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1.  Divergent transcriptional response to thermal stress by Anopheles gambiae larvae carrying alternative arrangements of inversion 2La 
Molecular ecology  2011;20(12):2567-2580.
The African malaria mosquito Anopheles gambiae is polymorphic for chromosomal inversion 2La, whose frequency strongly correlates with degree of aridity across environmental gradients. Recent physiological studies have associated 2La with resistance to desiccation in adults and thermal stress in larvae, consistent with its proposed role in aridity tolerance. However, the genetic basis of these traits remains unknown. To identify genes that could be involved in the differential response to thermal stress, we compared global gene expression profiles of heat hardened 2La or 2L+a larvae at three time points, for up to eight hours following exposure to the heat stress. Treatment and control time series, replicated four times, revealed a common and massive induction of a core set of heat shock genes regardless of 2La orientation. However, clear differences between the 2La and 2L+a arrangements emerged at the earliest (0.25 h) time point, in the intensity and nature of the stress response. Overall, 2La was associated with the more aggressive response: larger numbers of genes were heat responsive and up-regulated. Transcriptionally induced genes were enriched for functions related to ubiquitin-proteasomal degradation, chaperoning, and energy metabolism. The more muted transcriptional response of 2L+a was largely repressive, including genes involved in proteolysis and energy metabolism. These results may help explain the maintenance of the 2La inversion polymorphism in An. gambiae, as the survival benefits offered by high thermal sensitivity in harsh climates could be offset by the metabolic costs of such a drastic response in more equable climates.
doi:10.1111/j.1365-294X.2011.05114.x
PMCID: PMC3686099  PMID: 21535279
Anopheles gambiae; chromosomal inversion; heat hardening; malaria vector; microarray; thermal stress; transcriptional profiling
2.  Behavioural divergence of sympatric Anopheles funestus populations in Burkina Faso 
Malaria Journal  2014;13:65.
Background
In Burkina Faso, two chromosomal forms of the malaria vector Anopheles funestus, Folonzo and Kiribina, are distinguished by contrasting frequencies of shared polymorphic chromosomal inversions. Sympatric and synchronous populations of Folonzo and Kiribina mate assortatively, as indicated by a significant deficit of heterokaryotypes, and genetic associations among inversions on independently segregating chromosome arms. The present study aimed to assess, by intensive longitudinal sampling, whether sympatric Folonzo and Kiribina populations are characterized by behavioural differences in key malaria vectorial parameters.
Methods
The study was conducted in two adjacent villages near Ouagadougou, in the dry savanna of central Burkina Faso. Mosquito adult resting behaviour of both forms was compared based on parallel indoor/outdoor collections across six breeding seasons; 8,235 fully karyotyped samples of half-gravid females were analysed in total. Additionally, indoor/outdoor human biting behaviour, host selection, and Plasmodium falciparum sporozoite rate was assessed and compared between chromosomal forms.
Results
The Kiribina form was numerically predominant in the area. However, the Folonzo form was significantly over-represented in indoor resting collections and showed stronger post-prandial endophily, while Kiribina predominated outdoors. Neither form was statistically distinguishable in human biting behaviour, and both were more likely to seek human blood meals indoors than outside. The human blood index and sporozoite rate were comparably high in both chromosomal forms in indoor collections (>89% and >8%, respectively).
Conclusions
Both Kiribina and Folonzo chromosomal forms are formidable malaria vectors in Burkina Faso. However, the significantly greater tendency for the Kiribina form to rest outdoors despite its pronounced anthropophily suggests that uniform exposure of the overall An. funestus population to indoor-based vector control tools cannot be expected; Kiribina is more likely to evade indoor interventions and escape unharmed outdoors, reducing the efficacy of malaria control. Accordingly, more efficient methods to detect Kiribina and Folonzo, and a more complete understanding of their distribution and behaviour in Africa are advocated.
doi:10.1186/1475-2875-13-65
PMCID: PMC3937823  PMID: 24559382
Anopheles funestus; Anthropophily; Behavioural divergence; Chromosomal forms; Exophily; Folonzo; Kiribina; Malaria vector; West Africa
3.  Mario Coluzzi (1938–2012) 
Malaria Journal  2014;13:10.
doi:10.1186/1475-2875-13-10
PMCID: PMC3914365  PMID: 24498979
4.  The Evolution of the Anopheles 16 Genomes Project 
G3: Genes|Genomes|Genetics  2013;3(7):1191-1194.
We report the imminent completion of a set of reference genome assemblies for 16 species of Anopheles mosquitoes. In addition to providing a generally useful resource for comparative genomic analyses, these genome sequences will greatly facilitate exploration of the capacity exhibited by some Anopheline mosquito species to serve as vectors for malaria parasites. A community analysis project will commence soon to perform a thorough comparative genomic investigation of these newly sequenced genomes. Completion of this project via the use of short next-generation sequence reads required innovation in both the bioinformatic and laboratory realms, and the resulting knowledge gained could prove useful for genome sequencing projects targeting other unconventional genomes.
doi:10.1534/g3.113.006247
PMCID: PMC3704246  PMID: 23708298
comparative; assembly; vector; malaria; collaboration
5.  Genetic association of physically unlinked islands of genomic divergence in incipient species of Anopheles gambiae 
Molecular ecology  2010;19(5):925-939.
Previous efforts to uncover the genetic underpinnings of ongoing ecological speciation of the M and S forms of the African malaria vector Anopheles gambiae revealed two centromere-proximal islands of genetic divergence on X and chromosome 2. Under the assumption of considerable ongoing gene flow between M and S, these persistently divergent genomic islands were widely considered to be “speciation islands”. In the course of microarray-based divergence mapping, we discovered a third centromere-associated island of divergence on chromosome 3, which was validated by targeted re-sequencing. To test for genetic association between the divergence islands on all three chromosomes, SNP-based assays were applied in four natural populations of M and S spanning West, Central and East Africa. Genotyping of 517 female M and S mosquitoes revealed nearly complete linkage disequilibrium between the centromeres of the three independently assorting chromosomes. These results suggest that despite the potential for inter-form gene flow through hybridization, actual (realized) gene flow between M and S may be substantially less than commonly assumed, and may not explain most shared variation. Moreover, the possibility of very low gene flow calls into question whether diverged pericentromeric regions-- characterized by reduced levels of variation and recombination-- are in fact instrumental rather than merely incidental to the speciation process.
doi:10.1111/j.1365-294X.2010.04531.x
PMCID: PMC3683534  PMID: 20149091
centromeres; divergent adaptation; ecological speciation; malaria vector; reproductive isolation; speciation islands
6.  Differential gene expression in incipient species of Anopheles gambiae 
Molecular ecology  2008;17(10):2491-2504.
A speciation process is ongoing in the primary vector of malaria in Africa, Anopheles gambiae. Assortatively mating incipient species known as the M and S forms differentially exploit larval breeding sites associated with different ecological settings. However, some ongoing gene flow between M and S limits significant genomic differentiation mainly to small centromere-proximal regions on chromosomes X and 2L, termed “speciation islands” with the expectation that they contain the genes responsible for reproductive isolation. As the speciation islands exhibit reduced recombination and low polymorphism, more detailed genetic analysis using fine-scale mapping is impractical. We measured global gene expression differences between M and S using oligonucleotide microarrays, with the goal of identifying candidate genes that could be involved in this ongoing speciation process. Gene expression profiles were examined in two independent colonies of both forms at each of three developmental periods of interest: fourth instar larvae, virgin females, and gravid females. Patterns were validated on a subset of genes using quantitative real-time reverse transcription PCR of RNA samples from laboratory colonies and wild mosquitoes collected from Cameroon and Burkina Faso. Considered across all three developmental periods, differentially expressed genes represented ~1-2% of all expressed genes. Although disproportionately represented in the X speciation island, the vast majority of genes were located outside any speciation island. Compared to samples from the other developmental periods, virgin females were characterized by more than twice as many differentially expressed genes, most notably those implicated in olfaction and potentially, mate recognition.
doi:10.1111/j.1365-294X.2008.03774.x
PMCID: PMC3670799  PMID: 18430144
7.  Genetic structure of Anopheles gambiae populations on islands in northwestern Lake Victoria, Uganda 
Malaria Journal  2005;4:59.
Background
Alternative means of malaria control are urgently needed. Evaluating the effectiveness of measures that involve genetic manipulation of vector populations will be facilitated by identifying small, genetically isolated vector populations. The study was designed to use variation in microsatellite markers to look at genetic structure across four Lake Victoria islands and two surrounding mainland populations and for evidence of any restriction to free gene flow.
Methods
Four Islands (from 20–50 km apart) and two surrounding mainland populations (96 km apart) were studied. Samples of indoor resting adult mosquitoes, collected over two consecutive years, were genotyped at microsatellite loci distributed broadly throughout the genome and analysed for genetic structure, effective migration (Nem) and effective population size (Ne).
Results
Ne estimates showed island populations to consist of smaller demes compared to the mainland ones. Most populations were significantly differentiated geographically, and from one year to the other. Average geographic pair-wise FST ranged from 0.014–0.105 and several pairs of populations had Ne m < 3. The loci showed broad heterogeneity at capturing or estimating population differences.
Conclusion
These island populations are significantly genetically differentiated. Differences reoccurred over the study period, between the two mainland populations and between each other. This appears to be the product of their separation by water, dynamics of small populations and local adaptation. With further characterisation these islands could become possible sites for applying measures evaluating effectiveness of control by genetic manipulation.
doi:10.1186/1475-2875-4-59
PMCID: PMC1327676  PMID: 16336684
8.  Physiological correlates of ecological divergence along an urbanization gradient: differential tolerance to ammonia among molecular forms of the malaria mosquito Anopheles gambiae 
BMC Ecology  2013;13:1.
Background
Limitations in the ability of organisms to tolerate environmental stressors affect their fundamental ecological niche and constrain their distribution to specific habitats. Evolution of tolerance, therefore, can engender ecological niche dynamics. Forest populations of the afro-tropical malaria mosquito Anopheles gambiae have been shown to adapt to historically unsuitable larval habitats polluted with decaying organic matter that are found in densely populated urban agglomerates of Cameroon. This process has resulted in niche expansion from rural to urban environments that is associated with cryptic speciation and ecological divergence of two evolutionarily significant units within this taxon, the molecular forms M and S, among which reproductive isolation is significant but still incomplete. Habitat segregation between the two forms results in a mosaic distribution of clinally parapatric patches, with the M form predominating in the centre of urban agglomerates and the S form in the surrounding rural localities. We hypothesized that development of tolerance to nitrogenous pollutants derived from the decomposition of organic matter, among which ammonia is the most toxic to aquatic organisms, may affect this pattern of distribution and process of niche expansion by the M form.
Results
Acute toxicity bioassays indicated that populations of the two molecular forms occurring at the extremes of an urbanization gradient in Yaounde, the capital of Cameroon, differed in their response to ammonia. The regression lines best describing the dose-mortality profile differed in the scale of the explanatory variable (ammonia concentration log-transformed for the S form and linear for the M form), and in slope (steeper for the S form and shallower for the M form). These features reflected differences in the frequency distribution of individual tolerance thresholds in the two populations as assessed by probit analysis, with the M form exhibiting a greater mean and variance compared to the S form.
Conclusions
In agreement with expectations based on the pattern of habitat partitioning and exposure to ammonia in larval habitats in Yaounde, the M form showed greater tolerance to ammonia compared to the S form. This trait may be part of the physiological machinery allowing forest populations of the M form to colonize polluted larval habitats, which is at the heart of its niche expansion in densely populated human settlements in Cameroon.
doi:10.1186/1472-6785-13-1
PMCID: PMC3548750  PMID: 23294940
Local adaptation; Fundamental ecological niche; Environmental stressor; Evolution of tolerance; Urbanization; Malaria; Mosquito
9.  Patterns of Genomic Differentiation between Ecologically Differentiated M and S Forms of Anopheles gambiae in West and Central Africa 
Genome Biology and Evolution  2012;4(12):1202-1212.
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.
doi:10.1093/gbe/evs095
PMCID: PMC3542583  PMID: 23132896
divergent selection; genome scan; introgression; population genomics; SNP genotyping; speciation islands
10.  Adaptation to Aridity in the Malaria Mosquito Anopheles gambiae: Chromosomal Inversion Polymorphism and Body Size Influence Resistance to Desiccation 
PLoS ONE  2012;7(4):e34841.
Chromosomal inversions are thought to confer a selective advantage in alternative habitats by protecting co-adapted alleles from recombination. The frequencies of two inversions (2La and 2Rb) of the afro-tropical malaria mosquito Anopheles gambiae change gradually along geographical clines, increasing in frequency with degree of aridity. Such clines can result from gene flow and local selection acting upon alternative karyotypes along the cline, suggesting that these inversions may be associated with tolerance to xeric conditions. Since water loss represents a major challenge in xeric habitats, it can be supposed that genes inside these inversions are involved in water homeostasis. To test this hypothesis, we compared the desiccation resistance of alternative karyotypes from a colonised 2Rb/2La polymorphic population of A. gambiae from Cameroon. The strain included only the molecular form S, one of the genetic units marking incipient speciation in this taxon. Day-old mosquitoes of both sexes were assayed individually for time to death in a dry environment and the karyotype of each was determined post-mortem using molecular diagnostic assays for each inversion. In agreement with expectations based on their eco-geographical distribution, we found that 2La homokaryotypes survived significantly longer (1.3 hours) than the other karyotypes. However, there was weak support for the effect of 2Rb on desiccation resistance. Larger mosquitoes survived longer than smaller ones. Median survival of females was greater than males, but the effect of sex on desiccation resistance was weakly supported, indicating that differential survival was correlated to differences between sexes in average size. We found weak evidence for a heterotic effect of 2La karyotype on size in females. These results support the notion that genes located inside the 2La inversion are involved in water balance, contributing towards local adaptation of A. gambiae to xeric habitats, beyond the adaptive value conferred by a larger body size.
doi:10.1371/journal.pone.0034841
PMCID: PMC3325948  PMID: 22514674
11.  Spatially Explicit Analyses of Anopheline Mosquitoes Indoor Resting Density: Implications for Malaria Control 
PLoS ONE  2012;7(2):e31843.
Background
The question of sampling and spatial aggregation of malaria vectors is central to vector control efforts and estimates of transmission. Spatial patterns of anopheline populations are complex because mosquitoes' habitats and behaviors are strongly heterogeneous. Analyses of spatially referenced counts provide a powerful approach to delineate complex distribution patterns, and contributions of these methods in the study and control of malaria vectors must be carefully evaluated.
Methodology/Principal Findings
We used correlograms, directional variograms, Local Indicators of Spatial Association (LISA) and the Spatial Analysis by Distance IndicEs (SADIE) to examine spatial patterns of Indoor Resting Densities (IRD) in two dominant malaria vectors sampled with a 5×5 km grid over a 2500 km2 area in the forest domain of Cameroon. SADIE analyses revealed that the distribution of Anopheles gambiae was different from regular or random, whereas there was no evidence of spatial pattern in Anopheles funestus (Ia = 1.644, Pa<0.05 and Ia = 1.464, Pa>0.05, respectively). Correlograms and variograms showed significant spatial autocorrelations at small distance lags, and indicated the presence of large clusters of similar values of abundance in An. gambiae while An. funestus was characterized by smaller clusters. The examination of spatial patterns at a finer spatial scale with SADIE and LISA identified several patches of higher than average IRD (hot spots) and clusters of lower than average IRD (cold spots) for the two species. Significant changes occurred in the overall spatial pattern, spatial trends and clusters when IRDs were aggregated at the house level rather than the locality level. All spatial analyses unveiled scale-dependent patterns that could not be identified by traditional aggregation indices.
Conclusions/Significance
Our study illustrates the importance of spatial analyses in unraveling the complex spatial patterns of malaria vectors, and highlights the potential contributions of these methods in malaria control.
doi:10.1371/journal.pone.0031843
PMCID: PMC3279417  PMID: 22348131
12.  No evidence for biased co-transmission of speciation islands in Anopheles gambiae 
Genome-scale scans have revealed highly heterogeneous levels of divergence between closely related taxa in many systems. Generally, a small number of regions show high differentiation, with the rest of the genome showing no or only low levels of divergence. These patterns have been interpreted as evidence for ongoing speciation-with-gene-flow, with introgression homogenizing the whole genome except loci involved in reproductive isolation. However, as the number of selected loci increases, the probability of introgression at unselected loci decreases unless there is a transmission ratio distortion causing an over-representation of specific combinations of alleles. Here we examine the transmission of three ‘speciation islands’ that contain fixed differences between the M and S forms of the mosquito, Anopheles gambiae. We made reciprocal crosses between M and S parents and genotyped over 2000 F2 individuals, developing a hierarchical likelihood model to identify specific genotypes that are under- or over-represented among the recombinant offspring. Though our overall results did not match the expected number of F2 genotypes, we found no biased co-transmission among M or S alleles in the three islands. Our likelihood model did identify transmission ratio distortion at two of the three islands, but this distortion was small (approx. 3%) and in opposite directions for the two islands. We discuss how our results impinge on hypotheses of current gene flow between M and S and ongoing speciation-with-gene-flow in this system.
doi:10.1098/rstb.2011.0188
PMCID: PMC3233708  PMID: 22201167
Anopheles gambiae; speciation; centromeric drive
13.  A behavioral mechanism underlying ecological divergence in the malaria mosquito Anopheles gambiae 
Behavioral Ecology  2010;21(5):1087-1092.
Disruptive selection mediated by predation on aquatic immature stages has been proposed as a major force driving ecological divergence and fostering speciation between the M and S molecular forms of the African malaria mosquito, Anopheles gambiae. In the dry savannahs of West Africa where both molecular forms co-occur, the S form thrives in temporary pools filled with rainwater, whereas the M form preferentially breeds in permanent freshwater habitats where predator pressure is higher. Here, we explored the proximal mechanisms by which predation may contribute to habitat segregation between molecular forms using progeny of female mosquitoes captured in Burkina Faso. We show that the S form suffers higher predation rates than the M form when simultaneously exposed to the widespread predator, Anisops jaczewskii in an experimental arena. Furthermore, behavioral plasticity induced by exposure to the predator was observed in the M form, but not in the S form, and may partially explain its habitat use and ecological divergence from the S form. We discuss the role of adaptive phenotypic plasticity in allowing successful colonization of a new ecological niche by the M form and highlight further research areas that need to be addressed for a better understanding of the ultimate mechanisms underlying ecological speciation in this pest of major medical importance.
doi:10.1093/beheco/arq114
PMCID: PMC2920295  PMID: 22476108
adaptation; Anopheles gambiae; behavior; habitat divergence; mosquito; notonectidae; phenotypic plasticity, predation; speciation
14.  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.
doi:10.1186/1471-2148-9-298
PMCID: PMC2805638  PMID: 20028549
15.  Seasonal distribution of Anopheles funestus chromosomal forms from Burkina Faso 
Malaria Journal  2009;8:239.
Background
Previous studies of Anopheles funestus chromosomal inversion polymorphisms in Burkina Faso showed large departures from Hardy-Weinberg equilibrium and linkage disequilibrium among inversions located on different chromosomes, implying the existence of two taxonomic units ("chromosomal forms") with limited genetic flow. One chromosomal form, named Folonzo, is highly polymorphic for alternative rearrangements of 3Ra, 3Rb, 2Ra, and 3La; the other, Kiribina, is predominantly characterized by the standard arrangement of these inversions. To investigate the temporal distribution of these chromosomal forms, further collections were carried out in two villages near Ouagadougou where they are found in sympatry.
Methods
Chromosomal karyotypes were determined from indoor-resting, half-gravid females sampled within and across six breeding seasons, from December 1998 to April 2007.
Results
As expected, the pattern of chromosomal polymorphism in An. funestus was consistent with assortatively mating Folonzo and Kiribina forms. When samples were assigned to each chromosomal form, their relative abundance varied within successive breeding seasons in a repeating pattern of temporal variability. Relative abundance of the Folonzo form was correlated with climatic variables related to temperature and rainfall.
Conclusion
The relative abundance of Folonzo and Kiribina forms of An. funestus likely reflects different larval ecologies that are linked to varying climatic conditions. Further analysis of the bionomics of these vectors is recommended in light of its relevance to vector control.
doi:10.1186/1475-2875-8-239
PMCID: PMC2775746  PMID: 19857258
16.  Inversion 2La is associated with enhanced desiccation resistance in Anopheles gambiae 
Malaria Journal  2009;8:215.
Background
Anopheles gambiae, the principal vector of malignant malaria in Africa, occupies a wide range of habitats. Environmental flexibility may be conferred by a number of chromosomal inversions non-randomly associated with aridity, including 2La. The purpose of this study was to determine the physiological mechanisms associated with the 2La inversion that may result in the preferential survival of its carriers in hygrically-stressful environments.
Methods
Two homokaryotypic populations of A. gambiae (inverted 2La and standard 2L+a) were created from a parental laboratory colony polymorphic for 2La and standard for all other known inversions. Desiccation resistance, water, energy and dry mass of adult females of both populations were compared at several ages and following acclimation to a more arid environment.
Results
Females carrying 2La were significantly more resistant to desiccation than 2L+a females at emergence and four days post-emergence, for different reasons. Teneral 2La females had lower rates of water loss than their 2L+a counterparts, while at four days, 2La females had higher initial water content. No differences in desiccation resistance were found at eight days, with or without acclimation. However, acclimation resulted in both populations significantly reducing their rates of water loss and increasing their desiccation resistance. Acclimation had contrasting effects on the body characteristics of the two populations: 2La females boosted their glycogen stores and decreased lipids, whereas 2La females did the contrary.
Conclusion
Variation in rates of water loss and response to acclimation are associated with alternative arrangements of the 2La inversion. Understanding the mechanisms underlying these traits will help explain how inversion polymorphisms permit exploitation of a heterogeneous environment by this disease vector.
doi:10.1186/1475-2875-8-215
PMCID: PMC2754996  PMID: 19772577
17.  2La chromosomal inversion enhances thermal tolerance of Anopheles gambiae larvae 
Malaria Journal  2009;8:147.
Background
The mosquito Anopheles gambiae is broadly distributed throughout sub-Saharan Africa and this contributes to making it the most efficient vector of malaria on the continent. The pervasiveness of this species is hypothesized to originate in local adaptations facilitated by inversion polymorphisms. One inversion, named 2La, is strongly associated with aridity clines in West and Central Africa: while 2La is fixed in arid savannas, the 2L+a arrangement is predominantly found in the rainforest. Ability to survive high temperature exposure is an essential component of aridity tolerance, particularly in immature stages that are restricted to shallow puddles. Toward deciphering the role of the 2La inversion in local adaptation, the present investigation focused on variation in larval and pupal thermo-tolerance in two populations dissimilar solely in 2La arrangement.
Methods
A laboratory colony of A. gambiae that is polymorphic for 2La but standard for all other known inversions was used to create 2 homokaryotypic populations (2L+a and 2La). The survival of 4th instar larvae and pupae from both populations was then tested following exposure to thermal stress with and without prior heat hardening.
Results
Larvae responded identically to a 40°C heat stress, with about 50% of larvae dying after 1.5–2 h and few larvae surviving a 3 h stress. When heat hardened prior to the thermal stress, thermo-tolerance of both larval populations increased, with 2La 24 h survival significantly exceeding that of 2L+a. Pupae were generally more thermo-tolerant than larvae, although 2La pupae were less so than 2L+a. Heat hardening had no positive effect on pupal thermo-tolerance.
Conclusion
The increased thermo-tolerance observed in 2La larvae following heat hardening suggests higher responsiveness (i.e., thermal sensitivity) of the inverted karyotype. By responding more drastically to the heat shock, 2La larvae are better equipped to resist the potentially lethal temperatures that occur in arid habitats. The lower survival of 2La pupae compared with 2L+a may reflect the cost of this sensitivity, whereby the thermal resistance mechanisms prevent successful completion of metamorphosis. The costs and benefits of thermal resistance are discussed in light of the climates characterizing either end of the 2La frequency cline.
doi:10.1186/1475-2875-8-147
PMCID: PMC2712477  PMID: 19573238
18.  VectorBase: a data resource for invertebrate vector genomics 
Nucleic Acids Research  2008;37(Database issue):D583-D587.
VectorBase (http://www.vectorbase.org) is an NIAID-funded Bioinformatic Resource Center focused on invertebrate vectors of human pathogens. VectorBase annotates and curates vector genomes providing a web accessible integrated resource for the research community. Currently, VectorBase contains genome information for three mosquito species: Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, a body louse Pediculus humanus and a tick species Ixodes scapularis. Since our last report VectorBase has initiated a community annotation system, a microarray and gene expression repository and controlled vocabularies for anatomy and insecticide resistance. We have continued to develop both the software infrastructure and tools for interrogating the stored data.
doi:10.1093/nar/gkn857
PMCID: PMC2686483  PMID: 19028744
19.  Localization of Candidate Regions Maintaining a Common Polymorphic Inversion (2La) in Anopheles gambiae 
PLoS Genetics  2007;3(12):e217.
Chromosomal inversion polymorphisms are thought to play a role in adaptive divergence, but the genes conferring adaptive benefits remain elusive. Here we study 2La, a common polymorphic inversion in the African malaria vector Anopheles gambiae. The frequency of 2La varies clinally and seasonally in a pattern suggesting response to selection for aridity tolerance. By hybridizing genomic DNA from individual mosquitoes to oligonucleotide microarrays, we obtained a complete map of differentiation across the A. gambiae genome. Comparing mosquitoes homozygous for the 2La gene arrangement or its alternative (2L+a), divergence was highest at loci within the rearranged region. In the 22 Mb included within alternative arrangements, two ∼1.5 Mb regions near but not adjacent to the breakpoints were identified as being significantly diverged, a conclusion validated by targeted sequencing. The persistent association of both regions with the 2La arrangement is highly unlikely given known recombination rates across the inversion in 2La heterozygotes, thus implicating selection on genes underlying these regions as factors responsible for the maintenance of 2La. Polymorphism and divergence data are consistent with a model in which the inversion is maintained by migration-selection balance between multiple alleles inside these regions, but further experiments will be needed to fully distinguish between the epistasis (coadaptation) and local adaptation models for the maintenance of 2La.
Author Summary
A chromosomal inversion occurs when part of the chromosome breaks, rotates 180 degrees, and rejoins the broken chromosome. The result is a chromosome carrying a segment whose gene order is reversed. Whereas the physical rearrangement itself may have no direct consequences on gene function, recombination between alleles in the rearranged and wild type segments is suppressed. If multiple alleles inside the inverted or original orientations are well adapted to contrasting environmental conditions, suppressed recombination provides a mechanism to keep beneficial allelic combinations from being shuffled between different genetic backgrounds. Working with wild populations of flies, Dobzhansky provided the first evidence that selection was key to maintaining inversion polymorphism. Subsequently, examples of inversion polymorphisms under selection in other organisms have been found, notably in the mosquito that transmits most cases of human malaria, Anopheles gambiae. However, the genes or gene regions conferring fitness advantages have yet to be discovered. In this study, the authors used modern genomics tools to map such regions in an inversion at an unprecedented level of detail, and show that these regions are likely to be responsible for the maintenance of the inversion polymorphism in natural populations. This study lays the groundwork for future efforts to identify the genes themselves and their role in adaptation.
doi:10.1371/journal.pgen.0030217
PMCID: PMC2134946  PMID: 18069896
20.  Localization of Candidate Regions Maintaining a Common Polymorphic Inversion (2La) in Anopheles gambiae 
PLoS Genetics  2007;3(12):e217.
Chromosomal inversion polymorphisms are thought to play a role in adaptive divergence, but the genes conferring adaptive benefits remain elusive. Here we study 2La, a common polymorphic inversion in the African malaria vector Anopheles gambiae. The frequency of 2La varies clinally and seasonally in a pattern suggesting response to selection for aridity tolerance. By hybridizing genomic DNA from individual mosquitoes to oligonucleotide microarrays, we obtained a complete map of differentiation across the A. gambiae genome. Comparing mosquitoes homozygous for the 2La gene arrangement or its alternative (2L+a), divergence was highest at loci within the rearranged region. In the 22 Mb included within alternative arrangements, two ∼1.5 Mb regions near but not adjacent to the breakpoints were identified as being significantly diverged, a conclusion validated by targeted sequencing. The persistent association of both regions with the 2La arrangement is highly unlikely given known recombination rates across the inversion in 2La heterozygotes, thus implicating selection on genes underlying these regions as factors responsible for the maintenance of 2La. Polymorphism and divergence data are consistent with a model in which the inversion is maintained by migration-selection balance between multiple alleles inside these regions, but further experiments will be needed to fully distinguish between the epistasis (coadaptation) and local adaptation models for the maintenance of 2La.
Author Summary
A chromosomal inversion occurs when part of the chromosome breaks, rotates 180 degrees, and rejoins the broken chromosome. The result is a chromosome carrying a segment whose gene order is reversed. Whereas the physical rearrangement itself may have no direct consequences on gene function, recombination between alleles in the rearranged and wild type segments is suppressed. If multiple alleles inside the inverted or original orientations are well adapted to contrasting environmental conditions, suppressed recombination provides a mechanism to keep beneficial allelic combinations from being shuffled between different genetic backgrounds. Working with wild populations of flies, Dobzhansky provided the first evidence that selection was key to maintaining inversion polymorphism. Subsequently, examples of inversion polymorphisms under selection in other organisms have been found, notably in the mosquito that transmits most cases of human malaria, Anopheles gambiae. However, the genes or gene regions conferring fitness advantages have yet to be discovered. In this study, the authors used modern genomics tools to map such regions in an inversion at an unprecedented level of detail, and show that these regions are likely to be responsible for the maintenance of the inversion polymorphism in natural populations. This study lays the groundwork for future efforts to identify the genes themselves and their role in adaptation.
doi:10.1371/journal.pgen.0030217
PMCID: PMC2134946  PMID: 18069896
21.  Segmental Duplication Implicated in the Genesis of Inversion 2Rj of Anopheles gambiae 
PLoS ONE  2007;2(9):e849.
The malaria vector Anopheles gambiae maintains high levels of inversion polymorphism that facilitate its exploitation of diverse ecological settings across tropical Africa. Molecular characterization of inversion breakpoints is a first step toward understanding the processes that generate and maintain inversions. Here we focused on inversion 2Rj because of its association with the assortatively mating Bamako chromosomal form of An. gambiae, whose distinctive breeding sites are rock pools beside the Niger River in Mali and Guinea. Sequence and computational analysis of 2Rj revealed the same 14.6 kb insertion between both breakpoints, which occurred near but not within predicted genes. Each insertion consists of 5.3 kb terminal inverted repeat arms separated by a 4 kb spacer. The insertions lack coding capacity, and are comprised of degraded remnants of repetitive sequences including class I and II transposable elements. Because of their large size and patchwork composition, and as no other instances of these insertions were identified in the An. gambiae genome, they do not appear to be transposable elements. The 14.6 kb modules inserted at both 2Rj breakpoint junctions represent low copy repeats (LCRs, also called segmental duplications) that are strongly implicated in the recent (∼0.4Ne generations) origin of 2Rj. The LCRs contribute to further genome instability, as demonstrated by an imprecise excision event at the proximal breakpoint of 2Rj in field isolates.
doi:10.1371/journal.pone.0000849
PMCID: PMC1952172  PMID: 17786220
22.  Polymorphism at the defensin gene in the Anopheles gambiae complex: testing different selection hypotheses 
Genetic variation in defensin, a gene encoding a major effector molecule of insects immune response was analyzed within and between populations of three members of the Anopheles gambiae complex. The species selected included the two anthropophilic species, An. gambiae and An. arabiensis and the most zoophilic species of the complex, An. quadriannulatus. The first species was represented by four populations spanning its extreme genetic and geographical ranges, whereas each of the other two species was represented by a single population. We found (i) reduced overall polymorphism in the mature peptide region and in the total coding region, together with specific reductions in rare and moderately frequent mutations (sites) in the coding region compared with non coding regions, (ii) markedly reduced rate of nonsynonymous diversity compared with synonymous variation in the mature peptide and virtually identical mature peptide across the three species, and (iii) increased divergence between species in the mature peptide together with reduced differentiation between populations of An. gambiae in the same DNA region. These patterns suggest a strong purifying selection on the mature peptide and probably the whole coding region. Because An. quadriannulatus is not exposed to human pathogens, identical mature peptide and similar pattern of polymorphism across species implies that human pathogens played no role as selective agents on this peptide.
doi:10.1016/j.meegid.2006.11.004
PMCID: PMC1876661  PMID: 17161659
Anopheles gambiae; Africa; malaria; vector; arthropod; immunity; Defensin; evolution; polymorphism; selection
23.  VectorBase: a home for invertebrate vectors of human pathogens 
Nucleic Acids Research  2006;35(Database issue):D503-D505.
VectorBase () is a web-accessible data repository for information about invertebrate vectors of human pathogens. VectorBase annotates and maintains vector genomes providing an integrated resource for the research community. Currently, VectorBase contains genome information for two organisms: Anopheles gambiae, a vector for the Plasmodium protozoan agent causing malaria, and Aedes aegypti, a vector for the flaviviral agents causing Yellow fever and Dengue fever.
doi:10.1093/nar/gkl960
PMCID: PMC1751530  PMID: 17145709
24.  Effective population size of Anopheles funestus chromosomal forms in Burkina Faso 
Malaria Journal  2006;5:115.
Background
As Anopheles funestus is one of the principal Afro-tropical malaria vectors, a more complete understanding of its population structure is desirable. In West and Central Africa, An. funestus population structure is complicated by the coexistence of two assortatively mating chromosomal forms. Effective population size (Ne) is a key parameter in understanding patterns and levels of intraspecific variation, as it reflects the role of genetic drift. Here, Ne was estimated from both chromosomal forms, Kiribina and Folonzo, in Burkina Faso.
Methods
Short-term Ne was estimated by evaluating variation at 16 microsatellite loci across temporal samples collected annually from 2000–2002. Estimates were based on standardized variance in allele frequencies or a maximum likelihood method. Long-term Ne was estimated from genetic diversity estimates using mtDNA sequences and microsatellites.
Results
For both forms, short-term and long-term Ne estimates were on the order of 103 and 105, respectively. Long-term Ne estimates were larger when based on loci from chromosome 3R (both inside and outside of inversions) than loci outside of this arm.
Conclusion
Ne values indicate that An. funestus is not subject to seasonal bottlenecks. Though not statistically different because of large and overlapping confidence intervals, short-term Ne estimates were consistently smaller for Kiribina than Folonzo, possibly due to exploitation of different breeding sites: permanent for Folonzo and intermittent for Kiribina. The higher long-term Ne estimates on 3R, the arm carrying the two inversions mainly responsible for defining the chromosomal forms, give natural selection broader scope and merit further study.
doi:10.1186/1475-2875-5-115
PMCID: PMC1676016  PMID: 17125511
25.  Anthropogenic Habitat Disturbance and Ecological Divergence between Incipient Species of the Malaria Mosquito Anopheles gambiae 
PLoS ONE  2012;7(6):e39453.
Background
Anthropogenic habitat disturbance is a prime cause in the current trend of the Earth’s reduction in biodiversity. Here we show that the human footprint on the Central African rainforest, which is resulting in deforestation and growth of densely populated urban agglomerates, is associated to ecological divergence and cryptic speciation leading to adaptive radiation within the major malaria mosquito Anopheles gambiae.
Methodology/Principal Findings
In southern Cameroon, the frequency of two molecular forms–M and S–among which reproductive isolation is strong but still incomplete, was correlated to an index of urbanisation extracted from remotely sensed data, expressed as the proportion of built-up surface in each sampling unit. The two forms markedly segregated along an urbanisation gradient forming a bimodal cline of ∼6-km width: the S form was exclusive to the rural habitat, whereas only the M form was present in the core of densely urbanised settings, co-occurring at times in the same polluted larval habitats of the southern house mosquito Culex quinquefasciatus–a species association that was not historically recorded before.
Conclusions/Significance
Our results indicate that when humans create novel habitats and ecological heterogeneities, they can provide evolutionary opportunities for rapid adaptive niche shifts associated with lineage divergence, whose consequences upon malaria transmission might be significant.
doi:10.1371/journal.pone.0039453
PMCID: PMC3382172  PMID: 22745756

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