Molecular mechanisms underlying the formation of larval green color and camouflage patterns in swallowtail butterfly, Papilio memnon

Insects have various strategies like mimicry or camouflage to avoid predation. Swallowtail butterfly larvae switch from a black and white pattern mimicking bird droppings to a green camouflage pattern in the fifth (final) instar. This larval pattern switch is regulated during the juvenile hormone (JH)-sensitive period, when JH titer declines rapidly, and clawless (cll), abdominal-A (abd-A), and Abdominal-B (Abd-B) function during this period. However, the molecular mechanism behind the background green color, a crucial aspect of the camouflage pattern, remains poorly understood. Here, we used Papilio memnon, which switches to the camouflage pattern in the fifth instar but is greenish from the third instar, to investigate the mechanism of camouflage pattern formation, particularly the larval green coloration. Through RNA sequencing, we found that BBPs forming a gene cluster are upregulated in the green regions of P. memnon larvae during the fourth instar, whereas P. xuthus larvae, which have not yet turned green, showed minimal BBPs expression. When BBP1 and BBP2, which were particularly highly expressed, were knocked down by RNAi, there was a phenotypic change in green to yellow in both fourth and fifth instar larvae. Expression analysis and knockdown experiments were conducted also for JHBP, which had been previously reported, and confirmed that it is involved in the synthesis of yellow pigment. Furthermore, knockdown of Ubx resulted in no phenotypic change in fourth instar larvae, but in fifth instar larvae, the eyespots pattern characteristic of the camouflage pattern almost entirely disappeared, suggesting that Ubx is also functional only during JH-sensitive period. Our results indicate that the switch from mimetic to camouflage patterns resulted from the function of cll, abd-A, Abd-B, and Ubx prepatterning genes during the JH-sensitive period. And the increased expression of BBPs and JHBPs, independent of the JH-sensitive period, contributed to the development of green coloration.