Chromosomal rearrangements in mosquitoes: from micro- to macroevolution

Abstract

Chromosomal rearrangements are believed to play a critical role in shaping the eco-evolutionary dynamics of diverse organisms, including mosquitoes. The availability of high-quality genome assemblies of multiple mosquito species has revolutionized the study of these rearrangements. Researchers can now employ genomic approaches to identify chromosomal rearrangements and gain new insights into their role in both microevolutionary and macroevolutionary processes. Autosomal inversions are emerging as possible key drivers of adaptation in mosquitoes. By maintaining standing genetic variation, inversions enable mosquito populations to rapidly adapt to environmental pressures, including insecticide-based vector control methods. X-chromosomal inversions may also play a role in speciation by reducing gene flow between diverging populations. Since mosquitoes are primary vectors of many human diseases, understanding the genomic basis of their adaptation and evolution is important from an epidemiological perspective. Studying chromosomal rearrangements can help identify of chromosomal regions associated with adaptation to environmental stresses or insecticide resistance, thereby improving the efficacy of disease management programs.

Introduction

Mosquitoes are vectors of dangerous human pathogens including malaria parasites, dengue, Zika, and West Nile viruses, as well as filarial worms [1]. Mosquito species diverged from other dipterans ~250 million years ago (MYA), giving a rise to the mosquito family Culicidae [2]. About 147–213 million years ago, a major split occurred in the mosquito family, resulting in the formation of two clades, or groups of species that share a common ancestor, with distinct abilities to transmit pathogens: the Anophelinae and Culicinae subfamilies. The Anophelinae mosquitoes are major vectors of malaria parasites, while the Culicinae mosquitoes are mostly vectors of arboviruses. The Culicinae subfamily further diverged into two tribes, Culicini and Aedini which share a common ancestor ~87–104 MYA. Unlike Drosophila [3] and many other insects [4], mosquitoes have a remarkably conserved karyotype. The number of chromosomes (2n) is equal to six in all mosquito species, except for Chagasia bathana from the subfamily Anophlenae, which has eight chromosomes [5]. However, the chromosome composition within the karyotypes differs between the two mosquito subfamilies [6]. The chromosomal complement of the subfamily Anophelinae consists of two heteromorphic sex chromosomes (X and Y) and two pairs of metacentric or submetacentric autosomes. In contrast, Culicinae mosquitoes, have three pairs of metacentric autosomes, one of which contains a sex-determining locus on the shortest autosome in Aedes aegypti [7,8] and Culex quinquefasciatus [6] or on the longest autosome in Culex tarsalis [9]. Another striking difference between the two mosquito subfamilies is the size of their genomes [6], which is reflected in chromosome length [10–12]. Some Culicinae mosquito genomes are up to five times larger than Anophelinae genomes due to dramatic expansions of transposable elements and other repeats in Culicinae [6].

Chromosomal rearrangements are genetic mutations that are usually detectable cytogenetically under a microscope. They can alter the number of chromosomes (fusions and fissions), the chromosome composition (deletions, duplications, and translocations), or the order of genetic material (inversions) [13]. Recent advances in genomic technologies have enabled the development of high-quality, chromosome-scale assemblies for multiple mosquito species from the both subfamilies Anophelinae [14–17] and Culicinae [6,7]. These genome assemblies provide new opportunities to identify chromosomal rearrangements and reassess their potential role in mosquito micro- and macroevolution. In this review, we discuss classical and recent approaches to discovering and detecting chromosomal rearrangements in different mosquito lineages, as well as the potential impact of these rearrangements on micro- and macroevolution.

Methods for discovery and detection of chromosomal rearrangements

The study of chromosomal rearrangements requires reliable methods for their discovery and detection in natural populations. Inversions are the most common type of rearrangement observed in natural mosquito populations. Large multi-megabase (Mb)-long polymorphic and fixed chromosomal inversions can be detected in mosquito polytene chromosomes by light microscopy. In this cse, heterozygous karyotypes appear as loops because homologous chromosomes are pared (Figure 1a). The further analysis involves reading the banding patterns of polytene chromosomes by comparing the observed chromosome images with the standard cytogenetic maps. This method has been widely used to study chromosomal inversions in several species groups including the Anopheles gambiae complex [18], the Maculipennis group [19–21], Anopheles funestus [22], Anopheles stephensi [23], Anopheles darlingi [24], and others.

Figure 1.

A workflow for the discovery, validation, characterization and detection of chromosomal inversions in mosquito populations. Inversions are discovered either by reading of the banding pattern of polytene chromosomes (a) or by performing Hi-C on multiple mosquito individuals (b). Panel (a) shows a heterozygous inversion loop in polytene chromosome. Pannel (b) shows an example of the Hi-C heat map. The inversion appears as a butterfly pattern outside of the main diagonal. The presence of the inversion is validated by FISH using DNA probes on chromosomes (c). DNA probes hybridized to the chromosomes in the opposite sides of inversions are shown by blue and yellow colors. WGS of individual mosquitoes is used to identify SNPs fixed between alternative chromosomal arrangements (d). Diagnostic SNPs in standard and inverted chromosome arrangements are shown in red and green, respectively. Routine detection of polymorphic inversions in mosquito populations is performed using ARMS-PCR (e). The bottom panel shows result of the gel electrophoresis with standard (std), inverted (inv), and heterozygous (het) inversion arrangements.

The Hi-C method can be used to identify inversions in species with chromosome-scale reference genome assemblies [25]. This method measures the frequency of physical contacts between specific genomic loci by crosslinking and further sequencing regions that interact with each other in nuclear space. In the presence of an inversion, the breakpoint regions that interact in nuclear space appear as bright butterfly-shaped patterns outside the main diagonal line representing the high-frequency contacts in the Hi-C maps. The triangles of the butterfly wings connect at the breakpoints, with the color intensity increasing as a gradient from the base to the center of the triangles (Figure 1b). Hi-C has been used to map the breakpoint regions of the previously known polymorphic inversions 2Rbc and 2Rd in An. gambiae [26], and to discover new chromosomal inversions, such as 2L1 in Anopheles atroparvus and 2Rt Anopheles merus [16]. Using the Hi-C method, researchers found 20 novel chromosomal inversions in 25 wild strains of Ae. aegypti [27].

Fluorescence in situ hybridization (FISH) is used to map breakpoint regions in polytene chromosomes and to validate inversions in mitotic chromosomes [28,29]. Typically, two FISH probes are designed from the opposite sides within the inversion locus and labeled with different colors. The probes are then hybridized to chromosomes simultaneously. The presence or absence of the inversions is determined by the order of the signals on the chromosomes (Figure 1c).

Once, inversions are discovered and characterized at the molecular level using whole genome sequencing (WGS) (Figure 1d), they can be routinely detected in mosquito populations using various assays. If the sequences flanking the inversion breakpoints are known, primers can be designed to amplify DNA fragments of different sizes polymerase chain reaction (PCR) to distinguish between standard and inverted chromosome arrangements [28]. More recently developed molecular karyotyping assays do not require knowledge of inversion breakpoints. These assays are based on single nucleotide polymorphism (SNP) tags found within standard and inverted chromosome arrangements. Fixed SNPs accumulate between alternative arrangements due to the suppression of recombination within an inversion. Two PCR-restriction fragment length polymorphism (RFLP) assays have been developed for the molecular karyotyping of the two widespread inversions 2Rb and 2Rc in An. gambaie and Anopheles coluzzii in West Africa [30,31]. These methods rely on inversion-specific SNPs that overlap with restriction enzyme recognition sites and consist of two steps: 1) PCR amplification of the DNA fragment, and 2) subsequent cleavage of the DNA fragment using restriction enzymes that distinguish alternative allelic states at the tag SNPs.

WGS data can be used to detect inversions in individual mosquitoes using a Principal Component Analysis (PCA)-based method. This method identifies inversion-specific single nucleotide polymorphisms (SNPs) through sequence alignment with chromosome-scale reference genomes. It identifies chromosomal inversions in the WGS of individual mosquitoes. For instance, PCA classified all An. gambiae and An. coluzzii samples into three inversion genotypes: inverted homozygous, non-inverted (standard) homozygous and heterozygous by including the subset of samples associated with cytogenetic data in the Anopheles Gambiae 1000 Genome Project (Ag1000G) WGS dataset [32]. A total of 50 to 200 tag SNPs were identified for each of six cytogenetically described chromosomal inversions, 2La, 2Rb, 2Rc, 2Rd, 2Rj, and 2Ru. The performance of the tag SNPs using molecular genotyping approaches was consistent with traditional cytogenetic karyotyping on the same 960 individual An. gambiae and An. coluzzii mosquitoes. Two high-throughput molecular genotyping approaches, the TaqMan OpenArray (OA) and Genotyping-in-Thousands by Sequencing (GT-seq), successfully predicted inversion genotypes for the common polymorphic inversions in these species [33]. The inversion-specific SNP identification was further optimized and tested against a new benchmark derived from three publicly available insect datasets, including D. melanogaster, An. gambiae, and An. coluzzii [34].

The routine detection of polymorphic inversions in natural mosquito populations would benefit from the availability of a simple, inexpensive assay. Recently, a novel tetra-primer amplification refractory mutation system (ARMS)-PCR assay based on a tag SNP has recently been developed for genotyping the 2Ru inversions in An. gambiae and An. coluzzii [35]. Five tag SNPs previously have been shown to be strongly correlated with the 2Ru arrangement. One tag SNP was selected for the final assay because it was highly predictive of the 2Ru inversion. The assay requires only one PCR with four primers followed by electrophoresis. The four primers include two outer primers that amplify the region containing the tag SNP, and two SNP-specific inner primers that targeted alternative alleles of the 2Ru inversion. The tetra-primer ARMS-PCR assay was validated using 454 specimens that were karyotyped either by traditional cytogenetic analysis or by a previously developed multilocus GT-seq method [33]. The ARMS-PCR assay was successful overall in 98.5% of the specimens and showed 96.7% concordance with the established methods [35], suggesting that similar assays can be developed to detect other polymorphic inversions (Figure 1e).

Based on the currently available state-of-the-art approaches, we present a workflow for the discovery, validation, characterization and detection of chromosomal inversions in mosquito populations (Figure 1a-e).

Microevolutionary impact of chromosomal rearrangements

Microevolutionary studies focus on the impact of chromosomal rearrangements on processes that influence divergence between populations, which may or may not result in speciation [13]. These rearrangements are typically polymorphic within species and vary in frequencies among natural populations. Chromosomal inversions are important contributors to microevolutionary processes in mosquitoes. Since inversions result in the reverse order of the genetic material within a chromosome, these regions may be partly protected from normal recombination [36]. By capturing different sets of alleles, inversions can have dramatic effects on the ecological, behavioral, and physiological adaptations of their carriers [37]. Of the more than 90 chromosomal inversions identified in the African malaria vector An. gambiae [38], five inversions ranging in size from 5 to 22 Mb are considered the most common and are shared between the sister taxa An. gambiae and An. coluzzii 2R (b, c, d, u) and 2La [32]. Ancient 2La inversion polymorphism in the An. gambiae complex [39] has been associated with anthropophilic behavior, drought tolerance, and Plasmodium transmission [40,41]. The 2Rb and 3Ra inversions have been associated with host preference and resting behavior in An. arabiensis [42].

Several recent studies have examined on understanding the relationship between chromosomal inversions and the rapid evolution of insecticide resistance in An. gambiae and An. coluzzii. The 2La inversion has been shown to correlate with the knockdown resistance (kdr) mutation involved in resistance to DDT and pyrethroids in An. gambiae in Kenya through association with outdoor resting behavior of mosquitoes [43]. Relationships between resistance to dieldrin and the 2La inversion polymorphism have also been demonstrated in An. coluzzii in Nigeria [44]. WGS data for natural populations of An. gambiae, An. coluzzii, and An. arabiensis across the Africa available through the Ag1000G were analyzed for selective sweeps [45]. Of the two independent mutations at the insecticide dieldrin resistance locus (Rdl), one was located within of the 2La inversion. The data support the hypothesis that this mutation originated in An. gambiae with 2L+^a chromosomes, and then, after interspecific introgression into An. coluzzii and interkaryotypic introgression into 2La chromosome, it spread through West and Central African populations via two independent hard selective sweeps [45]. A recent analysis of insecticide resistance to deltamethrin was performed experimentally using a scalable, genome-based Snakemake workflow, called RNA-Seq-Pop [46]. During the selection of An. gambiae for resistance to deltamethrin over multiple generations, the 2La chromosomal inversion increased in frequency from 33% to 86%. This result supports a link between this inversion and pyrethroid resistance. The Cyp4J5-L43F mutation, associated with insecticide resistance, lies within the 2La inversion. However, it is unclear whether the Cyp4J5 gene is the cause, or if there are other alleles on the 2La haplotype that cause the shift in the 2La frequency. Associations between the 2La inversion and insecticide resistance have also been reported in East African populations of An. gambiae [47]. In another study, An. coluzzii mosquitoes from colonies with different pyrethroid resistance phenotypes were sequenced using RNA-seq and WGS approaches [48]. The study found several blocks in the An. coluzzii genome that were clearly associated with resistance, including the one within the 2Rb inversion.

An interesting insight into the role of autosomal chromosomal inversions in the rapid adaptation of a major African malaria vector, An. funestus, to a new anthropogenic larval habitat such as rice fields was observed [49]. Previous studies conducted in Burkina Faso found significant heterozygote deficits for four autosomal inversions in An. funestus (2Ra, 2Rs, 3Ra, and 3Rb) [50,51]. This observation led to the hypothesis that An. funestus populations consist of two stable, sympatric entities, the “Kiribina” and “Folonzo” chromosomal forms with an uncertain taxonomic status. The PCA analysis of 168 individuals WGS from 11 locations in Burkina Faso revealed the presence of two clusters, K and F, which were identified as Kiribina and Folonzo based on karyotyping. These two clusters diverged only ~1,300 years ago following the massive expansion of rice cultivation ~1,850 years ago. Despite ongoing hybridization between the two forms, the study identified genome-wide differentiation with the highest divergence concentrated in chromosomal inversions. Thus, chromosomal inversions in An. funestus facilitated adaptive divergence between the ecotypes, which is consistent with their role in local adaptation to a new ecological environment. Importantly, the rapid diversification of the malaria vector was made possible by standing genetic variation present within evolutionarily ancient chromosomal rearrangements, rather than by de novo mutations [49].

Various theoretical models and empirical microevolutionary studies have suggested the role of chromosomal rearrangements in speciation [52]. Among members of the An. gambiae complex, extensive autosomal, but not X-chromosomal, introgression has been observed suggesting that the X chromosome likely harbors strong sterility factors that contribute to reducing gene flow between species [39,53]. Members of the An. gambiae complex are differentiated by at least ten fixed chromosomal inversions, five of which are located on the smallest X chromosome [18]. Similarly, most of the inversions fixed among members of the Maculipennis subgroup were identified on the X chromosome [54]. Physical genome mapping using FISH discovered two fixed inversions that distinguish the European mosquito An. atroparvus and the Eurasian mosquito An. messeae, both of which are members of the Maculipennis subgroup. One 10-Mb-long inversion was nested within the other 13-Mb-long inversion, resulting in only two small genomic blocks that changed position and orientation on the X chromosome between the two species. These rearranged chromosomal regions contain 181 annotated genes and are enriched for genes involved in immune regulation and mating behavior in mosquitoes [55]. These results provide insight into the possible role of X-chromosomal inversions in speciation of Anopheles mosquitoes.

A potential involvement of polymorphic X-chromosomal inversions in the divergence between two closely related sibling species has been observed in mosquitoes of the Maculipennis subgroup. The Anopheles messeae carries five widespread polymorphic chromosomal inversions X1, X2, 2R1, 3R1, and 3L1 [20]. The early studies showed that the frequencies of some inversion combinations were in linkage disequilibrium suggesting the presence of a cryptic taxon [56], which was later identified and named as An. daciae based on internal transcribed spacer 2 (ITS2) SNPs [57]. A recent study used cytogenetic karyotyping and ITS2 genotyping to assess the frequencies of 11 chromosomal inversions in 1932 specimens of An. messeae and An. daciae collected from 28 sites across Eurasia [58]. The frequencies of three polymorphic inversions in the X chromosome differed significantly between the two species. The X0 arrangement was present at a frequency of 2% or absent in An. messeae populations, whereas the X1 alternative arrangement was found at a frequency of 94% in the populations of this species. In contrast, both the X0 and X1 arrangements were found at nearly equal frequency in An. daciae populations. Two other inversions, X2 and X4, were endemic to An. messeae populations. Although the autosomal inversions 2R1, 3R1, 3L1 were shared between the species, their frequencies at the same locations differed by 13%-45% between the species, indicating restricted gene flow between the two taxa. The PCA and hierarchical cluster analyses, conducted based on chromosomal inversion frequencies, separated An. messeae and An. daciae into distinct clusters. Moreover, three additional clusters were identified within An. messeae suggesting that this species is more ancient and has been able to accumulate more genetic differences between populations [58]. The more recent evolution of An. daciae is likely related to the origin and spread of the X0 chromosomal arrangement in its ancestral population.

Macroevolutionary impact of chromosomal rearrangements

Macroevolutionary studies focus on identifying the patterns, rates and mechanisms of chromosomal rearrangements that are established between taxonomic groups at or above the species level. A recent study compared chromosome-level genome assemblies developed for An. albimanus, An. coluzzii, Cx. Quinquefasciatus and Ae. aegypti revealing details of chromosomal evolution in these mosquitoes [6]. Multiple paracentric inversions and whole arm translocations occurred in all mosquito lineages. A translocation between sex-determining chromosome 1 and an autosome separates the Anophelinae and Culicinae lineages. A pericentric inversion in chromosome 1 differentiates Cx. quinquefasciatus and Ae. aegypti. The X chromosome in Anopheles and both arms of the sex-determining chromosome 1 in Cx. quinquefasciatus and Ae. aegypti rearranged by inversions significantly faster than the autosomes in each species. The study revealed different patterns of the transposable element distribution in the mosquito genomes. These elements concentrate in the pericentromeric regions of the Anopheles genomes, but they spread along the chromosomal arms toward the telomeres in Cx. quinquefasciatus and are evenly distributed along the chromosomes in Ae. aegypti. Comparing the genome-wide locations of orthologous genes revealed that 6 chromosomal inversions in An. gambiae and 11 inversions in Ae. aegypti share similar sets of genes, suggesting that the two genera experienced parallel genome evolution in similar African environments [27].

A Hi-C study of five Anopheles species examined the evolutionary aspects of the 3D genome organization by exploring the relationships between topologically associated domains (TADs) and breakpoint regions of fixed rearrangements [16]. Anopheles chromatin is partitioned into ‘open’ (termed ‘A’) and ’closed’ (termed ‘B’) compartments within chromosomal territories that correspond to active and repressed TADs, respectively. The study revealed a conserved organization of TADs within syntenic blocks and a non-random positioning of breakpoint regions at TAD boundaries and transcriptionally active ‘open’ chromatin compartments (A-compartments). Interestingly, there was no enrichment of repetitive elements, but a strong enrichment of gene density was found around breakpoint regions. One possible mechanism of chromosomal rearrangements may involve increased DNA breakage in genomic regions containing open chromatin configurations and epigenetic features that promote DNA accessibility. At the same time rearrangements avoid disrupting essential genes and/or gene expression. This model explains why most mapped inversion breakpoints are localized to interbands of polytene chromosomes [38].

In summary, advances in genomics have transformed the way we investigate mosquito genome organization. Recent studies have provided new knowledge about chromosomal inversions and their role in mosquito evolution, offering critical insights into mosquito biology. Understanding how chromosomal inversions function could help identify the adaptive traits that enable mosquitoes to thrive in various environments, facilitating the design of targeted interventions to disrupt these adaptations. Ultimately, integrating genomics into vector control will significantly contribute to global efforts to control mosquito-borne diseases.

Highlights.

• Genomic approaches, including Hi-C proximity ligation and single-nucleotide polymorphism-specific polymerase chain reaction, are now being used to discover and detect chromosomal rearrangements in natural mosquito populations.

• Evolutionarily ancient autosomal inversions are associated with mosquito adaptation and the evolution of insecticide resistance based on standing genetic variation, while X-chromosome inversions may contribute to speciation in malaria mosquitoes.

• The X chromosomes of the Anophelinae and the sex-determining chromosomes of the Culicinae subfamiles have a significantly higher rate of rearrangements than the autosomes. The two subfamilies exhibit dramatic differences in the genome size and the chromosomal patterns of transposable element distribution.