Juvenile hormone receptor, Methoprene-tolerant, knockout induces black larval phenotype in the yellow fever mosquito, Aedes aegypti

The yellow fever mosquito, Aedes aegypti, vectors many human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life. The JH analogs are used for preventing the adult emergence in mosquitoes. Since RNA interference does not work efficiently in mosquitoes, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we developed a CRISPR/Cas9-based genome editing method to achieve complete knockout of genes in one generation and applied this method to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that krupple homolog 1, a key transcription factor in JH signal transduction, was downregulated but pupal/adult genes coding for proteins involved in cuticle synthesis and melanization, and digestion of blood meal were upregulated in L4 Met mutants. These data suggest that during the L3 and L4, Met mediates JH suppression of metamorphosis through suppression of pupal/adult genes. The methods developed and data obtained would help to advance our knowledge on JH regulation of larval development and mode of action of JH analogs in Ae. aegypti and other mosquitoes.