Germline transformation of the butterfly Bicyclus anynana

Abstract

Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

“…Delivering nucleases at the ''one nucleus'' stage in the embryo offers several advantages over classical approaches used in lepidopterans, in which material is commonly delivered at the syncytial preblastoderm stage and at the posterior pole of the egg (Peloquin et al 2000;Tamura et al 2000;Marcus et al 2004;Takasu et al 2010), when many cleavage nuclei are present. One advantage of our delivery system is that it circumvents the need to define the location of germ cell precursors during embryonic development in species for which development remains poorly characterized.…”

Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings

“…This system allows us to combine knowledge of ecology (often minimal for classical genetic model species) with experimental tractability, all the way through to the study of the molecular underpinnings of variation in eyespot morphology. Moreover, recently developed genomic resources (Beldade et al 2007) and gene expression manipulation techniques (Marcus et al 2004;Ramos et al 2006) can now be applied to analysing the phenotypically divergent mutant stocks and selection lines (Beldade et al 2005) available in our laboratory. This type of integrated analysis holds much promise for deepening our knowledge about the origin and diversification of the lineage-specific morphologies such as butterfly eyespots.…”

Heliconius wing patterns: an evo-devo model for understanding phenotypic diversity

“…Having exhausted the mapping resolution of crosses in each species, the involvement of the gene(s) identified from this region in colour pattern variation will be tested using various methods: expression studies on developing wings (eg Reed and Nagy, 2005) can test the up-or downregulation of genes identified from the BAC sequences, while association studies using BACderived markers in wild populations (eg Colosimo et al, 2005) can take advantage of historical recombination around the colour-pattern locus to identify narrower regions associated to specific genotypes. More targeted reverse genetics methods aimed at disrupting or enhancing specific gene expression, such as germline transformation (Peloquin et al, 2000;Marcus et al, 2004) and especially RNA interference (Fabrick et al, 2004; Eleftherianos et al, in press), have been successfully applied to lepidopteran species. Such techniques become increasingly transferable to diverse species (Marcus, 2005), and represent a promising way to test the involvement of genes in wing pattern phenotypes for species with rapid development such as Heliconius.…”