Genome-wide identification and gene-editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus

Abstract

Background Insect body coloration often functions as camouflage to survive from predators or mate selection. Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g. Rab protein). However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies. Results Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects. Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P. xuthus as a model and sequenced the transcriptomes of their morphological mutants. Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level. Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases. Conclusions Our data indicated these transporter proteins play an important role in body color of P. xuthus. Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

“…Because insects universally use one of these two pigment classes as filtering pigments in their compound eyes ( Summers et al., 1982 ), white loss-of-function mutations generate eye color defects that are easy to screen visually ( Ge et al., 2016 ), and played a foundational role in the early history of genetics ( Green, 1996 ). These features have made white a conventional target for developing targeted mutagenesis methods in insects ( Bai et al., 2019 ; Heu et al., 2020 ; Li et al., 2020 ; Liu et al., 2021 ; Ohde et al., 2018 ; Sim et al., 2019 ), though it should be noted that its knockout is recessive-lethal in a subset of tested species ( Khan et al., 2017 ; Reding and Pick, 2020 ).…”

Testing for fitness epistasis in a transplant experiment identifies a candidate adaptive locus inTimemastick insects

“…However, the most intriguing candidate gene ( predicted gene g6064) is located approximately 118 kilobase pairs away from outlier 2 and is homologous to the Punch (Pu) gene in D. melanogaster (table 1). This gene codes for a GTP cyclohydrase I enzyme, which is involved in the first step of the production of pteridine pigments in D. melanogaster and other insects, and is associated with eye and body coloration in multiple insect species [37][38][39][40]59]. This led us to hypothesize that this SNP could also be associated with T. chumash eye coloration, a trait that we observed to be quite variable but which is previously unstudied in this species (electronic supplementary material, figure S2).…”

“…Chitinases are associated with cold or heat stress tolerance in several insect species [35,36] suggesting that this locus could be associated with heat tolerance in Timema. However, the most intriguing candidate, Punch, controls the first step of pteridine pigments production and is associated with eye and body coloration in many insect species [37][38][39][40]. This led us to hypothesize that this locus could be primarily associated with eye colour variation in T. chumash.…”

CRISPR/Cas9‐mediated knockout of scarlet gene produces eye color mutants in the soybean looper, Chrysodeixis includens

“…In H. armigera, disrupting scarlet produces pigment-less first-instar larvae and yellow-eyed adults, with significantly lower ommochrome precursors and increased pteridine ekapterin (Khan et al, 2017). Similarly, mutant adults lacking scarlet exhibit bright yellow eyes in S. frugiperda (Cheng et al, 2023), and abnormal eyes with mosaic stripes of white and black/red-brown in Papilio xuthus (Liu et al, 2021). In addition, the scarlet mutant of Bactrocera oleae showed a striking yellow eye phenotype ( Koidou et al, 2020).…”