White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter

Christopher Ward ,František Marec,Irina Häcker,Katerina Nikolouli,Thu Nguyen ,Gary Leveque,Amanda Choo,Margaret Hughes ,Carlos E Cáceres-Barrios ,Sarah J Reiling,Sheina B Sim,Peter A Crisp ,Haïg Djambazian ,Georgia Gouvi,Scott M Geib,Jiannis Ragoussis,Kostas Bourtzis,Sam Whiteford,Elisabeth Fung,Mark Whitehead ,Roswitha A Aumann,Alistair C Darby,Simon W Baxter,Marc F Schetelig

doi:10.1038/s41467-020-20680-5

Abstract

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Highlights

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s
The B. dorsalis white pupae phenotype was introgressed into B. tryoni to generate a strain referred to as the Bactrocera introgressed line (BIL, Supplementary Fig. 1)
Full penetrance expressivity and recessive inheritance rendered wp the marker of choice for genetic sexing strains (GSS) construction in two additional tephritid species, B. dorsalis and Z. cucurbitae[11,12], allowing automated sex sorting based on pupal color

Summary

Introduction

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests. In the same series of experiments, the wp locus was shown to be tightly linked to a temperature-sensitive lethal (tsl) gene (position 59B–61C), which is the second selectable marker of the VIENNA 7 and VIENNA 8 GSS currently used in all medfly SIT operational programs worldwide[7,15]

Methods

Results

Conclusion