Applications & Publications
Technical Notes
Automated Analysis and Sorting of 1st instar Larvae of the Malaria Mosquito Anopheles sp. (QTN-012)
Publications
Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi
Larrosa-Godall et al. February 07, 2025 Parasit Vectors. 2025; 18: 46. Published online 2025 Feb 7. doi: 10.1186/s13071-025-06688-0
View AbstractChallenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi
Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx).
Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi
Weng et al. November 11, 2024 Infect Dis Poverty. 2024; 13: 83. Published online 2024 Nov 11. doi: 10.1186/s40249-024-01256-7
View AbstractEstablishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi
Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.
Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance
Morianou et al. October 21, 2024 bioRxiv preprint doi: https://doi.org/10.1101/2024.10.21.618489
Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance
Establishing a Male-Positive Genetic Sexing Strain in the Asian Malaria Vector Anopheles stephensi
Weng et al. July 18, 2024 Version 1. bioRxiv. Preprint. 2024 Jul 18. doi: 10.1101/2024.07.17.603997Published in: Infect Dis Poverty. 2024; 13: 83.
View AbstractEstablishing a Male-Positive Genetic Sexing Strain in the Asian Malaria Vector Anopheles stephensi
Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit human pathogens. This reduces the risk of spreading pathogens while enabling effective population control. Robust sex sorting methods to enable early larval sorting in mosquitoes need to be developed to allow for scalable sex sorting for genetic biocontrol interventions. This study applies the SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) system, previously developed for Aedes aegypti, to the Asian malaria vector Anopheles stephensi. We hypothesized that the intron from the doublesex gene in Anopheles gambiae would function in An. stephensi due to evolutionary conservation. Our results confirm that the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species. This system enables reliable positive male selection from first instar larval to pupal stages. RT-PCR analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. The SEPARATOR system's independence from sex-chromosome linkage confers resistance to meiotic recombination and chromosomal rearrangements. This approach may facilitate the mass release of males, and the cross-species portability of SEPARATOR establishes it as a valuable tool for genetic biocontrol interventions across various pest species.
Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter
Vitale et al. June 20, 2024 PLoS Genet. 2024 Jun; 20(6): e1011303. Published online 2024 Jun 7. doi:10.1371/journal.pgen.1011303
View AbstractY chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter
Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.
Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
D'Amato et al. February 21, 2024 Nat Commun 15, 952 (2024). https://doi.org/10.1038/s41467-024-44907-x
Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
Anderson et al. January 25, 2024 Nat Commun. 2024; 15: 729. Published online 2024 Jan 25. doi: 10.1038/s41467-024-44956-2
View AbstractA multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.
A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes
Green et al. December 05, 2023 eLife. 2023; 12: e93142. Published online 2023 Dec 5. doi: 10.7554/eLife.93142
View AbstractA population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes
Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this antimalarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.
Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti
Weng et al. November 27, 2023 PLoS Genet 19(11): e1011065. https://doi.org/10.1371/journal.pgen.1011065
View AbstractEfficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti
Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.
Efficient Sex Separation by Exploiting Differential Alternative Splicing of a Dominant Marker in Aedes aegypti
Weng et al. November 19, 2023 Version 2. bioRxiv. Preprint. 2023 Jun 16 [revised 2023 Jul 12]. doi: 10.1101/2023.06.16.545348Published in: PLoS Genet. 2023 Nov; 19(11): e1011065.
View AbstractEfficient Sex Separation by Exploiting Differential Alternative Splicing of a Dominant Marker in Aedes aegypti
Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.
Combining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control
Lutrat et al. June 16, 2023 Commun Biol. 2023; 6: 646. Published online 2023 Jun 16. doi: 10.1038/s42003-023-05030-7
View AbstractCombining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control
Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01–0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.
The salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites.
Klug et al. March 02, 2023 PLoS Pathog. 2023 Mar; 19(3): e1010538. Published online 2023 Mar 2. doi: 10.1371/journal.ppat.1010538
View AbstractThe salivary protein Saglin facilitates efficient midgut colonization of Anopheles mosquitoes by malaria parasites.
Malaria is caused by the unicellular parasite Plasmodium which is transmitted to humans through the bite of infected female Anopheles mosquitoes. To initiate sexual reproduction and to infect the midgut of the mosquito, Plasmodium gametocytes are able to recognize the intestinal environment after being ingested during blood feeding. A shift in temperature, pH change and the presence of the insect-specific compound xanthurenic acid have been shown to be important stimuli perceived by gametocytes to become activated and proceed to sexual reproduction. Here we report that the salivary protein Saglin, previously proposed to be a receptor for the recognition of salivary glands by sporozoites, facilitates Plasmodium colonization of the mosquito midgut, but does not contribute to salivary gland invasion. In mosquito mutants lacking Saglin, Plasmodium infection of Anopheles females is reduced, resulting in impaired transmission of sporozoites at low infection densities. Interestingly, Saglin can be detected in high amounts in the midgut of mosquitoes after blood ingestion, possibly indicating a previously unknown host-pathogen interaction between Saglin and midgut stages of Plasmodium. Furthermore, we were able to show that saglin deletion has no fitness cost in laboratory conditions, suggesting this gene would be an interesting target for gene drive approaches.
Life-history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background
Ntoyi et al. September 05, 2022 Malar J. 2022; 21: 254. Published online 2022 Sep 5. doi: 10.1186/s12936-022-04276-6
View AbstractLife-history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background
Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation
Fuchs et al. October 05, 2021 PLoS Genet 17(10): e1009740. https://doi.org/10.1371/journal. pgen.1009740
Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation
Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field
Hammond A. et al. July 28, 2021 Nature Commun.;12(1):4589 • PMID: 34321476 • DOI: 10.1038/s41467-021-24790-6
Gene-drive suppression of mosquito populations in large cages as a bridge between lab and field
Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance
Hammond et al. January 29, 2021 PLoS Genet 17(1): e1009321. https://doi.org/10.1371/journal. pgen.1009321
Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance
Efficient Production of Male Wolbachia-infected Aedes Aegypti Mosquitoes Enables Large-Scale Suppression of Wild Populations
Crawford et al. April 06, 2020 Nature Biotechnology volume 38, pages 482–492(2020) • PMID: 32265562 • DOI: 10.1038/s41587-020-0471-x
Efficient Production of Male Wolbachia-infected Aedes Aegypti Mosquitoes Enables Large-Scale Suppression of Wild Populations
A New Role of the Mosquito Complement-like Cascade in Male Fertility in Anopheles gambiae
Julien Pompon1 ,¤ and Elena A. Levashina1 ,2 September 22, 2015 PLoS Biol. 2015 Sep; 13(9): e1002255. Published online 2015 Sep 22. doi: 10.1371/journal.pbio.1002255
View AbstractA New Role of the Mosquito Complement-like Cascade in Male Fertility in Anopheles gambiae
Tools for Anopheles gambiae Transgenesis.
Volohonsky G1, Terenzi O11 Soichot J1, Naujoks DA2, Nolan T2, Windbichler N2, Kapps D1, Smidler AL1, Vittu A1, Costa G3, Steinert S1, Levashina EA3, Blandin SA1, Marois E 4 April 13, 2015 G3 (Bethesda). 2015 Apr 13;5(6):1151-63. doi: 10.1534/g3.115.016808.
View AbstractTools for Anopheles gambiae Transgenesis.
1) INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France.
2) Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London SW7 2AZ, United Kingdom.
3) Department of Vector Biology, Max-Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, Germany.
4) INSERM U963, CNRS UPR9022, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, 67084 Strasbourg, France
A synthetic sex ratio distortion system for the control of the human malaria mosquito
Roberto Galizi¹,², Lindsey A. Doyle³, Miriam Menichelli¹, Federica Bernardini¹, Anne Deredec¹, Austin Burt¹, Barry L. Stoddard³, Nikolai Windbichler¹,* & Andrea Crisanti¹,²,* June 10, 2014 NATURE COMMUNICATIONS | 5:3977 | DOI: 10.1038/ncomms4977 |www.nature.com/naturecommunications
View AbstractA synthetic sex ratio distortion system for the control of the human malaria mosquito
1) Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. 2) Centro di Genomica Funzionale, University of Perugia, Dipartimento di Medicina Sperimentale Via Gambuli, Edificio D, 3 Piano, 06132 Perugia, Italy. 3) Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. * These authors contributed equally to this work.
Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases
Andrea L. Smidler, Olivier Terenzi, Julien Soichot, Elena A. Levashina, Eric Marois August 15, 2013 Published: August 15, 2013 • DOI: 10.1371/journal.pone.0074511
Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases
High-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions
Eric Marois¹,Christina Scali²,Julien Soichot¹,Christine Kappler¹, Elena A Levashina¹,³,*,†,Flaminia Catteruccia4,5,**,† August 28, 2012 Malaria Journal 2012, 11:302 doi:10.1186/1475-2875-11-302 1) Institut de Biologie Moléculaire et Cellulaire, INSERM U963, CNRS UPR9022,15 rue René Descartes, 67084 Strasbourg, France 2) Division of Cell and Molecular Biology, Imperial College London, Imperial College Road, London SW7 2AZ, UK 3) Department of Vector Biology, Max-Planck Institute for Infection Biology,Chariteplatz 1, 10117 Berlin, Germany 4) Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università degli Studi di Perugia, Terni 05100, Italy 5) Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA * Corresponding author. Department of Vector Biology, Max-Planck Institute for Infection Biology, Chariteplatz 1, 10117 Berlin, Germany ** Corresponding author. Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA † Equal contributors.
View AbstractHigh-throughput sorting of mosquito larvae for laboratory studies and for future vector control interventions
Background: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures.
Methods: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females.
Results: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers.
Conclusions: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors.
An Anopheles transgenic sexing strain for vector control
Flaminia Catteruccia1, 2, Jason P Benton1, 2 & Andrea Crisanti1 October 09, 2005 Nature Biotechnology 1) Imperial College London, Department of Biological Sciences, Imperial College Road, London SW7 2AZ, UK. 2) These authors contributed equally to this work.
View AbstractAn Anopheles transgenic sexing strain for vector control
Genetic manipulation of mosquito species that serve as vectors for human malaria is a prerequisite to the implementation of gene transfer technologies for the control of vector-borne diseases. Here we report on the development of transgenic sexing lines for the mosquito Anopheles stephensi, the principal vector of human malaria in Asia. Male mosquitoes, expressing enhanced green fluorescent protein (EGFP) under the control of the β2-tubulin promoter, are identified by their fluorescent gonads in as early as their 3rd instar larval stage, and can be efficiently separated from females using both manual methods and automated sorting machines. Importantly, β2-EGFP males are not impaired in their mating ability and viable fluorescent spermatozoa are also detected in spermathecae of wild-type females mated with transgenic males. The transgenic mosquito lines described here combine most of the features desired and required for a safe application of transgenic methodologies to malaria-control programs.