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. 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 pupation 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.
Highlights
More than 150 years ago, pioneering evolutionary biologists identified the phenomenon of mimicry, in which prey deceives its predator by adopting a color, shape, or other characteristic to mimic other objects (Bates, 1862; Wallace, 1865)
With reference to the detailed conditions for selective pupal coloration in P. xuthus (Hiraga, 2006), we attempted to establish these conditions in P. polytes to produce green and brown pupae
When last-instar larvae just before gut purge (GP) were placed on a Kimtowel wiper in a plastic cup and positioned on the shelf exposed to the least amount of light in the incubator, 49 of 50 pre-pupae (98%) developed into brown pupae (Supplementary Figure S1)
Summary
More than 150 years ago, pioneering evolutionary biologists identified the phenomenon of mimicry, in which prey deceives its predator by adopting a color, shape, or other characteristic to mimic other objects (Bates, 1862; Wallace, 1865). Pupae of many butterfly species belonging to the families Papilionidae, Pieridae, Nymphalid, and Lycaenid exhibit pupal cryptic colors, which fit with the background color to enable the avoidance of predators (Maisch and Bückmann, 1987; Jones et al, 2007). Pupae of most species in the Papilio family show green or brown colors in response to the environment (Figure 1). Hidaka et al (1959) reported that brown and green pupae of Papilio xuthus were preyed upon less in lawns with the same background color, suggesting that pupal color dimorphism is effective for protection against predators. It has been suggested that some environmental factors such as temperature, relative humidity, wavelength of light, and photoperiod perceived during the larval growth period influence the protective pupal coloration in some Papilionid and Lycaenid species (Ishizaki and Kato, 1956; Bückmann, 1960; Smith, 1978; Honda, 1981; Yamamoto et al, 2011). In P. xuthus and P. protenor, it was shown that the pupal color was not determined by the background color (Ohnishi and Hidaka, 1956)
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