Molecular Mechanisms Underlying Pupal Protective Color Switch in Papilio polytes Butterflies

Abstract

Mimicry is a survival strategy in which organisms deceive their predators by imitating other organisms or the surrounding environment. One example of this involves pupal color polymorphism, which is widely observed in butterflies and moths. It has been suggested that the pupal colors of Papilio butterflies are selected according to the tactile stimulation experienced before pupation (P). Specifically, larvae crawling on smooth leaves become green pupae, but those crawling on rough stems become brown pupae. These protective colors fit with the surrounding environment. However, the detailed molecular mechanisms underlying pupal polymorphism have generally remained unknown. To reveal these mechanisms, we first established control over the green and brown pupal coloration in Papilio polytes under laboratory conditions and clarified temporal and spatial changes of pupal pigments under both conditions. We also analyzed the expression of coloration genes during the pre-pupal stage and at P in the epidermis under the green and brown conditions, by RNA sequencing and quantitative RT-PCR. These analyses revealed that the brown pupal color is regulated mainly by melanin synthesis genes, tyrosine hydroxylase (TH) and laccase 2. In contrast, the green pupal color was suggested to be formed mainly through the expression of both multiple bilin binding protein (BBP)-related genes responsible for blue pigmentation and multiple juvenile hormone binding protein (JHBP)-related genes responsible for yellow pigmentation. Electroporation-mediated RNAi showed that the knockdown of TH or laccase 2 blocked the brown pupal coloration, and that the knockdown of BBP-or JHBP-related genes caused yellow or blue coloration in the green-conditioned pupae, respectively, supporting the above hypothesis. We here report how genes involved in the pupal coloration of P. polytes are regulated, which sheds light on the evolutionary process of pupal protective colors among Lepidoptera.

RELATIONSHIP BETWEEN PUPAL COLOUR AND SEX RATIO OF THE COMMON MORMON, Papilio polytes romulus CRAMER, 1775 (INSECTA: LEPIDOPTERA), SRI LANKA

“…Any variation in pupal colour plasticity in different zones also needs to be investigated. The molecular mechanism behind pupal colour dimorphism in P. polytes in Japan revealed that the brown colour was determined by melanin synthesis genes, tyrosine hydroxylase and laccase 2 genes while the green colour was determined by expression of both multiple bilin binding protein-related genes (blue pigmentation) and multiple juvenile hormone binding protein-related genes (yellow pigmentation) together (Yoda et al 2020). These genetic factors have not been studied in P. p. romulus in Sri Lanka previously or in this study.…”

Genetic Color Polymorphism of the Whitelined Sphinx Moth larva (Lepidoptera: Sphingidae)

“…These supergenes have the potential to be coupled with life history-related traits ( Cain and Shepard 1954 ), resulting in significant variation of fitness between color morphs ( Abbott and Svensson 2005 ). Recent genomic work has revealed such supergenes associated with coloration during various life stages in several species, including those of the Heliconus and Papilio genera ( Joron et al 2011 , Kunte et al 2014 , Wellenreuther et al 2014 , Saenko et al 2019 , Yoda et al 2020 ). Furthermore, the different supergene complexes that have been found to control coloration at different life stages have also been found to be decoupled from each other ( Medina et al 2020 ).…”

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies

“…Pigments, cuticular protein, and chitin are the main components of puparium (Futahashi et al, 2012;Liu et al, 2018;Yoda et al, 2020). So, we identified the genes related to these components.…”

“…Cuticle is mainly composed of chitin and many kinds of cuticular proteins (Liu et al, 2018). The pigment of green pupae was a mixture of blue (bile pigment) and yellow (carotenoids) chromoproteins (Ohnishi, 1959;Riley et al, 1984;Yoda et al, 2020), while melanin, canthaxanthine, papilioerythrin (oxidized lutein) were found in brown pupae (Ohnishi, 1959). Physiologically, ligatured abdomens experiment reveals that neuroendocrine mechanism involved in the pupal color dimorphism in Papilio xuthus (Awiti and Hidaka, 1982).…”

“…Neuropeptides, commonly signalled by GPCRs, play a central role in the physiological processes around whole life of insects, such as controlling of development, reproduction, behavior, feeding and others (Tanaka et al, 2014;Bauknecht and Jekely, 2015;Yeoh et al, 2017). For molecular basis of pupal coloration, the only report was on pigmentation genes involved in the pupal coloration of P. polytes, including melanin synthesis genes, tyrosine hydroxylase and laccase 2 responsible for the formation of brown pupae, and multiple bilin binding protein (BBP)-related genes and multiple juvenile hormone binding protein (JHBP)-related genes responsible for the formation of green pupae (Yoda et al, 2020). Especially, the knowledge of the expression profiles of PCMH/PMRF and related genes/metabolic pathways between green and brown pupae are very limited.…”