A Fluorescent Sex-Sorting Technique for Insects with the Demonstration in <i>Drosophila melanogaster</i>

Liu, Junru; Rayes, Danny; Akbari, Omar S.

doi:10.1089/genbio.2023.0041

Cited by 3 publications

(1 citation statement)

References 55 publications

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“…While manual F0 sorting was performed in this study, a technique effective for small-scale field trials ( 87 ), F0 sorting by optical sorters could be adapted to the current iteration of pgSIT to immediately enable larger-scale trials and possibly even release campaigns. Future iterations of pgSIT, termed pgSIT2.0, could consolidate the two gRNA transgenes and introduce sex-specific fluorescent reporters to enable higher-throughput F0 sorting by COPAS ( 54 , 95 – 97 ). With a COPAS sorting capacity at conservatively 40,000 larvae per hour ( 54 ), F0 sorting a pgSIT2.0 system could yield 2 million F1 sterilized males in the next generation with one machine ( SI Appendix , Text S1 ), facilitating production required for releases on a continental scale.…”

Section: Discussionmentioning

confidence: 99%

Eliminating malaria vectors with precision-guided sterile males

Apte,

Smidler,

Pai

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Controlling the principal African malaria vector, the mosquito Anopheles gambiae , is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

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Section: Discussionmentioning

confidence: 99%

Eliminating malaria vectors with precision-guided sterile males

Apte,

Smidler,

Pai

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Establishing a Male-Positive Genetic Sexing Strain in the Asian Malaria VectorAnopheles stephensi

Weng,

Chen,

et al. 2024

Preprint

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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.

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Fluorescent-based sex-separation technique in major invasive crop pest,Drosophila suzukii

Liu,

Rayes,

Yang

et al. 2024

Preprint

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Insect population biocontrol methods such as the sterile insect technique (SIT), represent promising alternatives to traditional pesticide-based control applications. To use these strategies efficiently requires scalable sex separation techniques which are currently lacking inDrosophila suzukii, a prominent crop pest species. Having previously characterized a fluorescence-based sex-sorting technique in other pests, termed SEPARATOR (SexingElementProduced byAlternativeRNA-splicing ofA TransgenicObservableReporter), here we explore its potential applicability toDrosophila suzukii. Here, we engineer several strains ofDrosophila suzukiiencoding SEPARATOR constructs that allow for efficient sex selection in early larval stages.

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A Fluorescent Sex-Sorting Technique for Insects with the Demonstration in Drosophila melanogaster

Cited by 3 publications

References 55 publications

Eliminating malaria vectors with precision-guided sterile males

Eliminating malaria vectors with precision-guided sterile males

Establishing a Male-Positive Genetic Sexing Strain in the Asian Malaria VectorAnopheles stephensi

Fluorescent-based sex-separation technique in major invasive crop pest,Drosophila suzukii

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