The white gene as a transgenesis marker for the cricket Gryllus bimaculatus.

Abstract

The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G. bimaculatus, we have designed a transformation marker gene inspired from the widespread Drosophila mini-white+. Using CRISPR/Cas9, we first generated a loss-of-function mutant allele of the Gb-white gene (Gb-w), which exhibits a white eye coloration at all developmental stages. We then demonstrate that transgenic insertions of a piggyBac vector containing a 3xP3-Gb-w+ cassette rescue eye pigmentation. As an application, we used this vector to generate G. bimaculatus lines expressing a centromeric histone H3 variant (CenH3.1) fused to EGFP and validated EGFP-CenH3.1 detection at cricket centromeres. Finally, we demonstrate that Minos-based germline transformation and site-specific plasmid insertion with the ΦC31 integrase system function in G. bimaculatus.