Identification of stage-specific larval camouflage associated genes in the swallowtail butterfly, Papilio xuthus

The swallowtail butterfly Papilio xuthus changes its larval body pattern dramatically during the fourth ecdysis. The penultimate (fourth) instar larvae mimic bird droppings that have many tubercle structures on their surface, whereas the final (fifth) instar larvae have a green camouflage color. To identify the genes involved in stage-specific larval mimicry markings, we compared the epidermal messenger ribonucleic acid expression between the third and fourth molts of P. xuthus using a complementary deoxyribonucleic acid subtraction method. After analyzing 2,072 clones from two subtractive libraries, we obtained 31 and 64 candidate genes for final (Fsg) or penultimate instar-specific genes (Psg), respectively. The expression pattern of each gene was examined by reverse transcriptase polymerase chain reaction and in situ hybridization. Among Fsgs, Fsg02 and Fsg20 were expressed in the presumptive green region only during the fourth molt, suggesting that these two genes were correlated with green coloration in the final instar. Among Psgs, 11 cuticular protein genes were expressed specifically in the tubercle structures during the third molt. These genes are likely to be involved in the formation of the unique tubercle structures observed in the juvenile instar. We found that genes with similar expression patterns do not necessarily share the same protein motifs and vice versa. This study provides novel molecular markers and insights into the molecular mechanisms of the larval color pattern and body shape.