Inversion 2La Research Articles

Polymorphic inversion 2La frequencies associated with ecotypes in populations of Anopheles coluzzii from Southwest Nigeria

Chromosomal inversion 2La play a vital role in local adaptation of the malaria vector Anopheles. This polymorphic inversion, linked with ecological and behavioural plasticity, ensures adaptation of these species and continued transmission of the malaria parasite. However, the adaptive significance of this inversion in several Anopheles populations is still not well understood. Here, we determined the frequencies of polymorphic inversion 2La in natural populations of Anopheles coluzzii, the second major malaria vector in Southwest Nigeria, and its association with ecotypes. Anopheles larvae were collected from eleven localities across the mangrove/forest and forest/savannah ecozones of Southwest Nigeria. Adults were identified morphologically and with Polymerase Chain Reaction (PCR). Chromosomal inversion frequencies were determined by PCR. Relationship between genotype frequencies and ecotypes was determined using linear regression analysis and chi square. A total of 333 Anopheles coluzzii were scored for inversion 2La karyotypes. Frequencies of the 2La/2La homokaryotype increased progressively Northwards (F = 10.9; p = 0.01; R2=0.54) corresponding to a significant decline in the frequencies of 2L+a/2L+a (linear regression: F = 7.03; p = 0.03; R2=0.44), an indication of association with ecotypes. The heterokaryotype (2L+a/2La) however, showed high frequencies across ecotypes (F = 0.15; p = 0.71; R2=0.02). There was no significant deviation of the karyotype frequencies from Hardy-Weinberg estimates, an indication of a possible heterozygous advantage.

Preliminary evidence from microsatellite and 2La inversion polymorphism suggests DDT resistance drives genetic differentiation between populations of Anopheles gambiae s.s. in South West Nigeria

Background Resistance to the insecticide DDT has emerged in several Anopheles gambiae populations which has stalled its use in malaria vector control programs in Nigeria. Data on insecticide resistance in Nigeria are still patchy with consequent lack of understanding of resistance mechanisms. This study was therefore designed to investigate the relationship between DDT resistance, inversion 2La and microsatellite loci in Anopheles gambiae s.s. populations from Lagos and Oyo State, South West, Nigeria.

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

BackgroundPrincipal 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.MethodsTo 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.ResultsAfter 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.ConclusionsOur 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.

A New Chromosomal Phylogeny Supports the Repeated Origin of Vectorial Capacity in Malaria Mosquitoes of the Anopheles gambiae Complex

Understanding phylogenetic relationships within species complexes of disease vectors is crucial for identifying genomic changes associated with the evolution of epidemiologically important traits. However, the high degree of genetic similarity among sibling species confounds the ability to determine phylogenetic relationships using molecular markers. The goal of this study was to infer the ancestral–descendant relationships among malaria vectors and nonvectors of the Anopheles gambiae species complex by analyzing breakpoints of fixed chromosomal inversions in ingroup and several outgroup species. We identified genes at breakpoints of fixed overlapping chromosomal inversions 2Ro and 2Rp of An. merus using fluorescence in situ hybridization, a whole-genome mate-paired sequencing, and clone sequencing. We also mapped breakpoints of a chromosomal inversion 2La (common to An. merus, An. gambiae, and An. arabiensis) in outgroup species using a bioinformatics approach. We demonstrated that the “standard” 2R+p arrangement and “inverted” 2Ro and 2La arrangements are present in outgroup species Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The data indicate that the ancestral species of the An. gambiae complex had the 2Ro, 2R+p, and 2La chromosomal arrangements. The “inverted” 2Ro arrangement uniquely characterizes a malaria vector An. merus as the basal species in the complex. The rooted chromosomal phylogeny implies that An. merus acquired the 2Rp inversion and that its sister species An. gambiae acquired the 2R+o inversion from the ancestral species. The karyotype of nonvectors An. quadriannulatus A and B was derived from the karyotype of the major malaria vector An. gambiae. We conclude that the ability to effectively transmit human malaria had originated repeatedly in the complex. Our findings also suggest that saltwater tolerance originated first in An. merus and then independently in An. melas. The new chromosomal phylogeny will facilitate identifying the association of evolutionary genomic changes with epidemiologically important phenotypes.

Genome-Wide Transcriptional Analysis of Genes Associated with Acute Desiccation Stress in Anopheles gambiae

Malaria transmission in sub-Saharan Africa varies seasonally in intensity. Outbreaks of malaria occur after the beginning of the rainy season, whereas, during the dry season, reports of the disease are less frequent. Anopheles gambiae mosquitoes, the main malaria vector, are observed all year long but their densities are low during the dry season that generally lasts several months. Aestivation, seasonal migration, and local adaptation have been suggested as mechanisms that enable mosquito populations to persist through the dry season. Studies of chromosomal inversions have shown that inversions 2La, 2Rb, 2Rc, 2Rd, and 2Ru are associated with various physiological changes that confer aridity resistance. However, little is known about how phenotypic plasticity responds to seasonally dry conditions. This study examined the effects of desiccation stress on transcriptional regulation in An. gambiae. We exposed female An. gambiae G3 mosquitoes to acute desiccation and conducted a genome-wide analysis of their transcriptomes using the Affymetrix Plasmodium/Anopheles Genome Array. The transcription of 248 genes (1.7% of all transcripts) was significantly affected in all experimental conditions, including 96 with increased expression and 152 with decreased expression. In general, the data indicate a reduction in the metabolic rate of mosquitoes exposed to desiccation. Transcripts accumulated at higher levels during desiccation are associated with oxygen radical detoxification, DNA repair and stress responses. The proportion of transcripts within 2La and 2Rs (2Rb, 2Rc, 2Rd, and 2Ru) (67/248, or 27%) is similar to the percentage of transcripts located within these inversions (31%). These data may be useful in efforts to elucidate the role of chromosomal inversions in aridity tolerance. The scope of application of the anopheline genome demonstrates that examining transcriptional activity in relation to genotypic adaptations greatly expands the number of candidate regions involved in the desiccation response in this important malaria vector.

Divergent transcriptional response to thermal stress by Anopheles gambiae larvae carrying alternative arrangements of inversion 2La

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.

Evolutionary Dynamics of the Ty3/Gypsy LTR Retrotransposons in the Genome of Anopheles gambiae

Ty3/gypsy elements represent one of the most abundant and diverse LTR-retrotransposon (LTRr) groups in the Anopheles gambiae genome, but their evolutionary dynamics have not been explored in detail. Here, we conduct an in silico analysis of the distribution and abundance of the full complement of 1045 copies in the updated AgamP3 assembly. Chromosomal distribution of Ty3/gypsy elements is inversely related to arm length, with densities being greatest on the X, and greater on the short versus long arms of both autosomes. Taking into account the different heterochromatic and euchromatic compartments of the genome, our data suggest that the relative abundance of Ty3/gypsy LTRrs along each chromosome arm is determined mainly by the different proportions of heterochromatin, particularly pericentric heterochromatin, relative to total arm length. Additionally, the breakpoint regions of chromosomal inversion 2La appears to be a haven for LTRrs. These elements are underrepresented more than 7-fold in euchromatin, where 33% of the Ty3/gypsy copies are associated with genes. The euchromatin on chromosome 3R shows a faster turnover rate of Ty3/gypsy elements, characterized by a deficit of proviral sequences and the lowest average sequence divergence of any autosomal region analyzed in this study. This probably reflects a principal role of purifying selection against insertion for the preservation of longer conserved syntenyc blocks with adaptive importance located in 3R. Although some Ty3/gypsy LTRrs show evidence of recent activity, an important fraction are inactive remnants of relatively ancient insertions apparently subject to genetic drift. Consistent with these computational predictions, an analysis of the occupancy rate of putatively older insertions in natural populations suggested that the degenerate copies have been fixed across the species range in this mosquito, and also are shared with the sibling species Anopheles arabiensis.

Relationship of larval desiccation to Anopheles gambiae Giles and An. arabiensis Patton survival

Relationship of larval desiccation to Anopheles gambiae Giles and An. arabiensis Patton survival

Inversion 2La is associated with enhanced desiccation resistance in Anopheles gambiae

BackgroundAnopheles 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.MethodsTwo 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.ResultsFemales 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.ConclusionVariation 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.

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.

Dieldrin resistance in the malaria vector Anopheles gambiae in Ghana

Anopheles gambiae Giles s.s. (Diptera: Culicidae) is one of the principal vectors of malaria in the Ashanti region of central Ghana. High levels of resistance to dieldrin were recorded in a wild-caught sample from Obuasi (south of Kumasi) as well as a laboratory colony established using material from the wild population. Cytogenetic analysis of wild-caught and laboratory samples revealed chromosomal polymorphism for inversions 2La and 2Rb. Although inversion 2La has previously been shown to be associated with dieldrin resistance in certain other laboratory strains originating from West Africa, there was no obvious association between inversion karyotype assortment and the resistance phenotype in the Obuasi population. In addition, polymerase chain reaction analysis indicated the presence of the alanine296 to glycine mutation in the GABA (gamma amino-butyric acid) receptor (which has been mapped to a chromosomal position within inversion 2La). This mutation has previously been shown to be associated with dieldrin resistance in the same An. gambiae laboratory strains of West African origin. Our data show only a weak association between the dieldrin resistance phenotype and the presence of this mutation, suggesting that another dieldrin resistance mechanism is operational in the Obuasi population. Biochemical and synergist exposure assays suggest a metabolic component, probably mediated by monooxygenase P450 enzymes. We conclude that dieldrin resistance in the An. gambiae population of the Obuasi region occurs at a high level - most likely in the absence of selection - and that control of the resistance phenotype is polyfactorial and must include components other than mutations in the GABA receptor locus.

Breakpoint structure reveals the unique origin of an interspecific chromosomal inversion (2La) in the Anopheles gambiae complex.

Paracentric chromosomal inversions are major architects of organismal evolution and have been associated with adaptations relevant to malaria transmission in anopheline mosquitoes. The processes responsible for their origin and maintenance, still poorly understood, can be illuminated by analysis of inversion breakpoint sequences. Here, we report the breakpoint structure of chromosomal inversion 2La from the principal malaria vector Anopheles gambiae and its relatives in the A. gambiae complex. The distal and proximal breakpoints of the standard (2L+a) arrangement contain gene duplications: full-length genes and their truncated copies at opposite ends. Intact genes without pseudogene copies in the alternative arrangement (2La) imply that 2L+a is derived and was viable despite damage to genes, because duplication preserved gene function. A unique origin for the interspecific 2La inversion was challenged previously by indirect genetic evidence, but breakpoint sequences determined from members of the A. gambiae complex strongly suggest their descent from a single event. The derived position of 2L+a, long considered ancestral in this medically important group, has significant implications for the phylogenetic history and the evolution of vectorial capacity in the A. gambiae complex.

Stable chromosomal inversion polymorphisms and insecticide resistance in the malaria vector mosquito Anopheles gambiae (Diptera: Culicidae).

Anopheles gambiae Giles has been implicated as a major vector of malaria in Africa. A number of paracentric chromosomal inversions have been observed as polymorphisms in wild and laboratory populations of this species. These polymorphisms have been used to demonstrate the existence of five reproductive units in West African populations that are currently described as incipient species. They have also been correlated with various behavioral characteristics such as adaptation to aridity and feeding preference and have been associated with insecticide resistance. Two paracentric inversions namely 2La and 2Rb are highly ubiquitous in the wild and laboratory populations sampled. Both inversions are easily conserved during laboratory colonization of wild material and one shows significant positive heterosis with respect to Hardy-Weinberg proportions. Inversion 2La has previously been associated with dieldrin resistance and inversion 2Rb shows an association with DDT resistance based on this study. The stability and maintenance of these inversions as polymorphisms provides an explanation for the transmission and continued presence of DDT and dieldrin resistance in a laboratory strain of An. gambiae in the absence of insecticide selection pressure. This effect may also be operational in wild populations. Stable inversion polymorphism also provides a possible mechanism for the continual inheritance of suitable genetic factors that otherwise compromise the fitness of genetically modified malaria vector mosquitoes.

Resistance to dieldrin + fipronil assorts with chromosome inversion 2La in the malaria vector Anopheles gambiae.

Cyclodiene insecticide resistance in malaria vector mosquitoes of the Anopheles gambiae species complex (Diptera: Culicidae) has been reported previously from several parts of Africa. We report resistance to dieldrin, a cyclodiene, in two laboratory strains of An. gambiae Giles sensu stricto code-named Ian P20 from Nigeria (1979) and CIG from Cote d'Ivoire (1997). Dieldrin resistance levels were high in adult female mosquitoes (40-75% survived exposure to 4% dieldrin for 1 h) and was closely linked with chromosomal paracentric inversion 2La. This inversion did not occur in Hardy-Weinberg proportions, but showed an excess of heterozygotes in both strains, which may account for the high levels of resistance. This linkage also suggests that dieldrin resistance in Ian P20 is dominant. After subsamples of strain Ian P20 were exposed for 1 h to dieldrin 4% or fipronil 2% (discriminating concentrations), the resultant mortality-rates (61% and 65%) were not significantly different. Most survivors after fipronil treatment also survived subsequent exposure to dieldrin (46/50=92%). This apparent cross-resistance between insecticides of two classes (cyclodiene and phenyl pyrazole) has implications for the management of insecticide resistance in wild populations of the An. gambiae complex.