Next Article in Journal
Subcellular-Level Mitochondrial Energy Metabolism Response in the Fat Body of the German Cockroach Fed Abamectin
Next Article in Special Issue
Genetic Polymorphism and Phylogenetics of Aedes aegypti from Sudan Based on ND4 Mitochondrial Gene Variations
Previous Article in Journal
Microbiome of the Aphid Genus Dysaphis Börner (Hemiptera: Aphidinae) and Its Relation to Ant Attendance
Previous Article in Special Issue
Invasion Pattern of Aedes aegypti in the Native Range of Ae. albopictus in Vietnam Revealed by Biogeographic and Population Genetic Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Insects
Volume 13
Issue 12
10.3390/insects13121090
insects-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

High Levels of Admixture in Anopheles gambiae Populations from Côte d’Ivoire Revealed by Multilocus Genotyping

by
Naminata Tondossama
1,2,
Zanakoungo I. Coulibaly
2,
Issouf Traoré
1,2,
Bérenger A. Ako
3,
Danielle D. Zoh
1,4,
Chiara Virgillito
5,
Négnorogo Guindo-Coulibaly
1,
Paola Serini
5,
Fabrice K. Assouho
4,
Ibrahima Dia
6,
Andre O. Touré
3,
Maurice A. Adja
1,4,
Beniamino Caputo
5,
Alessandra della Torre
5 and
Verena Pichler
5,*
1
Laboratoire de Biologie et Santé, UFR Biosciences, Université Félix Houphouet Boigny Cocody, Abidjan 01 BP V34, Côte d’Ivoire
2
Entomology and Herpetology Unit, Institut Pasteur de Côte d’Ivoire, Abidjan 01 PB 490, Côte d’Ivoire
3
Malaria Unit, Institut Pasteur de Côte d’Ivoire, Abidjan 01 PB 490, Côte d’Ivoire
4
Institut Pierre Richet/Institut National de Santé Publique, Bouaké 01 BP 1500, Côte d’Ivoire
5
Dipartimento di Sanità Pubblica e Malattie Infettive, Istituto Pasteur Italia-Fondazione Cenci-Bolognetti, Università di Roma “La Sapienza,” Piazzale Aldo Moro, 5, 00185 Rome, Italy
6
Pôle de Zoologie Médicale, Institut Pasteur de Dakar, 36 Avenue Pasteur, Dakar BP 220, Senegal
*
Author to whom correspondence should be addressed.
Insects 2022, 13(12), 1090; https://doi.org/10.3390/insects13121090
Submission received: 29 October 2022 / Revised: 22 November 2022 / Accepted: 23 November 2022 / Published: 26 November 2022
(This article belongs to the Special Issue Biology, Genetics and Evolution of Mosquitoes)
Browse Figure
Review Reports Versions Notes

Abstract

Simple Summary

Anopheles gambiae and An. coluzzii are two mosquito species with the most prominent role in transmitting malaria parasites to humans in the Afrotropical region. They are morphologically indistinguishable, but their ecological and behavioral differences affect their geographical distribution and may impact their role as malaria vectors and their response to malaria control interventions. A few genomic markers differentiate the two species and allow them to be consistently identified across most of their range. We here report the presence of two populations in Côte d’Ivoire characterized by an admixed pattern of these markers and try to understand their nature. Results do not support the hypothesis that the observed patterns are due to the current crossing between the two species, highlighting the constraints of currently available markers in clarifying the origin of the “unusual” populations in the country. Further analysis exploiting a larger set of markers will eventually solve this puzzle and allow a better understanding of its potential impact on malaria transmission and control.

Abstract

Anopheles coluzzii and An. gambiae—the two most recently radiated species of the An. gambiae complex and the major Afrotropical malaria vector species—are identified by markers in the X-centromeric IGS rDNA region. Putative IGS-hybrids are rarely found in the field, except in restricted areas where genomic studies have led to the hypothesis that the observed IGS-patterns are due to cryptic taxa rather than to hybridization between the two species. We investigated the genome-wide levels of admixture in two villages in Côte d’Ivoire where high levels of IGS-hybrids have been detected, confirming unparalleled high frequencies in the coastal village. Genotyping of 24 Ancestry Informative Markers (AIMs) along the three chromosomes produced discordant results between the IGS-marker and the multilocus genotype obtained for AIMs across the whole genome (29%) as well as AIMs on chromosome-X (considered to be fundamental for species reproductive isolation) only (21%). Results highlight a complicated pattern of admixture that deserves deeper genomic analyses to understand better possible underlying causes (from extensive processes of hybridization to the existence of different cryptic taxa), and stress the need of developing advanced diagnostics for An. coluzzii, An. gambiae and putative new taxa, instrumental for assessing taxon-specific epidemiological characters.

1. Introduction

The Anopheles gambiae complex comprises some of the most important malaria vectors present in sub-Saharan Africa. The nine recognized species in the complex are morphologically indistinguishable, but exhibit important differences at the ecological, behavioral, and epidemiological level, including their ability to vector efficiently the Plasmodium parasite [1]. While initially only crossing experiments could highlight the existence of such cryptic species, during the past few decades the increasing availability of cytogenetic and molecular techniques has allowed the detection of important sub-structuring within some of the species of the complex, leading eventually to the description of new taxonomic units. This has led to the split of the species An. quadriannulatus into An. quadriannulatus and An. amharicus [2], An. bwambae into An. Bwambae, and An. fontenillei [3], as well as An. gambiae s.s. into An. gambiae (formerly defined as molecular form S) and An. coluzzii (formerly defined as molecular form M) [2,4]. The latter two species represent the most efficient malaria vectors within the complex, owing to their strong anthropophilic and anthropophagic tendencies.
The two species are known to freely mate and produce viable progeny under laboratory conditions but premating and post-mating mechanisms contribute to their reproductive isolation in the field [5]. However, residual hybridization between the two species has very relevant implications. First, it has affected the effectiveness of insecticide-based vector control interventions by allowing the transfer from one species to the other of adaptive mutations, the most well-studied being the knock-down-resistance (kdr) mutation L1014F (or L995F; [6,7,8]) within the vgsc gene, highly associated with resistance to pyrethroids. Second, knowledge on ongoing hybridization is relevant for the correct planning of malaria vector control strategies based on releases of genetically manipulated males [9,10].
Anopheles gambiae and An. coluzzii are defined based on species-specific SNPs within the IGS region of the X-centromeric rDNA (IGS-marker; [4,11]) and the discrimination of the two species is commonly performed by molecular methods able to identify such polymorphisms [12,13,14]. Additionally, the An. coluzzii-exclusive insertion of a SINE situated in the X-centromere close to the rDNA region (SINE200 X6.1, hereafter SINE-marker) is frequently used to identify the two species [15]. Individuals characterized by heterozygous patterns are considered as putative hybrids and their sporadic finding in the field (on average <0.2%; [5]) represented the main proof of reproductive isolation based on which they have been raised to different taxa. Nowadays, all evidence of ecological interspecific differentiation—from larval site preferences [16,17,18] to capacity of dispersal by wind [19,20]—are based on specimens’ identification by the above markers.
In the last years, the Anopheles gambiae 1000 Genome (Ag1000G) consortium (https://www.malariagen.net/mosquito/ag1000g accessed on 24 July 2022) has provided strong evidence that IGS-identified specimens are consistent with a panel of 506 SNPs across the whole species’ genomes that are virtually fixed between them in most sub-Saharan Africa and are thus defined as Ancestry Informative Markers (AIMs) [8,21]. Only coastal populations at the western extremes of the two species’ range were shown to be characterized by stable frequencies >20% of putative hybrids [22,23,24,25,26,27] as identified by the IGS-marker (hereafter, IGS-hybrids), leading to the definition of this geographical region as High Hybridization zone [28]. Further investigations on populations from Guinea Bissau allowed to describe patterns of apparent admixture across the whole genome, leading to hypothesize the existence of a further cryptic form carrying an An. gambiae-like X-chromosome and An. coluzzii-like autosomes [25]. Whole Genome Sequence (WGS) data from the Ag1000G consortium provide support to the admixed nature of these populations and attributed to them an ”uncertain species status” due to the presence of a mixture of species-specific AIMs across the genome [8,9].
At the local level, high frequencies of IGS-hybrids in larval samples from Burkina Faso led to the hypothesis of the existence of further cryptic taxa, named GOUNDRY-form [29,30]. This was later shown to represent an admixed population descended from both An. coluzzii and an additional cryptic taxon named Anopheles TENGRELA [31]. On the other hand, high frequencies of IGS-hybrids in Mali were shown to represent temporary break-downs of reproductive isolation resulting in introgressive hybridization of adaptive alleles conferring resistance to pyrethroid insecticides (e.g., kdr alleles) [6,32].
Overall, these pieces of evidence suggest that high frequencies of IGS-hybrids are indicative of relevant biological phenomena—from novel forms/taxa to introgressive hybridization—which could impact the success of malaria vector control strategies. For this reason, we are here following up the report of the presence of IGS-hybrids at frequencies >20% and ~5% in a coastal and in an inland village in Côte d’Ivoire, respectively [33]. The presence of these IGS-hybrids has been confirmed in different seasons of the year at frequencies varying from 21% to 33% in the coastal village, suggesting this may be a stable phenomenon and not only a temporary breakdown of reproductive isolation. Moreover, PCR-genotyping of two AIMs on chromosome-3 showed occurrence of autosomal introgression [33]. Herein, we present the results of genotyping of a panel of AIMs across the genome in specimens sampled during the same time period and in the same two villages.

2. Materials and Methods

The study was carried out in two villages of Côte d’Ivoire, both of which are sentinel sites of the National Malaria Control Program (NMCP): Ayame (5.60216° N and 3.09290° W) a coastal village in the southeast region, and Pétessou (7.55505° N and 5.06052° W), situated in the central region. Frequencies of 27% and 5% of An. coluzzii/An. gambiae hybrid IGS-genotypes were reported in Ayame and Pétessou, respectively [33].
Mosquitoes were collected during December 2018 and in March, May, and October 2019 by pyrethrum spray collections for four consecutive days/month in five randomly selected houses/village. The protocol for this study was reviewed and approved by the National Research Ethics Committee of Côte d’Ivoire (023-18/1VISHP/CNER-kp). Free and informed consent was obtained from the heads of households for collection in their rooms. The collected mosquitoes were identified as An. gambiae s.l using the key of Gillies and Coetzee [34] and kept dry in microtubes containing silica gel.
DNA of An. gambiae s.l. females was extracted from single legs using DNAzol (MRC. Inc., Cincinnati, OH, USA) following the protocol described by Rider et al. [35] and extracted DNA was stored at −20 °C for future analysis. All collected specimens were genotyped for species-specific SNPs in the IGS-rDNA region by the IMP-PCR approach described by Wilkins et al. [14]. A multilocus approach [28] was applied to genotype a panel of 24 Ancestry Informative Markers (AIMs) [8] in randomly selected specimens from Ayame and Pétessou. To this aim, DNA was extracted from whole body using the DNA Blood and Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer protocol.
Subsequent library preparation as well as sequencing and filtering of sequences was performed at the Polo d’Innovazione di Genomica, Genetica e Biologia Srl within the framework of the Infravec2 project. Libraries for the amplicons spanning the 24 AIMs (10 on chromosome-X centromere, 6 on chromosome-2L centromere, 2 on chromosome-2R, 4 on chromosome-3R telomere and 2 on chromosome-3L centromere; Supplementary Material: Table S1; [28]) were prepared in accordance with RhAmpseq Library preparation kit (©2019 Integrated DNA Technologies Inc., Coralville, IA, USA) using the primer sequences listed in Table S1, and sequencing was performed using the NextSeq platform (2 × 150 paired end). The subsequent bioinformatic analysis was performed following manufacturers guidelines developed to analyze the rhAmpSeq sequencing data. Each sequenced sample was trimmed for adapter sequences during demultiplexing with bcl2fastq v2.20.0.422 software (2019 Illumina, Inc. San Diego, CA, USA) aiming at a preliminary assessment on the overall quality of the produced sequences. Quality control was done using FastQC tool [36]. The reads were aligned to the AgamP4.12 reference genome using the program BWA v0.7.15- r1140 (Burrows-Wheeler Aligner tool, [37]). A sorted-by-position aligned BAM file was produced for each of the 88 samples. Recalibration of base quality scores was performed using two GATK4 v4.2.5.0 packages: the BaseRecalibrator and the ApplyBQRS in order to identify and correct possible systematic errors that arose during the sequencing step when calculating the base quality score. The function TrimPrimers of the fgbio v0.5.1 program was used to trim the primer sequences from the amplicons to avoid any contribution of these sequences to the wild-type variation. The GATK4 packages HaplotypeCaller, CombineGVCFs and GenotypeGVCFs [38] were used to detect the variants for the 24 AIMs described by Caputo et al. [28]. The variants were filtered based on quality by means of the GATK package VariantFiltration [38].

3. Results

A total of 277 specimens were successfully identified by IGS-PCR (106 IGS-An. coluzzii, 109 IGS-An. gambiae and 62 IGS-hybrids). IGS-hybrids were found at frequencies of 38% and 7% in the coastal and inland village, respectively (Table 1).
Eighty-eight specimens (61 and 27 from the coastal and inland village, respectively) were processed by amplicon sequencing. Genotyping was not successful for 40 specimens, likely due to low quality/quantity of available DNA. In the 48 successfully genotyped specimens (coastal: 10 IGS-An. coluzzii, 11 IGS-An. gambiae, 8 IGS-hybrids; inland: 8 IGS-An. coluzzii, 10 IGS-An. gambiae, 1 IGS-hybrid) the genotyping success was >90% for each of the 24 AIMs analyzed and thus none of them was excluded from the analysis (Figure 1, Supplementary Material: Table S2).
Following suggestions by Caputo et al. [28], we defined a minimum number of consensus between species-specific variants to define pure An. gambiae, An. coluzzii, F1 and admixed specimens. No discordant AIMs were allowed for loci on chromosome-X, while a flexibility of 1 discordant species-specific variant was allowed for the 8 autosomal loci on chromosome arms 2R, 3R and 3L, in order to account for low levels of intraspecific autosomal polymorphism. Loci on chromosomal arm 2L were not considered to define species due to the widespread introgression from An. gambiae into An. coluzzii of the 2L centromeric region where the vgsc gene—carrying possibly kdr mutations involved in insecticide resistance—is located.
In both villages, An. coluzzii specimens showed consistent IGS and mulitlocus genotypes (N = 18; Table 2). In the inland village, 2 out of 8 IGS-An. gambiae specimens and the only IGS-hybrid genotyped were discordantly identified by the two genotyping approaches: one IGS-An. gambiae was genotyped as An. coluzzii by the mulitlocus approach, one IGS-An. gambiae showed a highly admixed genome and the only IGS-hybrid was genotyped as An. gambiae by all species-specific AIMs. In the coastal village, 7 out of 11 IGS-An. gambiae successfully genotyped were identified discordantly: one was identified by all AIMs as An. coluzzii, while 6 were defined as admixed by the multilocus approach. Interestingly, of these 6 specimens, two showed signs of admixture on both autosomes and X-chromosome, while 4 carried an An. gambiae-like X-centromere and all An. coluzzii specific autosomal markers (with exception of markers on chromosomal arm 2L). Of the eight specimens identified as IGS-hybrids, four were genotyped as pure An. coluzzii by the multilocus approach, while the remaining four carried signs of admixture on chromosome-X and predominantly An. coluzzii-like autosomal AIMs.
Overall, 71% (34/48) of specimens identified based on IGS markers (100% of An. coluzzii; 57% of An. gambiae and 44% of IGS-hybrids) show consistent AIM-genotypes. On the other hand, IGS-An. gambiae are characterized by either admixed (33%, i.e., 7/21) or An. coluzzii-like (10%, i.e., 2/21) AIMs, while IGS-hybrids are characterized by either An. coluzzii-like (44%, i.e., 4/9) or An. gambiae-like AIMs (11%, i.e.,1/9).
Observing the data based on results of the multilocus genotype, the frequency of specimens identified as An. gambiae decreases from 44% (IGS; i.e., 21/48) to 27% (AIMs; i.e., 13/48), while the percentage of specimens identified as either An. coluzzii or admixed increases from 37% (18/48) to 50% (24/48) and from 19% (9/48) to 23% (11/48), respectively. The 11 specimens with admixed multilocus genotype are characterized by either admixed (64%) or An. gambiae-like (36%) chromosome-X AIMs, and in most cases by An. coluzzii-like autosomal AIMs.
Focusing on the X-centromeric region (considered instrumental for the two species reproductive isolation; [39]), 21% discordances are observed between IGS-genotypes and the genotype resulting from the 10 AIMs in the region: 5 IGS-hybrids characterized by either An. coluzzii AIMs (N = 4) or An. gambiae AIMs (N = 1) and 5 IGS-An. gambiae characterized by either admixed AIMs (N = 3) or An. coluzzii ones (N = 2). Interestingly both the IGS and the multilocus approach identify signature of admixture in the X-centromere in a similar number of specimens (i.e., 9 IGS-hybrids vs 7 specimens with admixed X-centromeres AIMs), but these are not the same individuals.
In 21% of specimens, chromosome-X AIM-genotypes and autosomal AIMs on chromosomal arms 2R, 3R, and 3L produce inconsistent results. These are due to (i) 4 individuals characterized by An. Gambiae AIMs on chromosome-X (and An. Gambiae IGS-genotype) and An. Coluzzii AIMs on autosomes; (ii) 6 individuals characterized by admixed AIMs on chromosome-X and An. Coluzzii (N = 5) or An. Gambiae (N = 1) autosomal AIMs.
Results of the genotyping of AIMs on chromosomal arms 2L show strong signals of introgression in all individuals characterized by An. Coluzzii IGS and AIMs, as well as in those showing hybrid/admixed genotypes.

4. Discussion

Herein, we report IGS-hybrids in Côte d’Ivoire at frequencies rarely observed across the species range, as well as incongruences between IGS-diagnostic markers and chromosome-X AIMs and between chromosome-X and autosomal AIMs.
The high IGS-hybrid frequency observed in the coastal village (38%) are unparalleled even by frequencies in the putative high hybridization zone at the westernmost extremes of the species range (<25%; [22,23,24,25,26,27]), but consistent with those resulting from CDC collections carried out in the same village in 2018–2019 (21–33%; [33]. Notably, the literature data describing species composition in Côte d’Ivoire (Supplementary Material: Table S3) either do no report presence of IGS-hybrids, or report them at frequencies ranging from <0.5% [40,41] to 11% [42]. Several explanations may account for this variability in the observed frequency of IGS-hybrids.
First, Ayame village may lay within a region of high hybridization triggered by balanced frequencies of An. coluzzii and An. gambiae [33], which may favor high levels of hybridization due to increased inter-specific contact, as hypothesized by Pombi et al. [5]. This may support the hypothesis of a temporary breakdown of reproductive barriers restricted to Cote d’Ivoire south-east region in 2018–2019, leading to bouts of hybridization and gene-flow and, thus, important variations in frequency of IGS-hybrids within close years [32]. Identification of further An. gambiae samples from different years could help to confirm or dispute this hypothesis.
However, differences between frequencies reported herein and the literature data could also be due to methodological biases which can lead to an underreporting of putative hybrids in scientific publications. First, reporting of a few IGS-hybrids may be considered a neglectable information (or even out of scope) in the case of papers focusing on investigating epidemiologically relevant factors—such as insecticide resistance or other aspects of An. coluzzii and An. gambiae bionomics directly linked to malaria transmission. Second, the choice of the diagnostic marker used to identify the two species can impact observed frequencies of putative hybrids. In fact, except for Caputo et al. [33], all recent papers reporting the presence of putative hybrids in Cote d’Ivoire (Supplementary Material: Table S3) use the PCR-amplification of an An. coluzzii-specific X-centromeric SINE insertion as identification method [15]. Although this method provides results mostly consistently with IGS-PCR, it has been highlighted that—being based on a single copy and irreversible SINE200 insertion—it is not subjected to peculiar evolutionary patterns which instead affect rDNA repeats carrying the IGS-marker, thus leading to a lower rate of heterozygote/hybrid patters compared to the IGS-PCR [43]. In fact, IGS-based identifications can overestimate frequencies of putative hybrids compared to SINE-based identifications, due to an incomplete homogenization of the rDNA arrays leading to the co-existence of An. coluzzii and An. gambiae specific IGS-sequences on single chromosomes, as shown in hemizygous males [44].
Results of genotyping of 18 AIMs on chromosomal arms X, 2R, 3R, and 3L allow to dispute the hypothesis of a current breakdown of reproductive barriers between An. coluzzii and An. gambiae since no individuals characterized by consistent heterozygous IGS and AIM patterns (expected for F1 hybrids) are observed. The presence instead of IGS-hybrids carrying concordant homozygous AIM patterns across the genome does support the hypothesis of the presence of admixed IGS arrays which have not been homogenized yet by the process of concerted evolution.
Discordances between the AIMs situated on chromosome-X and on chromosomal arms 2R, 3R, and 3L further complicate the picture. Patterns of admixture seem to be different from what is observed in coastal Guinea Bissau and The Gambia, where linkage among autosomal AIMs is apparently lower than in Côte d’Ivoire [28]. Anyway, it needs to be acknowledged that the sample size analyzed herein is low and genotyping of a higher number of specimens will be needed to provide a more reliable picture and to compare the observed patterns of admixture with that observed in the so-called high hybridization zone in far-West Africa.
From the methodological perspective, the consistency of results of the multilocus genotyping performed herein with those of the PCR-genotyping of only two AIMs on chromosome-3 (i.e., 3R:42848 and 3L:129051) reinforces results obtained by Caputo et al. [33], as well as the value of the two chromosome-3 AIMs in revealing signatures of genomic admixture when associated to IGS-diagnostics. However, it should be noted that the multilocus approach allows the genotyping of 10 AIMs in the chromosome-X centromeric region where also the IGS-marker is located. This allows a direct comparison between these markers and, possibly, a more reliable representation of this so called “speciation island”, where genes implicated in reproductive isolation between An. coluzzii and An. gambiae are believed to be located [39].

5. Conclusions

Overall, the present results highlight the growing problem of correctly interpreting high frequencies of heterozygous IGS-genotypes, conventionally defined as putative hybrids between An. coluzzii and An. gambiae and generally reported at frequencies <0.2% [5]. As in the cases of populations at the westernmost extreme of the species range [22,23,24,25,27,28] and of the Tengrela population in Burkina Faso [31], the finding of unparalleled frequencies of IGS-hybrids in the coastal village of Côte d’Ivoire (reported also by [33]) reveals a complicated pattern of admixture that deserves deeper genomic analyses to allow a better understanding of possible underlying causes, from extensive processes of hybridization to the existence of further cryptic taxa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects13121090/s1. Table S1: Information on Ancestry Informative SNPs genotyped and primer sequences used for the amplicon sequencing approach. Table S2: Genotyping results obtained for 24 Ancestry Informative SNPs for IGS-An.gambiae, IGS-An.coluzzii and IGS-hybrids from a coastal (Ayame) and an inland (Petessou) village Côte d’Ivoire. Table S3: Details on scientific publications reporting An.gambiae species composition in Côte d’Ivoire since 2010.

Author Contributions

Conceptualization, A.d.T., A.O.T., B.C., V.P., N.T., I.D., Z.I.C. and M.A.A.; methodology, B.A.A., B.C., C.V., D.D.Z., F.K.A., I.T., N.G.-C., N.T., P.S. and V.P.; formal analysis, B.C., C.V. and V.P.; data curation, V.P.; writing—original draft preparation, A.d.T., N.T. and V.P.; writing—review and editing, A.d.T., A.O.T., B.C., B.A.A., C.V., D.D.Z., F.K.A., I.D., I.T., M.A.A., N.G.-C., N.T., P.S., V.P. and Z.I.C.; supervision, A.d.T., A.O.T., B.C., I.D., M.A.A., V.P. and Z.I.C.; funding acquisition, B.C. All authors have read and agreed to the published version of the manuscript.

Funding

This publication was supported by the ExGenMal Institute Pasteur grant (ACIP No. 41–2017; PI: BC) and the project Research Infrastructures for the control of vector-borne diseases (Infravec2), which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 731060.

Data Availability Statement

All data are available within the article and its Supplementary Material.

Acknowledgments

We heartily thank inhabitants of the villages of Ayame and Petessou villages for their cooperation in the field part of study.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. White, B.J.; Lawniczak, M.K.N.; Cheng, C.; Coulibaly, M.B.; Wilson, M.D.; Sagnon, N.F.; Costantini, C.; Simard, F.; Christophides, G.K.; Besansky, N.J. Adaptive Divergence between Incipient Species of Anopheles Gambiae Increases Resistance to Plasmodium. Proc. Natl. Acad. Sci. USA 2011, 108, 244. [Google Scholar] [CrossRef] [PubMed]
  2. Coetzee, M.; Hunt, R.H.; Wilkerson, R.; Della Torre, A.; Coulibaly, M.B.; Besansky, N.J. Anopheles Coluzzii and Anopheles Amharicus, New Members of the Anopheles Gambiae Complex. Zootaxa 2013, 3619, 246–274. [Google Scholar] [CrossRef] [PubMed]
  3. Barrón, M.G.; Paupy, C.; Rahola, N.; Akone-Ella, O.; Ngangue, M.F.; Wilson-Bahun, T.A.; Pombi, M.; Kengne, P.; Costantini, C.; Simard, F.; et al. A New Species in the Major Malaria Vector Complex Sheds Light on Reticulated Species Evolution. Sci. Rep. 2019, 9, 14753. [Google Scholar] [CrossRef] [PubMed]
  4. della Torre, A.; Fanello, C.; Akogbeto, M.; Favia, G.; Petrarca, V.; Coluzzi, M. Molecular Evidence of Incipient Speciation within Anopheles Gambiae s. s. in West Africa. Insect Mol. Biol. 2001, 10, 9–18. [Google Scholar] [CrossRef] [PubMed]
  5. Pombi, M.; Kengne, P.; Tene-fossog, G.G.B.; Ayala, D.; Kamdem, C.; Santolamazza, F.; Moussa, W.; Falé, G.N.; Vincenzo, S.; Fontenille, D.; et al. Dissecting Functional Components of Reproductive Isolation among Closely Related Sympatric Species of the Anopheles Gambiae Complex. Evol. Appl. 2017, 10, 1102–1120. [Google Scholar] [CrossRef] [PubMed]
  6. Clarkson, C.S.; Weetman, D.; Essandoh, J.; Yawson, A.E.; Maslen, G.; Manske, M.; Field, S.G.; Webster, M.; Antão, T.; MacInnis, B.; et al. Adaptive Introgression between Anopheles Sibling Species Eliminates a Major Genomic Island but Not Reproductive Isolation. Nat. Commun. 2014, 5, 4248. [Google Scholar] [CrossRef]
  7. Clarkson, C.S.; Miles, A.; Harding, N.J.; O’Reilly, A.O.; Weetman, D.; Kwiatkowski, D.; Donnelly, M.J. The Genetic Architecture of Target-Site Resistance to Pyrethroid Insecticides in the African Malaria Vectors Anopheles Gambiae and Anopheles Coluzzii. Mol. Ecol. 2021, 30, 5303–5317. [Google Scholar] [CrossRef]
  8. The Anopheles gambiae 1000 Genomes Consortium Genetic Diversity of the African Malaria Vector Anopheles Gambiae. Nature 2017, 552, 96–100. [CrossRef]
  9. The Anopheles gambiae 1000 Genomes Consortium Genome Variation and Population Structure among 1142 Mosquitoes of the African Malaria Vector Species Anopheles Gambiae and Anopheles Coluzzii. Genome Res. 2018, 30, 1533–1546. [CrossRef]
  10. Kyrou, K.; Hammond, A.M.; Galizi, R.; Kranjc, N.; Burt, A.; Beaghton, A.K.; Nolan, T.; Crisanti, A. A CRISPR–Cas9 Gene Drive Targeting Doublesex Causes Complete Population Suppression in Caged Anopheles Gambiae Mosquitoes. Nat. Biotechnol. 2018, 36, 1062–1071. [Google Scholar] [CrossRef]
  11. Favia, G.; Lanfrancotti, A.; Spanos, L.; Sidén-Kiamos, I.; Louis, C. Molecular Characterization of Ribosomal DNA Polymorphisms Discriminating among Chromosomal Forms of Anopheles Gambiae s.S. Insect Mol. Biol. 2001, 10, 19–23. [Google Scholar] [CrossRef]
  12. Fanello, C.; Santolamazza, F.; della Torre, A. Simultaneous Identification of Species and Molecular Forms of the Anopheles Gambiae Complex by PCR-RFLP. Med. Vet. Entomol. 2002, 16, 461–464. [Google Scholar] [CrossRef] [PubMed]
  13. Santolamazza, F.; Della Torre, A.; Caccone, A. Short Report: A New Polymerase Chain Reaction − Restriction Fragment Length Polymorphism Method To Identify Anopheles Arabiensis From An. Gambiae and Its Two Molecular Forms From Degraded Dna Templates or Museum Samples. Trop. Med. 2004, 70, 604–606. [Google Scholar] [CrossRef]
  14. Wilkins, E.E.; Howell, P.I.; Benedict, M.Q. IMP PCR Primers Detect Single Nucleotide Polymorphisms for Anopheles Gambiae Species Identification, Mopti and Savanna RDNA Types, and Resistance to Dieldrin in Anopheles Arabiensis. Malar. J. 2006, 5, 125. [Google Scholar] [CrossRef] [PubMed]
  15. Santolamazza, F.; Mancini, E.; Simard, F.; Qi, Y.; Tu, Z.; della Torre, A. Insertion Polymorphisms of SINE200 Retrotransposons within Speciation Islands of Anopheles Gambiae Molecular Forms. Malar. J. 2008, 7, 163. [Google Scholar] [CrossRef] [PubMed]
  16. Lehmann, T.; Diabate, A. The Molecular Forms of Anopheles Gambiae: A Phenotypic Perspective. Infect. Genet. Evol. 2008, 8, 737–746. [Google Scholar] [CrossRef]
  17. Gimonneau, G.; Pombi, M.; Dabiré, R.K.; Diabaté, A.; Morand, S.; Simard, F. Behavioural Responses of Anopheles Gambiae Sensu Stricto M and S Molecular Form Larvae to an Aquatic Predator in Burkina Faso. Parasit. Vectors 2012, 5, 65. [Google Scholar] [CrossRef]
  18. Gimonneau, G.; Pombi, M.; Choisy, M.; Morand, S.; Dabiré, R.K.; Simard, F.; Sapienza, R.; Moro, P.A.; Fondazione, P. Larval Habitat Segregation between the Molecular Forms of the Mosquito, Anopheles Gambiae in a Rice Field Area of Burkina Faso, West Africa. Med. Vet. Entomol. 2012, 26, 9–17. [Google Scholar] [CrossRef]
  19. Lehmann, T.; Dao, A.; Yaro, A.S.; Diallo, M. Seasonal Variation in Spatial Distribution of Anopheles Gambiaein a Sahelina Village: Evidence for Aestivation. J. Med. Entomol. 2014, 29, 997–1003. [Google Scholar] [CrossRef]
  20. Huestis, D.L.; Dao, A.; Diallo, M.; Sanogo, Z.L.; Samake, D.; Yaro, A.S.; Ousman, Y.; Linton, Y.-M.; Krishna, A.; Veru, L.; et al. Windborne Long-Distance Migration of Malaria Mosquitoes in the Sahel. Nature 2019, 574, 404–408. [Google Scholar] [CrossRef]
  21. The Anopheles gambiae 1000 Genomes Consortium Natural Diversity of the Malaria Vector Anopheles Gambiae. BioRxiv 2016, 096289. [CrossRef]
  22. Niang, E.H.A.; Konaté, L.; Diallo, M.; Faye, O.; Dia, I. Reproductive Isolation among Sympatric Molecular Forms of An. Gambiae from Inland Areas of South-Eastern Senegal. PLoS ONE 2014, 9, e104622. [Google Scholar] [CrossRef]
  23. Nwakanma, D.C.; Neafsey, D.E.; Jawara, M.; Adiamoh, M.; Lund, E.; Rodrigues, A.; Loua, K.M.; Konate, L.; Sy, N.; Dia, I.; et al. Breakdown in the Process of Incipient Speciation in Anopheles Gambiae. Genetics 2013, 193, 1221–1231. [Google Scholar] [CrossRef]
  24. Oliveira, E.; Salgueiro, P.; Palsson, K.; Vicente, J.L.; Arez, P.; Jaenson, T.G.; Caccone, A.; Pinto, J. High Levels of Hybridization between Molecular Forms of Anopheles Gambiae from Guinea Bissau. J. Med. Entomol. 2008, 45, 1057–1063. [Google Scholar] [CrossRef]
  25. Vicente, J.L.; Clarkson, C.S.; Caputo, B.; Gomes, B.; Pombi, M.; Sousa, C.A.; Tiago, A.; Dinis, J.; Bottà, G.; Mancini, E.; et al. Massive Introgression Drives Species Radiation at the Range Limit of Anopheles Gambiae? Sci. Rep. 2017, 7, 46451. [Google Scholar] [CrossRef]
  26. Caputo, B.; Nwakanma, D.; Jawara, M.; Adiamoh, M.; Dia, I.; Konate, L.; Petrarca, V.; Conway, D.J.; della Torre, A. Anopheles Gambiae Complex along The Gambia River, with Particular Reference to the Molecular Forms of An. Gambiae s.S. Malar. J. 2008, 7, 182. [Google Scholar] [CrossRef] [PubMed]
  27. Caputo, B.; Santolamazza, F.; Vicente, J.L.; Nwakanma, D.C.; Jawara, M.; Palsson, K.; Jaenson, T.; White, B.J.; Mancini, E.; Petrarca, V.; et al. The “Far-West” of Anopheles Gambiae Molecular Forms. PLoS ONE 2011, 6, e16415. [Google Scholar] [CrossRef]
  28. Caputo, B.; Pichler, V.; Bottà, G.; De Marco, C.; Hubbart, C.; Perugini, E.; Pinto, J.; Rockett, K.A.; Miles, A.; della Torre, A. Novel Genotyping Approaches to Easily Detect Genomic Admixture between the Major Afrotropical Malaria Vector Species, Anopheles Coluzzii and An. Gambiae. Mol. Ecol. Resour. 2021, 21, 1504–1506. [Google Scholar] [CrossRef] [PubMed]
  29. Riehle, M.M.; Guelbeogo, W.M.; Gneme, A.; Eiglmeier, K.; Holm, I.; Bischoff, E.; Garnier, T.; Snyder, G.M.; Li, X.; Markianos, K.; et al. A Cryptic Subgroup of Anopheles Gambiae Is Highly Susceptible to Human Malaria Parasites. Science 2011, 331, 596–598. [Google Scholar] [CrossRef] [PubMed]
  30. Crawford, J.E.; Riehle, M.M.; Markianos, K.; Bischoff, E.; Guelbeogo, W.M.; Gneme, A.; Sagnon, N.; Vernick, K.D.; Nielsen, R.; Lazzaro, B.P. Evolution of GOUNDRY, a Cryptic Subgroup of Anopheles Gambiae s.l., and Its Impact on Susceptibility to Plasmodium Infection. Mol. Ecol. 2016, 25, 1494–1510. [Google Scholar] [CrossRef]
  31. Tennessen, J.A.; Ingham, V.A.; Toé, K.H.; Guelbéogo, W.M.; Sagnon, N.; Kuzma, R.; Ranson, H.; Neafsey, D.E. A Population Genomic Unveiling of a New Cryptic Mosquito Taxon within the Malaria-Transmitting Anopheles Gambiae Complex. Mol. Ecol. 2021, 30, 775–790. [Google Scholar] [CrossRef] [PubMed]
  32. Lee, Y.; Marsden, C.D.; Norris, L.C.; Collier, T.C.; Main, B.J.; Fofana, A.; Cornel, A.J.; Lanzaro, G.C. Spatiotemporal Dynamics of Gene Flow and Hybrid Fitness between the M and S Forms of the Malaria Mosquito, Anopheles Gambiae. Proc. Natl. Acad. Sci. USA. 2013, 110, 19854–19859. [Google Scholar] [CrossRef]
  33. Caputo, B.; Tondossoma, N.; Virgillito, C.; Pichler, V.; Serini, P.; Calzetta, M.; Manica, M.; Coulibaly, Z.I.; Dia, I.; Akré, M.A.; et al. Is Côte D’Ivoire a New High Hybridization Zone for the Two Major Malaria Vectors, Anopheles Coluzzii and An. Gambiae (Diptera, Culicidae)? Infect. Genet. Evol. 2022, 98, 105215. [Google Scholar] [CrossRef] [PubMed]
  34. Gillies, M.T.; Coetzee, M. A Supplement to the Anophelinae of Africa South of the Sahara; South African Institute for Medical Research: Johannesburg, South Africa, 1987; Volume 55, pp. 1–143. [Google Scholar]
  35. Rider, M.A.; Byrd, B.D.; Keating, J.; Wesson, D.M.; Caillouet, K.A. PCR Detection of Malaria Parasites in Desiccated Anopheles Mosquitoes Is Uninhibited by Storage Time and Temperature. Malar. J. 2012, 11, 193. [Google Scholar] [CrossRef]
  36. Andrews, S. FASTQC: A Quality Control Tool for High Throughput Sequence Data; ScienceOpen: Online, 2010. [Google Scholar]
  37. Li, H.; Durbin, R. Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef]
  38. Van der Auwera, G.; O’Connor, B. Genomics in the Cloud: Using Docker, GATK, and WDL in Terra (1st Edition), 1st ed.; O’Reilly Media: Sebastopol, CA, USA, 2020. [Google Scholar]
  39. Aboagye-antwi, F.; Alhafez, N.; Weedall, G.D.; Brothwood, J. Experimental Swap of Anopheles Gambiae ’ s Assortative Mating Preferences Demonstrates Key Role of X-Chromosome Divergence Island in Incipient Sympatric Speciation. PLoS Genet. 2015, 11, e1005141. [Google Scholar] [CrossRef]
  40. Wipf, N.C.; Duchemin, W.; Kouadio, F.P.A.; Fodjo, B.K.; Sadia, C.G.; Mouhamadou, C.S.; Vavassori, L.; Mäser, P.; Mavridis, K.; Vontas, J.; et al. Multi-Insecticide Resistant Malaria Vectors in the Field Remain Susceptible to Malathion, despite the Presence of Ace1 Point Mutations. PLoS Genet. 2022, 18, e1009963. [Google Scholar] [CrossRef]
  41. Pelloquin, B.; Kristan, M.; Edi, C.; Meiwald, A.; Clark, E.; Jeffries, C.L.; Walker, T.; Dada, N.; Messenger, L.A. Overabundance of Asaia and Serratia Bacteria Is Associated with Deltamethrin Insecticide Susceptibility in Anopheles Coluzzii from Agboville, Côte d’Ivoire. Microbiol. Spectr. 2021, 9, e0015721. [Google Scholar] [CrossRef]
  42. Edi, V.A.C.; N’Dri, B.P.; Chouaibou, M.; Kouadio, F.B.; Pignatelli, P.; Raso, G.; Weetman, D.; Bonfoh, B. First Detection of N1575Y Mutation in Pyrethroid Resistant Anopheles Gambiae in Southern Côte d’ Ivoire. Wellcome Open Res. 2017, 2, 71. [Google Scholar] [CrossRef]
  43. Santolamazza, F.; Caputo, B.; Calzetta, M.; Vicente, J.L.; Mancini, E.; Petrarca, V.; Pinto, J.; Della Torre, A. Comparative Analyses Reveal Discrepancies among Results of Commonly Used Methods for Anopheles Gambiaemolecular Form Identification. Malar. J. 2011, 10, 215. [Google Scholar] [CrossRef] [PubMed]
  44. Caputo, B.; Pichler, V.; Mancini, E.; Pombi, M.; Vicente, J.L.; Dinis, J.; Steen, K.; Petrarca, V.; Rodrigues, A.; Pinto, J.; et al. The Last Bastion? X Chromosome Genotyping of Anopheles Gambiae Species Pair Males from a Hybrid Zone Reveals Complex Recombination within the Major Candidate “Genomic Island of Speciation.” . Mol. Ecol. 2016, 25, 5719–5731. [Google Scholar] [CrossRef] [PubMed]
Insects 13 01090 g001
Figure 1. Results of multilocus genotyping of IGS-An. gambiae, IGS-An. coluzzii and IGS-hybrid specimens from the coastal (Ayame) and the inland (Petessou) villages in Côte d’Ivoire. Each row represents an individual mosquito and columns represent genotyped Ancestry Informative Markers [21]. The first column represents the species ID based on the IGS diagnostic marker. Below the figure the approximate position of AIMs on chromosomal arms. Blue = homozygote for An. gambiae specific alleles, red = homozygote for An. coluzzii specific alleles, yellow = heterozygote, grey = NA.
Figure 1. Results of multilocus genotyping of IGS-An. gambiae, IGS-An. coluzzii and IGS-hybrid specimens from the coastal (Ayame) and the inland (Petessou) villages in Côte d’Ivoire. Each row represents an individual mosquito and columns represent genotyped Ancestry Informative Markers [21]. The first column represents the species ID based on the IGS diagnostic marker. Below the figure the approximate position of AIMs on chromosomal arms. Blue = homozygote for An. gambiae specific alleles, red = homozygote for An. coluzzii specific alleles, yellow = heterozygote, grey = NA.
Insects 13 01090 g001
Table 1. Frequency of IGS-An. coluzzii, IGS-An. gambiae and IGS-hybrids (GA/CO) in a coastal (Ayame) and in an inland (Petessou) village in Cote d’Ivoire. N = number of specimens identified by IGS markers [14].
Table 1. Frequency of IGS-An. coluzzii, IGS-An. gambiae and IGS-hybrids (GA/CO) in a coastal (Ayame) and in an inland (Petessou) village in Cote d’Ivoire. N = number of specimens identified by IGS markers [14].
Sampling SiteNAn.coluzziiGA/COAn.gambiae
Coastal1390.340.380.28
Inland1380.430.070.51
Table 2. Results of multilocus genotyping of IGS-An. Gambiae, IGS-An. Coluzzii, and IGS-hybrid specimens from the coastal (Ayame) and the inland (Petessou) villages in Côte d’Ivoire. Left panel: results from genotyping of 18 Ancestry Informative Markers (AIMs, [21]) on chromosomal arms X, 2R, 3R, and 3L. Individuals are defined as admixed (adm) when at least 1 locus on chromosome- X or >1 out of 8 autosomal loci is not consistent with the other loci. Right panel: results from genotyping of 10 AIMs on chromosome-X. Individuals are defined as admixed (adm) when at least 1 locus is non consistent with the other loci. Italics = specimens identified discordantly by the IGS- and the multilocus approach.
Table 2. Results of multilocus genotyping of IGS-An. Gambiae, IGS-An. Coluzzii, and IGS-hybrid specimens from the coastal (Ayame) and the inland (Petessou) villages in Côte d’Ivoire. Left panel: results from genotyping of 18 Ancestry Informative Markers (AIMs, [21]) on chromosomal arms X, 2R, 3R, and 3L. Individuals are defined as admixed (adm) when at least 1 locus on chromosome- X or >1 out of 8 autosomal loci is not consistent with the other loci. Right panel: results from genotyping of 10 AIMs on chromosome-X. Individuals are defined as admixed (adm) when at least 1 locus is non consistent with the other loci. Italics = specimens identified discordantly by the IGS- and the multilocus approach.
Multilocus IDX- Multilocus ID
VillageIGS-IDCOadmGACOadmGATotal
CoastalCO10--10 10
GA/CO44-44 8
GA16412811
Total15104156829
InlandCO8--8 8
GA/CO--1 11
GA11811810
Total91991919
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Tondossama, N.; Coulibaly, Z.I.; Traoré, I.; Ako, B.A.; Zoh, D.D.; Virgillito, C.; Guindo-Coulibaly, N.; Serini, P.; Assouho, F.K.; Dia, I.; et al. High Levels of Admixture in Anopheles gambiae Populations from Côte d’Ivoire Revealed by Multilocus Genotyping. Insects 2022, 13, 1090. https://doi.org/10.3390/insects13121090

AMA Style

Tondossama N, Coulibaly ZI, Traoré I, Ako BA, Zoh DD, Virgillito C, Guindo-Coulibaly N, Serini P, Assouho FK, Dia I, et al. High Levels of Admixture in Anopheles gambiae Populations from Côte d’Ivoire Revealed by Multilocus Genotyping. Insects. 2022; 13(12):1090. https://doi.org/10.3390/insects13121090

Chicago/Turabian Style

Tondossama, Naminata, Zanakoungo I. Coulibaly, Issouf Traoré, Bérenger A. Ako, Danielle D. Zoh, Chiara Virgillito, Négnorogo Guindo-Coulibaly, Paola Serini, Fabrice K. Assouho, Ibrahima Dia, and et al. 2022. "High Levels of Admixture in Anopheles gambiae Populations from Côte d’Ivoire Revealed by Multilocus Genotyping" Insects 13, no. 12: 1090. https://doi.org/10.3390/insects13121090

APA Style

Tondossama, N., Coulibaly, Z. I., Traoré, I., Ako, B. A., Zoh, D. D., Virgillito, C., Guindo-Coulibaly, N., Serini, P., Assouho, F. K., Dia, I., Touré, A. O., Adja, M. A., Caputo, B., della Torre, A., & Pichler, V. (2022). High Levels of Admixture in Anopheles gambiae Populations from Côte d’Ivoire Revealed by Multilocus Genotyping. Insects, 13(12), 1090. https://doi.org/10.3390/insects13121090

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop