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.

Keywords: ATP-binding cassette (ABC) transporters; CRISPR/Cas9; Papilio xuthus; Rab transporters; Transcriptome.

Fig. 1

Phylogenetic tree of ATP-binding cassette (ABC) transporters of Papilio xuthus (Px), Bombyx mori (BGIBM) and Drosophila melanogaster (CG). The maximum likelihood tree was calculated on the basis of multiple alignments of the ABC transporter protein sequences. All ABCs were clustered into eight subfamilies (ABCA-H). The green pentagrams represent the genes belongs to the P. xuthus, the blue circles indicate the genes among B. mori, and the orange boxs show the genes in the genome of D. melanogaster. Four Px genes highlighted in grey in ABCG subfamily were selected to investigate their function in the development of P. xuthus via CRISPR/Cas9 gene-editing technology

Fig. 2

Phylogenetic tree of Rab family of Papilio xuthus (Px), Bombyx mori (BGIBM) and Drosophila melanogaster (CG). The green pentagrams represent the genes belongs to the P. xuthus, the blue circles indicate the genes among B. mori, and the orange boxs show the gene in the genome of D. melanogaster. Lightoid, highlighted in red in cluster D, was selected to investigate its function in the development of P. xuthus via CRISPR/Cas9 gene-editing technology

Fig. 3

CRISPR/Cas9 disruption of white gene resulted in mosaic depigmented phenotypes in larval epidermis, testes and adult eyes of P. xuthus. a The fourth instar larva (L4). b The fifth instar larva (L5). c Testes of the fifth instar larva. d Adult eyes. Left panel: wild types; right panel: white mutants. The area with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red circle in the panels of (a) and (d) and in red square (b). Testes with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red arrow (c). Scale bars: 1 mm. The photo credit is provided by Zhiwei Dong

Fig. 4

Scarlet mutants showed morphological mutation in adult eye color. a wild type of adult eyes. b G0 (the generation from injected eggs), mutant with white and black mosaic eyes. c G0 mutant with red and white mosaic eyes. d G2 (the second generation of G0 adults) mutant with white eyes. The photo credit is provided by Zhiwei Dong

Fig. 5

Brown and ok mutants showed morphological mutation in the fifth instar larva (L5). a wild type of L5. b brown mutant of L5 (c) ok mutant of L5. The area with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red square. The photo credit is provided by Zhiwei Dong

Fig. 6

Lightoid mutants showed morphological mutations in the fourth instar larvae (L4), the fifth instar larvae (L5) and the testis of L5. a L4. b L5. c Testes of L5. The area with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red circle (a) and red square (b). Testes with obviously morphological mutation in mutants and their corresponding part in wild-type were highlighted in red arrow (c). Scale bars: 1 mm. The photo credit is provided by Zhiwei Dong

Fig. 7

Expression level of the exons in which the target sites are located. a Px_03417_w (white). b Px_03415_st (scarlet). c Px_17845_w (brown). d Px_17844_st (ok), and (E) Px_17846_ltd (lightoid). Red, green and blue color indicates the expression level of normal transcripts in wild-types, normal transcripts in mutants and abnormal transcripts in mutants, respectively. The expression level is evaluated by FPKM (Fragments Per Kilobase of exon model per Million mapped reads). The number and marker above the line is the P-value, which is performed with t-test and are marked with * (less than 0.05) and ** (less than 0.01), respectively

Fig. 8

The number of differentially expressed genes (DEGs) among mutants of the five genes and their Venn diagram. a The number of DEGs between mutants of the five edited genes and their wild-types. b Venn diagram of DEGs of mutants of the five edited genes and their wild-types

Fig. 9

The functional enrichment of Gene Ontology (GO) term and KEGG pathway for the differentially expressed genes (DEGs) which were down and up expressed in the mutated groups. a and b represent the gene enrichment of GO term and KEGG pathway of down-regulated DEGs, separately. c and d represent the gene enrichment of GO term and KEGG pathway of up-regulated DEGs, separately