Targeting female flight for genetic control of mosquitoes

Abstract

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

Fig 1. AeAct4 gene disruption by homology-directed repair results in female-specific recessive lack of flight ability.

(A) Injected Cas9 protein and sgRNAs targeting AeAct4 induce a double stranded break (DSB) at the target site. A donor plasmid (AGG1070) having 2kb homology arms corresponding to the immediate upstream and downstream regions of the outermost cut sites was injected as a template for HDR. A 3xP3-mCherry-SV40 cassette serves as a marker for integration. Male-specific alternative splicing incorporates a number of early start and stop codons (marked by blue x) which ablate AeAct4 translation. Green arrows represent primer pair used for amplicons shown in (c). (B) 50 flightless females were identified out of a total of 156 marker-positive female progeny (32%). (C) Molecular confirmation of 6 flying and 4 flightless mCherry positive female adults, individually analyzed by PCR using the primers indicated in (A). AeAct4^hdr1 and WT amplicons were generated. Based on this PCR assay, of the marker-positive females analyzed, all fliers were heterozygous for AeAct4^hdr1 and all flightless females were homozygous.

Fig 2. Transmission electron microscope images of the indirect flight muscles of Ae. aegypti female adults arranged within the thorax.

Filaments in the myofibrils appear to be disrupted in AeAct4^hdr1 homozygotes (A, arrows and inset) but not in AeAct4^hdr1 heterozygotes (B, inset) or WT (C, inset). Individual myofibrils are indicated with double-headed arrows, and mitochondria (Mi) in the muscle fiber cells are clearly visible. Insert scale bars = 200 nm.