femaleless Controls Sex Determination and Dosage Compensation Pathways in Females of Anopheles Mosquitoes

Abstract

Summary The insect sex determination and the intimately linked dosage compensation pathways represent a challenging evolutionary puzzle that has been solved only in Drosophila melanogaster . Analyses of orthologs of the Drosophila genes identified in non-drosophilid taxa 1 , 2 revealed that evolution of sex determination pathways is consistent with a bottom-up mode, 3 where only the terminal genes within the pathway are well conserved. doublesex ( dsx ), occupying a bottom-most position and encoding sex-specific proteins orchestrating downstream sexual differentiation processes, is an ancient sex-determining gene present in all studied species. 2 , 4 , 5 With the exception of lepidopterans, its female-specific splicing is known to be regulated by transformer ( tra ) and its co-factor transformer-2 ( tra2 ). 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 Here we show that in the African malaria mosquito Anopheles gambiae , a gene, which likely arose in the Anopheles lineage and which we call femaleless ( fle ), controls sex determination in females by regulating splicing of dsx and fruitless ( fru ; another terminal gene within a branch of the sex determination pathway). Moreover, fle represents a novel molecular link between the sex determination and dosage compensation pathways. It is necessary to suppress activation of dosage compensation in females, as demonstrated by the significant upregulation of the female X chromosome genes and a correlated female-specific lethality, but no negative effect on males, in response to fle knockdown. This unexpected property, combined with a high level of conservation in sequence and function in anopheline mosquitoes, makes fle an excellent target for genetic control of all major vectors of human malaria.

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression

“…Similarly, transgenic A. gambiae lines that express fle dsRNA show masculinization and, in the strongest lines, death of XX mosquitoes [34]. In wild type males, the Y-linked sex determining gene Yob [41] is thought to somehow inactivate fle and thus promote dosage compensation [34,42]. In the related mosquito A. stephensi, expression of the Y-linked gene Guy1 in transgenic lines was lethal to females, which appears to be due to a significant increase in expression of X-linked genes compared to autosomal genes [43].…”

“…Sxl homozygous females die at the embryo stage due to hyperactivation of expression of X chromosome genes [40]. Similarly, transgenic A. gambiae lines that express fle dsRNA show masculinization and, in the strongest lines, death of XX mosquitoes [34]. In wild type males, the Y-linked sex determining gene Yob [41] is thought to somehow inactivate fle and thus promote dosage compensation [34,42].…”

“…In the related mosquito A. stephensi, expression of the Y-linked gene Guy1 in transgenic lines was lethal to females, which appears to be due to a significant increase in expression of X-linked genes compared to autosomal genes [43]. Guy1 is strong candidate for the male determining factor [44] and may also somehow inactivate fle as knockdown of fle expression in A. stephensi embryos caused female-specific lethality [34]. Our analyses of the DR3#6; LctraIR7 strain indicates that tra is essential for both somatic sexual development and repression of X chromosome gene expression in L. cuprina.…”

“…Alternatively, knockdown of Lctra mRNA could lead to hyperactivation of the X chromosomes and death of XX transformed males, as seen in An. gambaie females that express femaleless dsRNA [34]. To address these possibilities, RNAseq was conducted on mid-stage pupae that were sexed by PCR using Y-linked primer sets (S2 Fig) . Of the 59 X-linked genes previously identified [17], 54 were expressed above the threshold set for this analysis (S3 Table ).…”

Double drives and private alleles for localised population genetic control

“…The most powerful design we considered targets a female-specific haplo-insufficient gene, or otherwise causes dominant female sterility or lethality. Such genes are not common, but there are some possible candidates [57][58][59][60], and our modelling motivates the search for others. Finally, performance (in terms of being able to cope with ever higher frequencies of pre-existing resistance) could presumably also be improved by using a third construct, to construct a triple drive, though modelling would be required to explore the implications of the many different configurations this extension would allow.…”