Heryanto, C. | Search bioRxiv

bioRxiv2022

Efficient hyperactive piggyBac transgenesis in Plodia pantry moths

While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase (hyPBase) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella. Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5-4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G0 injected generation was detectable from 72-hr embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

genetics↗

bioRxiv2021

A macromutation eliminates colour patterning in captive butterflies

Captive populations often harbor variation that is not present in the wild due to artificial selection. Recent efforts to map this variation have provided insights into the genetic and molecular basis of variation. Heliconius butterflies display a large array of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here we sought to identify the genetic basis of an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78kb deletion at the cortex wing patterning locus as the ivory mutation. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5 region of the cortex gene that includes a facultative 5UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.

genetics↗