Gene drive in species complexes: defining target organisms
2022; Elsevier BV; Volume: 41; Issue: 2 Linguagem: Inglês
10.1016/j.tibtech.2022.06.013
ISSN0167-9430
AutoresJohn B. Connolly, Jörg Romeis, Yann Devos, Boet Glandorf, Geoff Turner, Mamadou B. Coulibaly,
Tópico(s)Transgenic Plants and Applications
ResumoEngineered gene drives share many environmental risk assessment considerations with other transgenes in genetically modified organisms, but they can differ significantly in their potential to spread, increase in frequency, and persist in target populations.Recently, introduction of mosquitoes with an engineered gene drive completely suppressed caged wild type laboratory populations of the malaria vector Anopheles gambiae, belonging to a species complex containing both vector and nonvector species that can produce fertile interspecific hybrids.As target sequences of the gene drive are conserved amongst all species of this complex, vertical gene drive transfer to both vectors and nonvectors is plausible. This challenges the notion of a simple dichotomy between target organism and nontarget organism.Using this gene drive as a specific case study, options on defining target organisms of engineered gene drives in species complexes are developed here, including proposal of the new concept of target species complex. Engineered gene drives, which bias their own inheritance to increase in frequency in target populations, are being developed to control mosquito malaria vectors. Such mosquitoes can belong to complexes of both vector and nonvector species that can produce fertile interspecific hybrids, making vertical gene drive transfer (VGDT) to sibling species biologically plausible. While VGDT to other vectors could positively impact human health protection goals, VGDT to nonvectors might challenge biodiversity ones. Therefore, environmental risk assessment of gene drive use in species complexes invites more nuanced considerations of target organisms and nontarget organisms than for transgenes not intended to increase in frequency in target populations. Incorporating the concept of target species complexes offers more flexibility when assessing potential impacts from VGDT. Engineered gene drives, which bias their own inheritance to increase in frequency in target populations, are being developed to control mosquito malaria vectors. Such mosquitoes can belong to complexes of both vector and nonvector species that can produce fertile interspecific hybrids, making vertical gene drive transfer (VGDT) to sibling species biologically plausible. While VGDT to other vectors could positively impact human health protection goals, VGDT to nonvectors might challenge biodiversity ones. Therefore, environmental risk assessment of gene drive use in species complexes invites more nuanced considerations of target organisms and nontarget organisms than for transgenes not intended to increase in frequency in target populations. Incorporating the concept of target species complexes offers more flexibility when assessing potential impacts from VGDT. Gene drives (see Glossary) can allow genes, transgenes, or genetic traits to be transmitted to offspring at greater than Mendelian frequencies, a property that offers the potential for a genetic modification of interest to spread and increase in frequency through interbreeding target populations [1.Burt A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations.Proc. Biol. Sci. 2003; 270: 921-928Crossref PubMed Scopus (442) Google Scholar, 2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar, 3.Wang G.H. et al.Symbionts and gene drive: two strategies to combat vector-borne disease.Trends Genet. 2022; 38: 708-723Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar]. In particular, there is considerable interest in harnessing gene drives to control mosquito species that are vectors of human diseases such as malaria (Box 1) [4.African Union Development Agency – New Partnership for Africa's Development Gene Drives for Malaria Control and Elimination in Africa.2018Google Scholar,5.World Health Organization Evaluation of Genetically Modified Mosquitoes for the Control of Vector-Borne Diseases. WHO, 2020Google Scholar].Box 1Malaria and engineered gene drivesThe World Health Organization (WHO) has reported that in 2020 there were 241 million cases of malaria worldwide, associated with an estimated 627 000 deaths [68.World Health Organization World Malaria Report. WHO, 2021Google Scholar]. Countries from the WHO African Region continue to carry a disproportionately high share of the global malaria burden, being associated with 95% of malaria cases and 96% of malaria deaths. Plasmodium falciparum is the major pathogen responsible for causing malaria in humans and is spread via the bites of infected female Anopheles mosquitoes as they blood-feed on hosts to provide essential nutrients for development of their eggs. As a result of the use of insecticide-treated bed nets and indoor insecticide spraying, as well as both prophylactic and therapeutic pharmaceutical treatments, there has been a steady decline in malaria prevalence over the last decade. However, progress has recently stalled and remains under further threat from insecticide resistance and behavioural adaptations, such as increased zoophilic responses, in mosquitoes, as well as Plasmodium resistance to drugs, invoking the need for complementary approaches to reduce the burden of this disease, including via the use of novel vector control tools such as engineered gene drives [3.Wang G.H. et al.Symbionts and gene drive: two strategies to combat vector-borne disease.Trends Genet. 2022; 38: 708-723Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4.African Union Development Agency – New Partnership for Africa's Development Gene Drives for Malaria Control and Elimination in Africa.2018Google Scholar, 5.World Health Organization Evaluation of Genetically Modified Mosquitoes for the Control of Vector-Borne Diseases. WHO, 2020Google Scholar,68.World Health Organization World Malaria Report. WHO, 2021Google Scholar].Current gene drive strategies include the use of transgenes encoding both the CRISPR-Cas9 endonuclease that is expressed under the control of a germline promoter, along with ubiquitously and constitutively expressed gRNAs, that together can target and cleave specific sequences in the genome [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar,69.Hammond A.M. Galizi R. Gene drives to fight malaria: current state and future directions.Pathog. Glob. Health. 2018; 111: 412-423Crossref Scopus (62) Google Scholar]. Once the transgene is introduced into its genomic target location on one of a pair of homologous chromosomes, the gRNA and Cas9 act in concert in germ cells to cause a double-stranded break in the target DNA site of the wild-type homologous chromosome. When this double-stranded lesion is repaired by homology directed repair using the transgenic homologous chromosome as a template, the entire transgene, along with flanking sequences either side of the transgene, is pasted into the target site of the homologous, formerly wild-type, chromosome. This process of homing can create mostly pairs of parental homologous chromosomes that are homozygous for the transgene, so that a greater proportion of parental germ cells are transgenic than would otherwise be the case, leading to super-Mendelian inheritance of the transgene in progeny. Thus, once introduced into a mating population, transgenes capable of homing will increase in frequency, or drive, within that population, assuming any fitness costs from the transgene do not outweigh its increased transmission from homing [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar]. The World Health Organization (WHO) has reported that in 2020 there were 241 million cases of malaria worldwide, associated with an estimated 627 000 deaths [68.World Health Organization World Malaria Report. WHO, 2021Google Scholar]. Countries from the WHO African Region continue to carry a disproportionately high share of the global malaria burden, being associated with 95% of malaria cases and 96% of malaria deaths. Plasmodium falciparum is the major pathogen responsible for causing malaria in humans and is spread via the bites of infected female Anopheles mosquitoes as they blood-feed on hosts to provide essential nutrients for development of their eggs. As a result of the use of insecticide-treated bed nets and indoor insecticide spraying, as well as both prophylactic and therapeutic pharmaceutical treatments, there has been a steady decline in malaria prevalence over the last decade. However, progress has recently stalled and remains under further threat from insecticide resistance and behavioural adaptations, such as increased zoophilic responses, in mosquitoes, as well as Plasmodium resistance to drugs, invoking the need for complementary approaches to reduce the burden of this disease, including via the use of novel vector control tools such as engineered gene drives [3.Wang G.H. et al.Symbionts and gene drive: two strategies to combat vector-borne disease.Trends Genet. 2022; 38: 708-723Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4.African Union Development Agency – New Partnership for Africa's Development Gene Drives for Malaria Control and Elimination in Africa.2018Google Scholar, 5.World Health Organization Evaluation of Genetically Modified Mosquitoes for the Control of Vector-Borne Diseases. WHO, 2020Google Scholar,68.World Health Organization World Malaria Report. WHO, 2021Google Scholar]. Current gene drive strategies include the use of transgenes encoding both the CRISPR-Cas9 endonuclease that is expressed under the control of a germline promoter, along with ubiquitously and constitutively expressed gRNAs, that together can target and cleave specific sequences in the genome [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar,69.Hammond A.M. Galizi R. Gene drives to fight malaria: current state and future directions.Pathog. Glob. Health. 2018; 111: 412-423Crossref Scopus (62) Google Scholar]. Once the transgene is introduced into its genomic target location on one of a pair of homologous chromosomes, the gRNA and Cas9 act in concert in germ cells to cause a double-stranded break in the target DNA site of the wild-type homologous chromosome. When this double-stranded lesion is repaired by homology directed repair using the transgenic homologous chromosome as a template, the entire transgene, along with flanking sequences either side of the transgene, is pasted into the target site of the homologous, formerly wild-type, chromosome. This process of homing can create mostly pairs of parental homologous chromosomes that are homozygous for the transgene, so that a greater proportion of parental germ cells are transgenic than would otherwise be the case, leading to super-Mendelian inheritance of the transgene in progeny. Thus, once introduced into a mating population, transgenes capable of homing will increase in frequency, or drive, within that population, assuming any fitness costs from the transgene do not outweigh its increased transmission from homing [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar]. Before any environmental release of an engineered gene drive mosquito could be considered by regulators, decision-makers and stakeholders, environmental risk assessment (ERA), whether probabilistic, qualitative, or a combination thereof, must be conducted to evaluate potential risks to human health, animal health and the environment [6.National Academies of Sciences, Engineering, and Medicine Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. National Academies Press, 2016Google Scholar, 7.Landis W.G. et al.A general risk-based adaptive management scheme incorporating the Bayesian Network Relative Risk Model with the South River, Virginia, as case study.Integr. Environ. Assess. Manag. 2017; 13: 115-126Crossref PubMed Scopus (26) Google Scholar, 8.James S. et al.Pathway to deployment of gene drive mosquitoes as a potential biocontrol tool for elimination of malaria in Sub-Saharan Africa: recommendations of a Scientific Working Group.Am. J. Trop. Med. Hyg. 2018; 98: 1-49Crossref PubMed Scopus (122) Google Scholar, 9.EFSA Panel on Genetically Modified Organisms et al.Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post-market environmental monitoring of genetically modified insects containing engineered gene drives.EFSA J. 2020; 18e06297Google Scholar, 10.James S.L. et al.Toward the definition of efficacy and safety criteria for advancing gene drive-modified mosquitoes to field testing.Vector Borne Zoonotic Dis. 2020; 20: 237-251Crossref PubMed Scopus (38) Google Scholar, 11.World Health Organization Guidance Framework for Testing Genetically Modified Mosquitoes.2nd edn. WHO, 2021Google Scholar, 12.Ickowicz A. et al.Predicting the spread and persistence of genetically modified dominant sterile male mosquitoes.Parasit. Vectors. 2021; 14: 480Crossref PubMed Scopus (4) Google Scholar, 13.Connolly J.B. et al.Recommendations for environmental risk assessment of gene drive applications for malaria vector control.Malar. J. 2022; 21: 152Crossref PubMed Scopus (9) Google Scholar]. A prerequisite for effective ERA of genetically modified organisms (GMOs) is to define intended and unintended effects of the intervention on target organisms (TOs) and nontarget organisms (NTOs) [9.EFSA Panel on Genetically Modified Organisms et al.Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post-market environmental monitoring of genetically modified insects containing engineered gene drives.EFSA J. 2020; 18e06297Google Scholar,10.James S.L. et al.Toward the definition of efficacy and safety criteria for advancing gene drive-modified mosquitoes to field testing.Vector Borne Zoonotic Dis. 2020; 20: 237-251Crossref PubMed Scopus (38) Google Scholar,14.Romeis J. et al.The value of existing regulatory frameworks for the environmental risk assessment of agricultural pest control using gene drives.Environ. Sci. Policy. 2020; 108: 19-36Crossref Scopus (23) Google Scholar, 15.Devos Y. et al.Gene drive-modified organisms: developing practical risk assessment guidance.Trends Biotechnol. 2021; 39: 853-856Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 16.Devos Y. et al.Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment.Crit. Rev. Biotechnol. 2021; : 1-17Google Scholar, 17.Devos Y. et al.Risk management recommendations for environmental releases of gene drive modified insects.Biotechnol. Adv. 2021; 107807Crossref PubMed Scopus (9) Google Scholar]. This paradigm is typically applied in the qualitative ERA of GM plants, where, in general, there is a clear distinction between the organisms to be targeted, the TOs, and those that are not intended to be targeted, the NTOs. While engineered gene drives share many of the same considerations as other transgenes in GMOs, such as non-gene drive genetically modified mosquitoes (GMMs), they differ in that they are designed to spread, increase in frequency, and persist in target organisms of wild populations. In addition, the most significant malaria vectors belong to species complexes that contain both vector and nonvector species [18.Davidson G. Anopheles gambiae, a complex of species.Bull. World Health Organ. 1964; 31: 625-634PubMed Google Scholar, 19.Besansky N.J. et al.Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10818-10823Crossref PubMed Scopus (170) Google Scholar, 20.Small S.T. et al.Radiation with reticulation marks the origin of a major malaria vector.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 31583-31590Crossref PubMed Scopus (13) Google Scholar, 21.Antonio-Nkondjio C. Simard F. Highlights on anopheles nili and anopheles moucheti, malaria vectors in Africa.in: Anopheles Mosquitoes - New insights into Malaria Vectors. IntechOpen, 2013Crossref Google Scholar, 22.Crawford J.E. et al.Reticulate speciation and barriers to introgression in the Anopheles gambiae species complex.Genome Biol. Evol. 2015; 7: 3116-3131Crossref PubMed Scopus (27) Google Scholar], some combinations of which are capable of hybridisation to produce fertile interspecific hybrids. Such semipermeable or porous species boundaries facilitate introgression [19.Besansky N.J. et al.Semipermeable species boundaries between Anopheles gambiae and Anopheles arabiensis: evidence from multilocus DNA sequence variation.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10818-10823Crossref PubMed Scopus (170) Google Scholar,20.Small S.T. et al.Radiation with reticulation marks the origin of a major malaria vector.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 31583-31590Crossref PubMed Scopus (13) Google Scholar,23.Thelwell N.J. et al.Evidence for mitochondrial introgression between Anopheles bwambae and Anopheles gambiae.Insect Mol. Biol. 2000; 9: 203-210Crossref PubMed Scopus (24) Google Scholar] and could plausibly lead to vertical gene drive transfer (VGDT) amongst sibling species, including nonvectors. This represents a challenge to the notion of a binary choice between TO and NTO for engineered gene drives in species complexes. Depending on how the TO and protection goals are defined, the potential impacts of VGDT could be evaluated in a number of different ways in the ERA. Here, a case study involving a population suppression gene drive in Anopheles gambiae sensu lato (s.l.) [24.Kyrou K. et al.A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.Nat. Biotechnol. 2018; 36: 1062-1066Crossref PubMed Scopus (425) Google Scholar,25.Hammond A. et al.Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.Nat. Commun. 2021; 12: 4589Crossref PubMed Scopus (39) Google Scholar] is used to illustrate these differing possibilities and their consequences (Box 2).Box 2Case study: a population suppression gene drive in Anopheles gambiaeThere are two principal strategies for use of engineered gene drives in malaria vector control. In population replacement gene drives, transgenes disrupt endogenous mosquito genes or contain cargo genes to prevent the development of pathogens in the mosquito or its transmission from mosquitoes to humans [69.Hammond A.M. Galizi R. Gene drives to fight malaria: current state and future directions.Pathog. Glob. Health. 2018; 111: 412-423Crossref Scopus (62) Google Scholar]. In population suppression gene drives, the transgene causes a decline in population density by introducing a fitness cost or sex bias. Here, for example, gRNAs can target haplosufficient female fertility genes. Transgenic heterozygous females are disrupted for one copy of the gene and so remain fertile. Via homing, the transgene is transmitted to offspring at super-Mendelian inheritance rates [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar], so that heterozygous transgenic males and females increase in frequency in the population. They are therefore increasingly likely to mate with one another, generating increasing proportions of homozygous transgenics, females of which are sterile. Thus, the number of progeny decreases and the population is suppressed.Early attempts to produce engineered gene drives to suppress populations of A. gambiae led to resistance to drive caused by mutations that simultaneously both disrupted the gRNA target site and retained functionality in the protein from the endogenous targeted gene [70.Hammond A. et al.A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.Nat. Biotechnol. 2016; 34: 78-83Crossref PubMed Scopus (733) Google Scholar]. Recently, the doublesex sex determination locus in A. gambiae s.l. has been investigated in the laboratory for a population suppression gene drive. The locus, which is highly conserved, encodes two transcripts, one of which is male specific (AgdsxM), the other female specific (AgdsxF) [24.Kyrou K. et al.A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.Nat. Biotechnol. 2018; 36: 1062-1066Crossref PubMed Scopus (425) Google Scholar]. The gRNA of the engineered gene drive spans an intron–exon boundary, mutation of which selectively disrupts the AgdsxF isoform, causing homozygous transgenic females to be intersex, sterile and unable to blood feed. In the laboratory, introduction of mosquitoes with this engineered gene drive into small cages of wild type populations of A. gambiae caused population eliminations after 9–11 generations [24.Kyrou K. et al.A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.Nat. Biotechnol. 2018; 36: 1062-1066Crossref PubMed Scopus (425) Google Scholar]. In recent large cage experiments that more closely mimic the feeding and reproductive environments of the field, introduction of the doublesex engineered gene drive into overlapping, age-structured generations of wild type A. gambiae caused population collapse within a year without any evidence for the development of resistance to drive [25.Hammond A. et al.Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.Nat. Commun. 2021; 12: 4589Crossref PubMed Scopus (39) Google Scholar]. There are two principal strategies for use of engineered gene drives in malaria vector control. In population replacement gene drives, transgenes disrupt endogenous mosquito genes or contain cargo genes to prevent the development of pathogens in the mosquito or its transmission from mosquitoes to humans [69.Hammond A.M. Galizi R. Gene drives to fight malaria: current state and future directions.Pathog. Glob. Health. 2018; 111: 412-423Crossref Scopus (62) Google Scholar]. In population suppression gene drives, the transgene causes a decline in population density by introducing a fitness cost or sex bias. Here, for example, gRNAs can target haplosufficient female fertility genes. Transgenic heterozygous females are disrupted for one copy of the gene and so remain fertile. Via homing, the transgene is transmitted to offspring at super-Mendelian inheritance rates [2.Burt A. et al.Gene drive to reduce malaria transmission in sub-Saharan Africa.J. Responsible Innov. 2018; 5: S66-S80Crossref Scopus (44) Google Scholar], so that heterozygous transgenic males and females increase in frequency in the population. They are therefore increasingly likely to mate with one another, generating increasing proportions of homozygous transgenics, females of which are sterile. Thus, the number of progeny decreases and the population is suppressed. Early attempts to produce engineered gene drives to suppress populations of A. gambiae led to resistance to drive caused by mutations that simultaneously both disrupted the gRNA target site and retained functionality in the protein from the endogenous targeted gene [70.Hammond A. et al.A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.Nat. Biotechnol. 2016; 34: 78-83Crossref PubMed Scopus (733) Google Scholar]. Recently, the doublesex sex determination locus in A. gambiae s.l. has been investigated in the laboratory for a population suppression gene drive. The locus, which is highly conserved, encodes two transcripts, one of which is male specific (AgdsxM), the other female specific (AgdsxF) [24.Kyrou K. et al.A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.Nat. Biotechnol. 2018; 36: 1062-1066Crossref PubMed Scopus (425) Google Scholar]. The gRNA of the engineered gene drive spans an intron–exon boundary, mutation of which selectively disrupts the AgdsxF isoform, causing homozygous transgenic females to be intersex, sterile and unable to blood feed. In the laboratory, introduction of mosquitoes with this engineered gene drive into small cages of wild type populations of A. gambiae caused population eliminations after 9–11 generations [24.Kyrou K. et al.A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.Nat. Biotechnol. 2018; 36: 1062-1066Crossref PubMed Scopus (425) Google Scholar]. In recent large cage experiments that more closely mimic the feeding and reproductive environments of the field, introduction of the doublesex engineered gene drive into overlapping, age-structured generations of wild type A. gambiae caused population collapse within a year without any evidence for the development of resistance to drive [25.Hammond A. et al.Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field.Nat. Commun. 2021; 12: 4589Crossref PubMed Scopus (39) Google Scholar]. The dominant malaria vectors in Africa are Anopheles coluzzii, Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, and Anopheles funestus (all Diptera: Culicidae) [26.Sinka M.E. et al.The dominant Anopheles vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution maps and bionomic precis.Parasit. Vectors. 2010; 3: 117Crossref PubMed Scopus (503) Google Scholar,27.Afrane Y. et al.Secondary malaria vectors of Sub-Saharan Africa: threat to malaria elimination on the continent?.in: Rodriguez-Morales A.J. Current Topics in Malaria. IntechOpen, 2016Crossref Google Scholar]. Engineered gene drives targeting A. coluzzii, A. gambiae s.s., and A. arabiensis are currently under active development [28.Burt A. Crisanti A. Gene drive: evolved and synthetic.ACS Chem. Biol. 2018; 13: 343-346Crossref PubMed Scopus (49) Google Scholar]. All three vectors are members of the A. gambiae s.l. complex (Box 3). However, A. gambiae s.l. also contains nonvector species. Most combinations of sibling species of the complex that have been examined in the laboratory are capable of producing fertile female hybrids [18.Davidson G. Anopheles gambiae, a complex of species.Bull. World Health Organ. 1964; 31: 625-634PubMed Google Scholar]. Some combinations of these species hybrids have also been found in field populations, albeit typically at low frequencies [23.Thelwell N.J. et al.Evidence for mitochondrial introgression between Anopheles bwambae and Anopheles gambiae.Insect Mol. Biol. 2000; 9: 203-210Crossref PubMed Scopus (24) Google Scholar,29.White G.B. Chromosomal evidence for natural interspecific hybridization by mosquitoes of the Anopheles gambiae complex.Nature. 1971; 231: 184-185Crossref PubMed Scopus (41) Google Scholar, 30.Costantini C. et al.Living at the edge: biogeographic patterns of habitat segregation conform to speciation by niche expansion in anopheles gambiae.BMC Ecol. 2009; 9: 16Crossref PubMed Scopus (156) Google Scholar, 31.Pombi M. et al.Dissecting functional components of reproductive isolation among closely related sympatric species of the anopheles gambiae complex.Evol. Appl. 2017; 10: 1102-1120Crossref PubMed Scopus (29) Google Scholar, 32.Epopa P.S. et al.Seasonal malaria vector and transmission dynamics in western Burkina Faso.Malar. J. 2019; 18: 113Crossref PubMed Scopus (26) Google Scholar], thus representing a potential VGDT route.Box 3Anopheles gambiae s.l. species complexThe species complex of A. gambiae s.l. is typically recognised as containing at least nine morphologically indistinguishable sibling species that shared a common ancestor less than 2 million years ago [71.Fontaine M.C. et al.Mosquito genomics. Extensive introgression in a malaria vector species complex revealed by phylogenomics.Science. 2015; 3471258524Crossref Scopus (337) Google Scholar]: Anopheles amharicus, Anopheles arabiensis, Anopheles bwambae, Anopheles coluzzii, Anopheles fontenillei; Anopheles gambiae s.s., Anopheles melas, Anopheles merus, and Anopheles quadriannulatus.A. gambiae s.s., A. coluzzii and A. arabiensis are dominant regional vector species of malaria in large sections of Africa [26.Sinka M.E. et al.The dominant Anopheles vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution maps and bionomic precis.Parasit. Vectors. 2010; 3: 117Crossref PubMed Scopus (503) Google Scholar,27.Afrane Y. et al.Secondary malaria vectors of Sub-Saharan Africa: threat to malaria elimination on the continent?.in: Rodriguez-Morales A.J. Current Topics in Malaria. IntechOpen, 2016Crossref Google Scholar,72.Sinka M.E. et al.A global map of dominant malaria vectors.Parasit. Vectors. 2012; 5: 69Crossref PubMed Scopus (395) Google Scholar]. A. melas, A. merus, and A. bwambae are more geographically restricted, local vectors of the disease [48.White G.B. Anopheles bwambae sp.n., a malaria vector in the Semliki Valley, Uganda, and its relationships with other sibling species of the An. gambiae complex (Diptera: Culicidae).Syst. Entomol. 1985; 10: 501-522Crossref Scopus (44) Google Scholar,72.Sinka M.E. et al.A global map of dominant malaria vectors.Parasit. Vectors. 2012; 5: 69Crossref
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