anopheles gambiae in West and Central Africa.

Anopheles gambiae M and S are thought to be undergoing ecological speciation by adapting to different larval habitats. Toward an improved understanding of the genetic determinants and evolutionary processes shaping their divergence, we used a 400,000 single-nucleotide polymorphism (SNP) genotyping array to characterize patterns of genomic differentiation between four geographically paired M and S population samples from West and Central Africa. In keeping with recent studies based on more limited genomic or geographic sampling, divergence was not confined to a few isolated "speciation islands." Divergence was both widespread across the genome and heterogeneous. Moreover, we find consistent patterns of genomic divergence across sampling sites and mutually exclusive clustering of M and S populations using genetic distances based on all 400,000 SNPs, implying that M and S are evolving collectively across the study area. Nevertheless, the clustering of local M and S populations using genetic distances based on SNPs from genomic regions of low differentiation is consistent with recent gene flow and introgression. To account for these data and reconcile apparent paradoxes in reported patterns of M-S genomic divergence and hybridization, we propose that extrinsic ecologically based postmating barriers vary in strength as environmental conditions fluctuate or change.

Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion.

BACKGROUND: Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies. METHODS: Sequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon. RESULTS: The breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations. CONCLUSIONS: The complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.

Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology.

BACKGROUND: Phylogenetic analyses provide a framework for examining the evolution of morphological and molecular diversity, interpreting patterns in biogeography, and achieving a stable classification. The generic and suprageneric relationships within mosquitoes (Diptera: Culicidae) are poorly resolved, making these subjects difficult to address. RESULTS: We carried out maximum parsimony and maximum likelihood, including Bayesian, analyses on a data set consisting of six nuclear genes and 80 morphological characters to assess their ability to resolve relationships among 25 genera. We also estimated divergence times based on sequence data and fossil calibration points, using Bayesian relaxed clock methods. Strong support was recovered for the basal position and monophyly of the subfamily Anophelinae and the tribes Aedini and Sabethini of subfamily Culicinae. Divergence times for major culicid lineages date to the early Cretaceous. CONCLUSIONS: Deeper relationships within the family remain poorly resolved, suggesting the need for additional taxonomic sampling. Our results support the notion of rapid radiations early in the diversification of mosquitoes.