Differential Elimination of Marked Sex Chromosomes Enables Production of Nontransgenic Male Mosquitoes in a Single Strain

Infectious Diseases Bioengineering Genetics Life Sciences Medicine
mosquito control sex-separation homomorphic sex chromosome recessive lethal sterile insect technique

Research Background

The Aedes aegypti mosquito is a primary vector for arboviruses such as dengue and Zika. Current genetic control strategies relying on the release of non-biting males (e.g., Sterile Insect Technique, SIT, or Wolbachia-based Incompatible Insect Technique, IIT) face a critical bottleneck: how to efficiently and cost-effectively isolate millions of male mosquitoes free from contamination by biting females. Traditional methods depend on morphological sorting or transgenic markers but suffer from low efficiency, high costs, or regulatory hurdles. This study addresses this challenge by leveraging the homomorphic sex chromosomes of Aedes aegypti to develop an innovative system called “Differential Elimination of Marked Sex Chromosomes” (DEMARK).

Sex determination in Aedes aegypti is controlled by the M/m locus on chromosome 1: the M chromosome carries male-determining genes (e.g., nix and myo-sex), but it is morphologically similar to the m chromosome. Previous studies revealed a ~100 Mb low-recombination region (recombination desert) surrounding the M/m locus, providing a theoretical foundation for developing sex-separation strategies based on sex-linked recessive lethal alleles (RLAs).

Source of the Paper

Led by Zhijian Tu’s team at Virginia Tech in collaboration with institutions such as the University of California, Riverside, this study was published on May 8, 2025, in PNAS (Proceedings of the National Academy of Sciences), titled Differential Elimination of Marked Sex Chromosomes Enables Production of Nontransgenic Male Mosquitoes in a Single Strain.

Research Process and Results

1. Precise Measurement of Sex Chromosome Recombination Rates

Experimental Design:
- Used three transgenic marker lines (C1-dsRed: 167.945 Mb; P14-GFP: 180.261 Mb; P10-GFP: 224.678 Mb)
- Calculated genetic distances between markers and the M locus through large-scale progeny screening (>10,000 larvae)

Key Findings:
- Recombination rate between C1 and the M locus: only 0.043% (16 Mb physical distance ≈ 0.043 cM)
- Recombination rate between P14 and the M locus: 0.079% (28 Mb ≈ 0.079 cM)
- Confirmed extreme recombination suppression in this region (~1,000-fold lower than in Drosophila)

Technical Highlight:
Employed Oxford Nanopore sequencing to precisely map transgenic insertion sites (Dataset S2) and developed a high-resolution genetic mapping method.

2. Discovery and Validation of Naturally Occurring Sex-Linked Recessive Lethal Alleles (RLAs)

Experimental Process:
1. Obtained recombinant chromosomes via natural recombination in the C1 marker line:
- mᶜ¹ᵃ (acquired C1 marker and lethal allele l)
- m¹ (m chromosome lacking l)
2. Designed complementation tests to validate lethality:
- mᶜ¹ᵃ/mᶜ¹ᵃ females: embryonic lethality
- mᶜ¹ᵃ/M males: embryonic lethality (rescuable by m¹)

Data Support:
- Crosses showed significant deviation from the expected 1:1 sex ratio (SI Appendix, Table S5)
- Digital droplet PCR (ddPCR) confirmed the recessive lethal nature of l

3. Development of the DEMARK System

(1) Differential Elimination of Two Marked Chromosomes (DE2mark)

System Design:
- Used chromosomes carrying the same l but different fluorescent markers (mᶜ¹ᵃ-dsRed and mᴾ¹⁴ᵈ-GFP)
- Self-sustaining cycle:
mᴾ¹⁴ᵈ females × mᶜ¹ᵃ males → non-transgenic males (m¹/m) + marked females/males
- Double-marked females (mᶜ¹ᵃ/mᴾ¹⁴ᵈ) die due to l homozygosity

Results:
- Among 13,186 individuals, only 1 double-positive recombinant female was detected (SI Appendix, Table S7)
- Non-transgenic male production efficiency: 34.5% (4,544⁄13,186)

(2) Differential Elimination of One Marked Chromosome (DE1mark)

Engineered RLAs:
- CRISPR/Cas9 knockout of essential but haplosufficient genes:
- amon (neuroendocrine convertase gene, Mb from M locus)
- bag (homeobox gene, 260 kb from P14 marker)

System Performance:
- DE1mark_bag: Produced only non-transgenic males (m/mᵇᵃᵍ⁻) and transgenic females (mᴾ¹⁴ᵈ/mᵇᵃᵍ⁻)
- Mating competitiveness assays showed no significant difference between DE1mark males and wild-type (GLMM analysis, p=0.493)

Research Conclusions and Impact

  1. Scientific Value:

    • First self-sustaining sex-separation system leveraging homomorphic sex chromosomes
    • Reveals new mechanisms in Aedes aegypti sex chromosome evolution (proto-Y/X hypothesis)

  2. Practical Applications:

    • Single-strain production of non-transgenic males (reducing costs by >50%)
    • Compatible with existing SIT/IIT systems, bypassing GMO regulatory hurdles

  3. Scalability:

    • Proposed DEMARK adaptations for Anopheles heteromorphic sex chromosomes (Fig. 6b)
    • Integratable with conditional lethality systems for screening-free production

Key Innovations

  • Methodological Novelty: Adapted “balancer chromosome” concepts for vector control
  • Technical Breakthrough: Achieved 0.001% purity in sex separation
  • Cross-Species Potential: Provides a universal framework for genetic pest control