The yellow fever mosquito, Aedes aegypti, vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect’s life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited a 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 a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1, a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti.
|Number of pages||7|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Oct 22 2019|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS. This work was supported by grants from the National Institutes of Health (GM070559-13) and the National Institute of Food and Agriculture, US Department of Agriculture (under HATCH Project 2351177000). We thank Jeff Howell for help with insect rearing and reading the earlier version of the manuscript and Robert A. Harrell II, Channa Aluvihare, and Omar S. Akbari for help with embryonic injections, Ae. aegypti rearing, and the AAEL010097-Cas9 strain.
© 2019 National Academy of Sciences. All rights reserved.
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