The Potential for a Released Autosomal X-Shredder Becoming a Driving-Y Chromosome and Invasively Suppressing Wild Populations of Malaria Mosquitoes

Abstract

Sex-ratio distorters based on X-chromosome shredding are more efficient than sterile male releases for population suppression. X-shredding is a form of sex distortion that skews spermatogenesis of XY males towards the preferential transmission of Y-bearing gametes, resulting in a higher fraction of sons than daughters. Strains harboring X-shredders on autosomes were first developed in the malaria mosquito Anopheles gambiae, resulting in strong sex-ratio distortion. Since autosomal X-shredders are transmitted in a Mendelian fashion and can be selected against, their frequency in the population declines once releases are halted. However, unintended transfer of X-shredders to the Y-chromosome could produce an invasive meiotic drive element, that benefits from its biased transmission to the predominant male-biased offspring and its effective shielding from female negative selection. Indeed, linkage to the Y-chromosome of an active X-shredder instigated the development of the nuclease-based X-shredding system. Here, we analyze mechanisms whereby an autosomal X-shredder could become unintentionally Y-linked after release by evaluating the stability of an established X-shredder strain that is being considered for release, exploring its potential for remobilization in laboratory and wild-type genomes of An. gambiae and provide data regarding expression on the mosquito Y-chromosome. Our data suggest that an invasive X-shredder resulting from a post-release movement of such autosomal transgenes onto the Y-chromosome is unlikely.

Keywords: gene drive; genetic control; malaria; risk assessment; sex-ratio distortion.

FIGURE 1

piggyBac transposase components in laboratory and wild type individuals. (A) Schematic of a wild type piggyBac (PB) transposon from Trichoplusia ni (middle; NCBI accession DQ236240.1), the Ag(PMB)1 transformation construct [(Galizi et al., 2014); top] and the PB helper plasmid [(Volohonsky et al., 2015); bottom]. Shown are the regions of the endogenous PB locus present in the two microinjected plasmids highlighting how both the transformation and helper plasmid lack the complete machinery required for transposon mobility. pBacL (left) and pBacR (right) arms present in the pBac[3xP3-DsRed]β2-eGFP:I-PpoI-124L transformation construct contain the entire flanking inverted terminal repeats (ITRs) and partial regions of the PB open reading frame (ORF). The helper PB plasmid containing the complete PB ORF driven from the vasa2 regulatory regions lacks the flanking ITRs. Sequences of the transformation construct that are integrated in the genome, and those present only transiently in injected individuals are shown in solid lines and dashed lines, respectively. (B) Mapping of whole genome sequencing reads from the G3 and Ag(PMB)1 controls (bottom in negative y-axis; grey and red) and the 81 wild type individuals collected in Burkina Faso villages (top; blue). The position of the An. gambiae vasa regulatory regions (yellow boxes) and the PB transposase ORF (black box) is also shown. Reads are normalized by scaling counts to the number of reads in the most abundant sample.

FIGURE 2

Transcriptional suppression of Y-linked X-shredder constructs abolishes sex ratio distortion. (A) Progeny analysis of males from the two Y linked X-shredder strains crossed to wild-type females. Shown is the average number of eggs laid per n females analyzed (±represents the standard error of the mean; SEM). Average percentage of larvae hatching from the eggs (±SEM), from n females analyzed. Average percentage of males in the progeny (±SEM) from n females. The total number of eggs or individuals counted in each experiment is given in parentheses. Sequences (20 bp each side) flanking the PB integration site (TTAA) of the transformation constructs are also shown. (B) eGFP fluorescence from dissected wild type (WT), Ag(PMB)1 and YpBac-β2-^gfp124L testis. (C) Quantitative RT-PCR showing the relative expression of eGFP:I-PpoI variants in autosomal X-shredder strains [Ag(PMB)1-3] and Y-linked X-shredder strains. Expression levels were normalized to G3 wild-type (RQ = 1) which contains no I-PpoI component. Expression of the X-shredder is undetectable in both Y-chromosome insertions compared to G3 wild-type (unpaired t-test p = 0.1669 for YpBac and p = 0.2509 for YattP). Expression levels from autosomal strains, Ag(PMB)1 (unpaired t-test p = 0.0078), Ag(PMB)2 (originally W124L-3) and Ag(PMB)3 (originally L111A-2) which led to sex ratio distortion are shown.