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Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)
Journal of Insect Physiology 57 (2011) 930–934
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae) Kazuaki Yamamoto a,1, Yuki Tsujimura b, Miwako Kometani b, Chisato Kitazawa c, Abu Taher Md. Fayezul Islam a,d, Akira Yamanaka a,b,* a
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan Biological Institute, Faculty of Education, Yoshida 1677-1, Yamaguchi University, Yamaguchi 753-8513, Japan d Institute of Food and Radiation Biology, AERA, Saver, GPO Box 3787, Dhaka 1000, Bangladesh b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2010 Received in revised form 1 April 2011 Accepted 4 April 2011
We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 8C under a 60 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 8C were transferred and allowed to reach pupation at 10 8C and 25 8C under a 60 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause lightyellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 8C, and 12.2, 48.8 and 39.0% at 25 8C, respectively. On the other hand, when mature larvae reared at 18 8C were transferred and allowed to reach pupation at 10 8C, 18 8C and 25 8C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 8C, 14.5, 26.9 and 58.6% at 18 8C, and 8.3, 21.2 and 70.5% at 25 8C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Byasa alcinous Diapause pupa Diphenism Papilionidae Pupal coloration
1. Introduction The butterfly Byasa alcinous, belonging to the family Papilionidae, is an Aristolochia-feeding species and is distributed in the East Asian region. The life cycle of this species on Honshu Island, Japan, depends on various environmental factors such as photoperiod, temperature and food plants, as well as geographic factors such as latitude and altitude during the larval stages (Kato, 2000, 2001, 2005; Kozuki and Takeda, 2004). The diapause trait represented by non-diapause or diapause periods in the pupal stages is affected by these factors (Kato, 2000, 2001, 2005). Larvae inhabiting central Japan develop into non-diapause pupae
* Corresponding author. Tel.: +81 83 933 5720; fax: +81 83 933 5720. E-mail address: [email protected] (A. Yamanaka). 1 Present address: Noda Gakuen Junior-High & High School, Noda 56, Yamaguchi 753-0094, Japan. 0022-1910/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.04.002
for generations or diapause pupae for overwintering as a result of photoperiod conditions experienced during larval stages; larvae reared under long-day (LD) photoperiods develop into nondiapause pupae with a bright-yellow coloration, while larvae reared under short-day (SD) photoperiods develop into diapause pupae of a light-brown type, which is regarded as a developmental profile and a morphological trait (Kato, 2000). Hence, Kato (2000) also reported that pupal colorations of light-brown types in the Honshu population are generally considered to represent a monochromatic coloration depending on pupal diapause. On the other hand, pupae of the pipevine swallowtail butterfly Battus philenor, which belongs to the family Papilionidae and is an Aristolochia-feeding species similar to B. alcinous, show pupal color diphenism comprising green and brown types, and the pupal color is affected by background coloration or texture of pupational sites as environmental cues; however, the diapause and pupal color response differs between Californian and Virginian populations of
K. Yamamoto et al. / Journal of Insect Physiology 57 (2011) 930–934
this species (Clarke and Sheppard, 1972; Hazel and West, 1983; Sims and Shapiro, 1983). Similarly, some pupae of other Papilionidae butterflies such as Papilio xuthus, P. protenor demetrius, P. demoleus and P. polytes, which are mainly Citrus-feeding species, exhibit pupal color diphenism involving green and brown types, and some environmental cues such as textures and odor of the pupational site also affect the development of pupal coloration (Ohnishi and Hidaka, 1956; Honda, 1979, 1981; Smith, 1978). In P. xuthus, non-diapause pupae of the brown type are controlled by the secretion of a neuroendocrine factor, named pupal-cuticle-melanizing hormone (PCMH), derived from brain, subesophageal ganglion and prothoracic ganglion complexes of the head-thoracic part during pharatepupal stages (Yamanaka et al., 1999). Additionally, diapause pupae of P. xuthus show pupal color polyphenism comprising diapause green, orange and orangebrown types (Ishizaki and Kato, 1956). Recent studies have revealed that diapause pupae of the orange types are produced by the secretion of a neuroendocrine factor, named orange-pupainducing factor (OPIF), derived from mesothoracic and metathoracic ganglia and all abdominal ganglia of 5th instar larvae (Yamanaka et al., 2004, 2006, 2007), although environmental cues producing orange pupae have not been identified clearly. In B. alcinous, there is no information concerning environmental cues affecting pupal coloration, except for photoperiod conditions experienced during larval stages. However, we initially found a small portion of larvae that developed into diapause pupae of yellowish types among the diapause pupae of B. alcinous when mass rearing was conducted under SD conditions in the laboratory. The developmental mechanism producing diapause pupae of yellowish types in this species is currently unknown. In this paper, we investigated the effect of temperature and humidity as environmental cues on diapause pupal coloration in B. alcinous in order to determine whether diapause pupae of B. alcinous exhibit pupal color diphenism (or polyphenism).
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Fig. 1. Pupal coloration of a diapause pupa of Byasa alcinous observed in the field in winter.
of individuals was counted and photographs were taken to classify pupal coloration. 2.3. Temperature and humidity conditions After a gut purge, mature larvae reared under SD conditions at 18 8C were divided into six groups, and transferred under SD conditions at 10, 18 or 25 8C under 60 5 or over 90% RH. For an examination of the influence of humidity, an over 90% RH was used in some experiments by lining a container with moistened paper. When non-moistened paper was placed in a container alone, RH inside the container was below 50%. To maintain a 60 5% RH inside a container for mature larvae after a gut purge, the number of individuals inside the container was limited to between 5 and 8 larvae, whereas the number of insects was limited to less than 5 larvae per container in experiments involving an over 90% RH. RH inside the container was measured by a hygrometer. Individuals were allowed to reach pupation under each condition. After individuals had pupated, the pupal colors were classified into one of five color types represented by brown, lightbrown, yellowish-brown, light-yellow and yellow, as shown in Fig. 2.
2. Materials and methods 3. Results 2.1. Insect 3.1. Diapause pupal coloration in the field Adults of the swallowtail butterfly B. alcinous were collected from suburbs of Yamaguchi or Hofu City, Yamaguchi Prefecture, Japan, from May to October. Female butterflies were fed with a 10% sugar solution daily to ensure reproductive success. Five–seven female butterflies were released in a plastic cage (16 cm 27 cm 18 cm) and allowed to lay eggs on the leaves of the larval food plant, Aristolochi adebilis. Newly hatched larvae kept in a transparent plastic container (13 cm 20 cm 6 cm) fed on the fresh leaves of A. debilis, which were exchanged during the light period on a daily basis. Fresh leaves of the larval food plant A. debilis were collected from suburbs of Yamaguchi and Hofu City, Japan, and kept at 4 8C until used. Larvae were reared under either LD conditions (alternating 16-h light and 8-h dark periods, 16L:8D) at 25 8C for successive generations, or SD conditions (8L:16D) at 18 8C under a 60–80% relative humidity (RH).
To investigate whether the diapause pupal coloration of B. alcinous is represented by a monochromatic color of brown types, field surveys were performed along the riverbank of the Fushino river, Hirai, Yamaguchi City, between January and February, 2008. As shown in Table 1, twenty-one individuals were found on the trunks and twigs of Cherry trees in this area, and all exhibited pupal coloration of dark-brown types (Fig. 1). This finding indicates that mature larvae grown under SD photoperiods during late October develop into diapause pupae, and their pupal colorations exhibit dark-brown colors in the field. 3.2. Classification of diapause pupal coloration To classify the diapause pupal coloration of B. alcinous in the laboratory, newly hatched larvae were reared under SD conditions
2.2. Field observations Field observations were carried out in the Hirai area (39890 N, 1318270 E), Yamaguchi City, Japan. The riverbank of this area is covered mostly with weeds and low grasses dominated by the food plant of B. alcinous larvae, A. debilis, during middle May to late October as described by Saito et al. (2005). Individual diapause pupae were observed on the trunks and twigs of Cherry trees within this area twice from January to February, 2008. The number
Table 1 Classification of pupal coloration types in diapause pupae of Byasa alcinous observed during winter in Yamaguchi, West-Honshu, Japan. N
21 N, number of insects.
Number of pupae classified by pupal coloration type Dark-brown
Brown
Yellow
21
0
0
K. Yamamoto et al. / Journal of Insect Physiology 57 (2011) 930–934
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Fig. 2. Classification of the degree of yellow coloration in diapause pupae of Byasa alcinous. All larvae reared under short-day conditions at 18 8C under a 60 5% relative humidity developed into diapause pupae, and they were graded and classified into 5 types according to pupal coloration. Grade 0 represents a brown type, whereas grade 4 represents a diapause yellow type. Intermediates were classified as either grade 1, 2 or 3 depending on the grade of yellowness on the dorsal side of the pupa.
at 18 8C under a 60–75% RH, and mature larvae after a gut purge were transferred and allowed to reach pupation under SD conditions at 18 8C under a 60 5% RH. All larvae reared under these conditions developed into diapause pupae, which did not start adult development for 30 days at room temperature (23–27 8C) after pupation (N = 100), and diapause pupal colorations were divided and classified into 5 grades according to the level of brownness/yellowness of pupal color: grades 0, 1, 2, 3 and 4 represent a brown, light-brown, yellowish-brown, diapause light-yellow and diapause yellow type, respectively (Fig. 2). Table 2 summarizes the developmental ratio of diapause pupal colorations of B. alcinous individuals pupated under SD conditions at 18 8C and 60 5% RH. One-hundred mature larvae developed into pupal color types of brown and light-brown (58.0%), yellowish-brown (21.0%) and diapause light-yellow and diapause yellow (21.0%), and the average grade score for yellowness (AGY) was 1.34. However, no mature larvae developed into diapause pupae of dark-brown types as shown in Fig. 1.
These results indicate that diapause pupae of B. alcinous show not only pupal coloration of brown types, but also pupal color diphenism (or polyphenism) involving light-brown, yellowishbrown, diapause light-yellow and diapause yellow types under SD conditions at 18 8C under a 60 5% RH. 3.3. Effect of temperature and humidity on diapause pupal coloration To investigate environmental cues affecting the diapause pupal coloration of B. alcinous, mature larvae reared under SD conditions at 18 8C were transferred and allowed to pupate under different temperature and humidity conditions after a gut purge. Table 3 summarizes the developmental ratio of diapause pupal coloration types in B. alcinous under different humidity and temperature conditions. Under a 60 5% RH, mature larvae kept under SD conditions at 10 8C after a gut purge developed into diapause pupae of brown and light-brown (91.2%), yellowish-brown (8.8%) and diapause light-yellow and diapause yellow (0.0%) types with an AGY score of 0.35, whereas mature larvae kept at 25 8C
Table 2 Developmental ratio of diapause pupal coloration of Byasa alcinous pupated under short-day conditions at 18 8C under a 60 5% RH. N
100
Number of pupae classified by grades of yellowness for diapause pupal coloration
AGY
0
1
2
3
4
33
25
21
14
7
1.34
After a gut purge, larvae reared under SD conditions at 18 8C under a 60–80% RH were transferred and allowed to reach pupation under SD conditions at 18 8C under a 60 5% RH. N: number of insects; AGY: average grade score for yellowness on the dorsal side of pupae. AGY = {(0 number of insects of grade 0) + (1 number of insects of grade 1) + (2 number of insects of grade 2) + (3 number of insects of grade 3) + (4 number of insects of grade 4)}/total number of insects.
Table 3 Effects of humidity and temperature on diapause pupal coloration of Byasa alcinous after a gut purge. Humidity (% RH)
Temperature (8C)
N
60 5
10 25 10 18 25
34 41 89 145 193
>90
Number of pupae classified by grades of yellowness for diapause pupal coloration 0
1
2
3
4
25 0 50 3 1
6 5 21 18 15
3 20 15 39 41
0 7 3 41 74
0 9 0 44 62
N: number of insects; AGY: average grade score for yellowness on the dorsal side of pupae. AGY was calculated as shown in Table 2.
AGY
0.35 2.49 0.67 2.72 2.94
K. Yamamoto et al. / Journal of Insect Physiology 57 (2011) 930–934
developed into diapause pupae of brown and light-brown (12.2%), yellowish-brown (48.8%) and diapause light-yellow and diapause yellow (39.0%) types with an AGY score of 2.49. Additionally, a large portion of diapause pupae of grade 0 that pupated at 10 8C under a 60 5% RH exhibited dark-brown colors similar to a pupa presented in Fig. 1 (data not shown). On the other hand, mature larvae kept under SD conditions at 10 8C under an over 90% RH developed into diapause pupae of brown and light-brown (79.8%), yellowish-brown (16.9%) and diapause light-yellow and diapause yellow (3.3%) types with an AGY score of 0.67, whereas mature larvae kept under SD conditions at 18 8C and 25 8C under an over 90% RH also developed into diapause pupae of brown and light-brown (14.5 and 8.3%), yellowish-brown (26.9 and 21.2%) and diapause light-yellow and diapause yellow (58.6 and 70.5%) types with AGY scores of 2.72 and 2.94, respectively. These results indicate that temperature and humidity experienced after a gut purge affect diapause pupal coloration in B. alcinous as environmental cues, and suggest that low-temperature and low-humidity conditions are more effective in inducing pupal color of darker types, whereas high-temperature and highhumidity conditions are more effective in producing yellow types in diapause pupae of B. alcinous. 4. Discussion In the present study, we found that diapause pupae of B. alcinous exhibit pupal color diphenism involving dark-brown and diapause-yellow color types (see Figs. 1 and 2), and that these diapause pupal colorations were regulated mainly by temperature and humidity as environmental cues. Studies on the developmental control of diapause pupal coloration have been performed using butterfly species belonging to different families such as P. xuthus, P. polyxenes (Papilionidae) and Pieris rapae crucivora (Pieridae) (Ishizaki and Kato, 1956; West et al., 1972; Sims, 2007; Yamanaka et al., 2004, 2010), whereas studies on the coloration of SD pupae developing under SD conditions have been performed using Parnara guttata guttata (Hesperiidae), Vanessa cardui (Nymphalidae) and Lycaena phlaeas daimio (Lycaenidae), which represent three butterfly species that undergo larval diapause (Ishii, 1977; Usui et al., 2004; Yamanaka et al., 2009). In regard to the Papilionidae, the family to which B. alcinous belongs, diapause pupae of P. xuthus exhibit pupal color polyphenism involving diapause-green, orange and orange-brown types, and 65% of larvae kept under constant darkness and low temperature (20 8C) conditions during larval and pharate pupal stages as an investigation of environmental cues developed into pupae of orange types, which may be a modification of brown color types (Ishizaki and Kato, 1956). We reported recently that over 95% of diapause pupae exhibited a coloration of orange types when mature larvae of P. xuthus kept under SD conditions at 25 8C after a gut purge were allowed to pupate in rough-surfaced paper containers, consisting of a transparent plastic box with the inner surfaces covered with white kitchen towel paper (Nepia, Oji-nepia Co. Ltd., Japan) (Yamanaka et al., 2004). However, it remains unclear whether the light intensity of the inside of the container, the texture of the paper, or other factors act to influence the development of pupae of orange types in this species as environmental cues. In other butterfly families, temperature-dependent regulation of pupal coloration has already been reported in pupae of two butterfly species, Pa. guttata guttata and V. cardui, independent of LD or SD photoperiods, and low-temperature exposure during the larval and pharate pupal stages has induced the development of pupae exhibiting black or dark color types in both species (Ishii,
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1977; Yamanaka et al., 2009). Additionally, pupal coloration of L. phlaeas daimio is determined by temperature and photoperioddependent regulation, and low-temperature also induces pupae of black types (Usui et al., 2004). In this paper, pupae of dark-brown types in B. alcinous were similarly induced by low-temperature (10 8C) and low-humidity (60 5% RH) (Table 3), which displayed a coloration similar to pupae of dark-brown types found in the field during winter as shown in Fig. 1. In the Yamaguchi region of Western-Honshu, Japan, the climate from November to March usually consists of low temperatures and dry conditions, and mean monthly data for air temperature and RH on November 2007, the period during which the last generation of this species entered diapause for wintering, were 11.6 8C (maximum: 17.7 8C; minimum: 6.8 8C) and 69% RH (minimum: 22% RH), which were taken from the Yamaguchi meteorological station, Yamaguchi Prefecture, of the Japan Meteorological Agency. This indicates that no pupae of diapause-yellow types appear in the field after November, and it has been thought that diapause pupae of B. alcinous show a monochromatic coloration of dark-brown types. In winter, the dark-brown coloration of diapause pupae of B. alcinous might possess a cryptic nature similar to the brown pupae of P. machaon (Wiklund, 1975), and may involve a role as a developmental accelerator by augmenting heat absorption during the winter and spring seasons, as investigated for pupae of black types in Pa. guttata guttata (Ishii, 1977). It is still unclear whether the dark-brown or diapause-yellow type is the original color of diapause pupae in B. alcinous, or whether it is an intermediate color between the brown and diapause-yellow types. It is also interesting that non-diapause pupae of B. alcinous also exhibit pupal color diphenism in response to environmental cues such as temperature and/or humidity as found in this report, although it has been thought that nondiapause pupae are represented by a monochromatic coloration of yellow types (Kato, 2000). Further study of B. alcinous should be conducted to clarify some of the above questions, and further comparative study between different butterfly families is necessary in order to reveal the diversity of pupal color diphenism (or polyphenism) in butterflies. Acknowledgements We thank Prof. K. Endo (Yamaguchi University), Dr. I. Kodama (Asa High School) and Mr. T. Uchiyama for encouragement throughout this work. References Clarke, C.A., Sheppard, P.M., 1972. Genetic and environmental factors influencing pupal colour in the swallowtail butterflies Battus philenor (L.) and Papilio polytes L. Journal of Entomology 46, 123–133. Hazel, W.N., West, D.A., 1983. The effect of larval photoperiod on pupal colour and diapause in swallowtail butterflies. Ecological Entomology 8, 37–42. Honda, K., 1979. Environmental factors affecting the pupal coloration in Papilio protenor demetrius Cr. (Lepidoptera: Papilionidae) I. Effect of chemical stimuli ˆ 47, 191–195. (odor). Kontyu Honda, K., 1981. Environmental factors affecting the pupal coloration in Papilio protenor demetrius Cr. (Lepidoptera: Papilionidae) II. Effect of physical stimuli. Applied Entomology and Zoology 16, 467–471. Ishii, M., 1977. Melanization of pupae caused by low temperature in the rice-plant skipper, Parnara guttata guttata Bremer et Grey (Lepidoptera, Hesperiidae). Kontyu´ 45, 271–275. Ishizaki, H., Kato, M., 1956. Environmental factors affecting the formation of orange pupae in Papilio xuthus. Memoirs of the College of Science, University of Kyoto, Series B 23, 11–18. Kato, Y., 2000. Interpopulational variation in pupal diapause of the butterfly Atrophaneura alcinous (Klug) (Lepidoptera, Papilionidae) in the Kanto District, eastern Japan. Transactions of the Lepidopterological Society of Japan 51, 233–242. Kato, Y., 2001. Seasonal occurrence of the Aristolochia-feeding butterfly Atrophaneura alcinous (Lepidoptera, Papilionidae): comparison between the lowland and mountain populations. Transactions of the Lepidopterological Society of Japan 52, 139–149.
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Kato, Y., 2005. Geographic variation in photoperiodic response for the induction of pupal diapause in the Aristolochia-feeding butterfly Atrophaneura alcinous. Applied Entomology and Zoology 40, 347–350. Kozuki, Y., Takeda, M., 2004. Split life cycle and differentiations in diapause characteristics in three host-habitat strains of Atrophaneura alcinous (Lepidoptera: Papilionidae). Japanese Journal of Environmental Entomology and Zoology 15, 157–168. Ohnishi, E., Hidaka, T., 1956. Effect of environmental factors on the determination of pupal types in some swallowtail, Papilio xuthus L. and P. protenor demetrius Cr. Zoological Magazine 65, 185–187 (in Japanese with English summary). Saito, M., Yamanaka, A., Masaki, H., Nishijima, A., Harada, Y., Kitazawa, C., Abe, H., Watanabe, M., Endo, K., 2005. Environmental factors affecting black/white coloration of the silken girdle in the swallowtail butterfly, Atrophaneura alcinous (Lepidoptera: Papilionidae). Zoological Science 22, 1259–1263. Sims, S.R., 2007. Diapause dynamics, seasonal phenology, and pupal color dimorphism of Papilio polyxenes in southern Florida, USA. Entomologia Experimentalis et Applicata 123, 239–245. Sims, S.R., Shapiro, A.M., 1983. Pupal colour dimorphism in California Battusphilenor: pupation sites, environmental control and diapause linkage. Ecological Entomology 8, 95–104. Smith, A.G., 1978. Environmental factors influencing pupal colour determination in Lepidoptera I. Experiments with Papilio polytes, Papilio demoleus and Papilio polyxenes. Proceedings of the Royal Society of London, Series B: Biological Science 200, 295–329. Usui, Y., Yamanaka, A., Islam, A.T.M.F., Shahjahan, R., Endo, K., 2004. Photoperiodand temperature-dependent regulation of pupal beige/black polymorphism in the small copper butterfly, Lycaena phlaeas daimio Seitz. Zoological Science 21, 835–839.
West, D.A., Snellings, W.N., Herbek, T.A., 1972. Pupal color dimorphism and its environmental control in Papilio polyxenes asterius Stoll (Lepidoptera: Papilionidae). Journal of the New York Entomological Society 80, 205–211. Wiklund, C., 1975. Pupal colour polymorphism in Papilio machaon L. and the survival in the field of cryptic versus non-cryptic pupae. Transactions of the Royal Entomological Society of London 127, 73–84. Yamanaka, A., Endo, K., Nishida, H., Kawamura, N., Hatase, Y., Kong, W., Kataoka, H., Suzuki, A., 1999. Extraction of partial characterizations of pupal-cuticle-melanizing hormone (PCMH) in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae). Zoological Science 16, 261–268. Yamanaka, A., Imai, H., Adachi, M., Komatsu, M., Islam, A.T.M.F., Kodama, I., Kitazawa, C., Endo, K., 2004. Hormonal control of the orange coloration of diapause pupae in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae). Zoological Science 21, 1049–1055. Yamanaka, A., Adachi, M., Imai, H., Uchiyama, T., Inoue, M., Islam, A.T.M.F., Kitazawa, C., Endo, K., 2006. Properties of orange-pupa-inducing factor (OPIF) in the swallowtail butterfly, Papilio xuthus L. Peptides 27, 534–538. Yamanaka, A., Uchiyama, T., Naotori, M., Adachi, M., Inoue, M., Kitazawa, C., Endo, K., 2007. Effect of Bombyx mori central nervous system extracts on diapause pupal coloration in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera, Papilionidae). Chugoku Kontyu 21, 55–59. Yamanaka, A., Kometani, M., Yamamoto, K., Tsujimura, Y., Motomura, M., Kitazawa, C., Endo, K., 2009. Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui. Journal of Insect Physiology 55, 512–517. Yamanaka, A., Nomura, Y., Fujita, T., Hayase, T., Yamashita, K., Fujishima, T., Kodama, I., Genda, T., Kitazawa, C., Endo, K., 2010. Environmental and cerebral factors affecting diapause pupal coloration in the cabbage small white butterfly Pieris rapae crucivora. Information 13B, 1090–1098.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae) Kazuaki Yamamoto a,1, Yuki Tsujimura b, Miwako Kometani b, Chisato Kitazawa c, Abu Taher Md. Fayezul Islam a,d, Akira Yamanaka a,b,* a
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan Biological Institute, Faculty of Education, Yoshida 1677-1, Yamaguchi University, Yamaguchi 753-8513, Japan d Institute of Food and Radiation Biology, AERA, Saver, GPO Box 3787, Dhaka 1000, Bangladesh b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2010 Received in revised form 1 April 2011 Accepted 4 April 2011
We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 8C under a 60 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 8C were transferred and allowed to reach pupation at 10 8C and 25 8C under a 60 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause lightyellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 8C, and 12.2, 48.8 and 39.0% at 25 8C, respectively. On the other hand, when mature larvae reared at 18 8C were transferred and allowed to reach pupation at 10 8C, 18 8C and 25 8C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 8C, 14.5, 26.9 and 58.6% at 18 8C, and 8.3, 21.2 and 70.5% at 25 8C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues. ß 2011 Elsevier Ltd. All rights reserved.
Keywords: Byasa alcinous Diapause pupa Diphenism Papilionidae Pupal coloration
1. Introduction The butterfly Byasa alcinous, belonging to the family Papilionidae, is an Aristolochia-feeding species and is distributed in the East Asian region. The life cycle of this species on Honshu Island, Japan, depends on various environmental factors such as photoperiod, temperature and food plants, as well as geographic factors such as latitude and altitude during the larval stages (Kato, 2000, 2001, 2005; Kozuki and Takeda, 2004). The diapause trait represented by non-diapause or diapause periods in the pupal stages is affected by these factors (Kato, 2000, 2001, 2005). Larvae inhabiting central Japan develop into non-diapause pupae
* Corresponding author. Tel.: +81 83 933 5720; fax: +81 83 933 5720. E-mail address: [email protected] (A. Yamanaka). 1 Present address: Noda Gakuen Junior-High & High School, Noda 56, Yamaguchi 753-0094, Japan. 0022-1910/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.04.002
for generations or diapause pupae for overwintering as a result of photoperiod conditions experienced during larval stages; larvae reared under long-day (LD) photoperiods develop into nondiapause pupae with a bright-yellow coloration, while larvae reared under short-day (SD) photoperiods develop into diapause pupae of a light-brown type, which is regarded as a developmental profile and a morphological trait (Kato, 2000). Hence, Kato (2000) also reported that pupal colorations of light-brown types in the Honshu population are generally considered to represent a monochromatic coloration depending on pupal diapause. On the other hand, pupae of the pipevine swallowtail butterfly Battus philenor, which belongs to the family Papilionidae and is an Aristolochia-feeding species similar to B. alcinous, show pupal color diphenism comprising green and brown types, and the pupal color is affected by background coloration or texture of pupational sites as environmental cues; however, the diapause and pupal color response differs between Californian and Virginian populations of
K. Yamamoto et al. / Journal of Insect Physiology 57 (2011) 930–934
this species (Clarke and Sheppard, 1972; Hazel and West, 1983; Sims and Shapiro, 1983). Similarly, some pupae of other Papilionidae butterflies such as Papilio xuthus, P. protenor demetrius, P. demoleus and P. polytes, which are mainly Citrus-feeding species, exhibit pupal color diphenism involving green and brown types, and some environmental cues such as textures and odor of the pupational site also affect the development of pupal coloration (Ohnishi and Hidaka, 1956; Honda, 1979, 1981; Smith, 1978). In P. xuthus, non-diapause pupae of the brown type are controlled by the secretion of a neuroendocrine factor, named pupal-cuticle-melanizing hormone (PCMH), derived from brain, subesophageal ganglion and prothoracic ganglion complexes of the head-thoracic part during pharatepupal stages (Yamanaka et al., 1999). Additionally, diapause pupae of P. xuthus show pupal color polyphenism comprising diapause green, orange and orangebrown types (Ishizaki and Kato, 1956). Recent studies have revealed that diapause pupae of the orange types are produced by the secretion of a neuroendocrine factor, named orange-pupainducing factor (OPIF), derived from mesothoracic and metathoracic ganglia and all abdominal ganglia of 5th instar larvae (Yamanaka et al., 2004, 2006, 2007), although environmental cues producing orange pupae have not been identified clearly. In B. alcinous, there is no information concerning environmental cues affecting pupal coloration, except for photoperiod conditions experienced during larval stages. However, we initially found a small portion of larvae that developed into diapause pupae of yellowish types among the diapause pupae of B. alcinous when mass rearing was conducted under SD conditions in the laboratory. The developmental mechanism producing diapause pupae of yellowish types in this species is currently unknown. In this paper, we investigated the effect of temperature and humidity as environmental cues on diapause pupal coloration in B. alcinous in order to determine whether diapause pupae of B. alcinous exhibit pupal color diphenism (or polyphenism).
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Fig. 1. Pupal coloration of a diapause pupa of Byasa alcinous observed in the field in winter.
of individuals was counted and photographs were taken to classify pupal coloration. 2.3. Temperature and humidity conditions After a gut purge, mature larvae reared under SD conditions at 18 8C were divided into six groups, and transferred under SD conditions at 10, 18 or 25 8C under 60 5 or over 90% RH. For an examination of the influence of humidity, an over 90% RH was used in some experiments by lining a container with moistened paper. When non-moistened paper was placed in a container alone, RH inside the container was below 50%. To maintain a 60 5% RH inside a container for mature larvae after a gut purge, the number of individuals inside the container was limited to between 5 and 8 larvae, whereas the number of insects was limited to less than 5 larvae per container in experiments involving an over 90% RH. RH inside the container was measured by a hygrometer. Individuals were allowed to reach pupation under each condition. After individuals had pupated, the pupal colors were classified into one of five color types represented by brown, lightbrown, yellowish-brown, light-yellow and yellow, as shown in Fig. 2.
2. Materials and methods 3. Results 2.1. Insect 3.1. Diapause pupal coloration in the field Adults of the swallowtail butterfly B. alcinous were collected from suburbs of Yamaguchi or Hofu City, Yamaguchi Prefecture, Japan, from May to October. Female butterflies were fed with a 10% sugar solution daily to ensure reproductive success. Five–seven female butterflies were released in a plastic cage (16 cm 27 cm 18 cm) and allowed to lay eggs on the leaves of the larval food plant, Aristolochi adebilis. Newly hatched larvae kept in a transparent plastic container (13 cm 20 cm 6 cm) fed on the fresh leaves of A. debilis, which were exchanged during the light period on a daily basis. Fresh leaves of the larval food plant A. debilis were collected from suburbs of Yamaguchi and Hofu City, Japan, and kept at 4 8C until used. Larvae were reared under either LD conditions (alternating 16-h light and 8-h dark periods, 16L:8D) at 25 8C for successive generations, or SD conditions (8L:16D) at 18 8C under a 60–80% relative humidity (RH).
To investigate whether the diapause pupal coloration of B. alcinous is represented by a monochromatic color of brown types, field surveys were performed along the riverbank of the Fushino river, Hirai, Yamaguchi City, between January and February, 2008. As shown in Table 1, twenty-one individuals were found on the trunks and twigs of Cherry trees in this area, and all exhibited pupal coloration of dark-brown types (Fig. 1). This finding indicates that mature larvae grown under SD photoperiods during late October develop into diapause pupae, and their pupal colorations exhibit dark-brown colors in the field. 3.2. Classification of diapause pupal coloration To classify the diapause pupal coloration of B. alcinous in the laboratory, newly hatched larvae were reared under SD conditions
2.2. Field observations Field observations were carried out in the Hirai area (39890 N, 1318270 E), Yamaguchi City, Japan. The riverbank of this area is covered mostly with weeds and low grasses dominated by the food plant of B. alcinous larvae, A. debilis, during middle May to late October as described by Saito et al. (2005). Individual diapause pupae were observed on the trunks and twigs of Cherry trees within this area twice from January to February, 2008. The number
Table 1 Classification of pupal coloration types in diapause pupae of Byasa alcinous observed during winter in Yamaguchi, West-Honshu, Japan. N
21 N, number of insects.
Number of pupae classified by pupal coloration type Dark-brown
Brown
Yellow
21
0
0
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Fig. 2. Classification of the degree of yellow coloration in diapause pupae of Byasa alcinous. All larvae reared under short-day conditions at 18 8C under a 60 5% relative humidity developed into diapause pupae, and they were graded and classified into 5 types according to pupal coloration. Grade 0 represents a brown type, whereas grade 4 represents a diapause yellow type. Intermediates were classified as either grade 1, 2 or 3 depending on the grade of yellowness on the dorsal side of the pupa.
at 18 8C under a 60–75% RH, and mature larvae after a gut purge were transferred and allowed to reach pupation under SD conditions at 18 8C under a 60 5% RH. All larvae reared under these conditions developed into diapause pupae, which did not start adult development for 30 days at room temperature (23–27 8C) after pupation (N = 100), and diapause pupal colorations were divided and classified into 5 grades according to the level of brownness/yellowness of pupal color: grades 0, 1, 2, 3 and 4 represent a brown, light-brown, yellowish-brown, diapause light-yellow and diapause yellow type, respectively (Fig. 2). Table 2 summarizes the developmental ratio of diapause pupal colorations of B. alcinous individuals pupated under SD conditions at 18 8C and 60 5% RH. One-hundred mature larvae developed into pupal color types of brown and light-brown (58.0%), yellowish-brown (21.0%) and diapause light-yellow and diapause yellow (21.0%), and the average grade score for yellowness (AGY) was 1.34. However, no mature larvae developed into diapause pupae of dark-brown types as shown in Fig. 1.
These results indicate that diapause pupae of B. alcinous show not only pupal coloration of brown types, but also pupal color diphenism (or polyphenism) involving light-brown, yellowishbrown, diapause light-yellow and diapause yellow types under SD conditions at 18 8C under a 60 5% RH. 3.3. Effect of temperature and humidity on diapause pupal coloration To investigate environmental cues affecting the diapause pupal coloration of B. alcinous, mature larvae reared under SD conditions at 18 8C were transferred and allowed to pupate under different temperature and humidity conditions after a gut purge. Table 3 summarizes the developmental ratio of diapause pupal coloration types in B. alcinous under different humidity and temperature conditions. Under a 60 5% RH, mature larvae kept under SD conditions at 10 8C after a gut purge developed into diapause pupae of brown and light-brown (91.2%), yellowish-brown (8.8%) and diapause light-yellow and diapause yellow (0.0%) types with an AGY score of 0.35, whereas mature larvae kept at 25 8C
Table 2 Developmental ratio of diapause pupal coloration of Byasa alcinous pupated under short-day conditions at 18 8C under a 60 5% RH. N
100
Number of pupae classified by grades of yellowness for diapause pupal coloration
AGY
0
1
2
3
4
33
25
21
14
7
1.34
After a gut purge, larvae reared under SD conditions at 18 8C under a 60–80% RH were transferred and allowed to reach pupation under SD conditions at 18 8C under a 60 5% RH. N: number of insects; AGY: average grade score for yellowness on the dorsal side of pupae. AGY = {(0 number of insects of grade 0) + (1 number of insects of grade 1) + (2 number of insects of grade 2) + (3 number of insects of grade 3) + (4 number of insects of grade 4)}/total number of insects.
Table 3 Effects of humidity and temperature on diapause pupal coloration of Byasa alcinous after a gut purge. Humidity (% RH)
Temperature (8C)
N
60 5
10 25 10 18 25
34 41 89 145 193
>90
Number of pupae classified by grades of yellowness for diapause pupal coloration 0
1
2
3
4
25 0 50 3 1
6 5 21 18 15
3 20 15 39 41
0 7 3 41 74
0 9 0 44 62
N: number of insects; AGY: average grade score for yellowness on the dorsal side of pupae. AGY was calculated as shown in Table 2.
AGY
0.35 2.49 0.67 2.72 2.94
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developed into diapause pupae of brown and light-brown (12.2%), yellowish-brown (48.8%) and diapause light-yellow and diapause yellow (39.0%) types with an AGY score of 2.49. Additionally, a large portion of diapause pupae of grade 0 that pupated at 10 8C under a 60 5% RH exhibited dark-brown colors similar to a pupa presented in Fig. 1 (data not shown). On the other hand, mature larvae kept under SD conditions at 10 8C under an over 90% RH developed into diapause pupae of brown and light-brown (79.8%), yellowish-brown (16.9%) and diapause light-yellow and diapause yellow (3.3%) types with an AGY score of 0.67, whereas mature larvae kept under SD conditions at 18 8C and 25 8C under an over 90% RH also developed into diapause pupae of brown and light-brown (14.5 and 8.3%), yellowish-brown (26.9 and 21.2%) and diapause light-yellow and diapause yellow (58.6 and 70.5%) types with AGY scores of 2.72 and 2.94, respectively. These results indicate that temperature and humidity experienced after a gut purge affect diapause pupal coloration in B. alcinous as environmental cues, and suggest that low-temperature and low-humidity conditions are more effective in inducing pupal color of darker types, whereas high-temperature and highhumidity conditions are more effective in producing yellow types in diapause pupae of B. alcinous. 4. Discussion In the present study, we found that diapause pupae of B. alcinous exhibit pupal color diphenism involving dark-brown and diapause-yellow color types (see Figs. 1 and 2), and that these diapause pupal colorations were regulated mainly by temperature and humidity as environmental cues. Studies on the developmental control of diapause pupal coloration have been performed using butterfly species belonging to different families such as P. xuthus, P. polyxenes (Papilionidae) and Pieris rapae crucivora (Pieridae) (Ishizaki and Kato, 1956; West et al., 1972; Sims, 2007; Yamanaka et al., 2004, 2010), whereas studies on the coloration of SD pupae developing under SD conditions have been performed using Parnara guttata guttata (Hesperiidae), Vanessa cardui (Nymphalidae) and Lycaena phlaeas daimio (Lycaenidae), which represent three butterfly species that undergo larval diapause (Ishii, 1977; Usui et al., 2004; Yamanaka et al., 2009). In regard to the Papilionidae, the family to which B. alcinous belongs, diapause pupae of P. xuthus exhibit pupal color polyphenism involving diapause-green, orange and orange-brown types, and 65% of larvae kept under constant darkness and low temperature (20 8C) conditions during larval and pharate pupal stages as an investigation of environmental cues developed into pupae of orange types, which may be a modification of brown color types (Ishizaki and Kato, 1956). We reported recently that over 95% of diapause pupae exhibited a coloration of orange types when mature larvae of P. xuthus kept under SD conditions at 25 8C after a gut purge were allowed to pupate in rough-surfaced paper containers, consisting of a transparent plastic box with the inner surfaces covered with white kitchen towel paper (Nepia, Oji-nepia Co. Ltd., Japan) (Yamanaka et al., 2004). However, it remains unclear whether the light intensity of the inside of the container, the texture of the paper, or other factors act to influence the development of pupae of orange types in this species as environmental cues. In other butterfly families, temperature-dependent regulation of pupal coloration has already been reported in pupae of two butterfly species, Pa. guttata guttata and V. cardui, independent of LD or SD photoperiods, and low-temperature exposure during the larval and pharate pupal stages has induced the development of pupae exhibiting black or dark color types in both species (Ishii,
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1977; Yamanaka et al., 2009). Additionally, pupal coloration of L. phlaeas daimio is determined by temperature and photoperioddependent regulation, and low-temperature also induces pupae of black types (Usui et al., 2004). In this paper, pupae of dark-brown types in B. alcinous were similarly induced by low-temperature (10 8C) and low-humidity (60 5% RH) (Table 3), which displayed a coloration similar to pupae of dark-brown types found in the field during winter as shown in Fig. 1. In the Yamaguchi region of Western-Honshu, Japan, the climate from November to March usually consists of low temperatures and dry conditions, and mean monthly data for air temperature and RH on November 2007, the period during which the last generation of this species entered diapause for wintering, were 11.6 8C (maximum: 17.7 8C; minimum: 6.8 8C) and 69% RH (minimum: 22% RH), which were taken from the Yamaguchi meteorological station, Yamaguchi Prefecture, of the Japan Meteorological Agency. This indicates that no pupae of diapause-yellow types appear in the field after November, and it has been thought that diapause pupae of B. alcinous show a monochromatic coloration of dark-brown types. In winter, the dark-brown coloration of diapause pupae of B. alcinous might possess a cryptic nature similar to the brown pupae of P. machaon (Wiklund, 1975), and may involve a role as a developmental accelerator by augmenting heat absorption during the winter and spring seasons, as investigated for pupae of black types in Pa. guttata guttata (Ishii, 1977). It is still unclear whether the dark-brown or diapause-yellow type is the original color of diapause pupae in B. alcinous, or whether it is an intermediate color between the brown and diapause-yellow types. It is also interesting that non-diapause pupae of B. alcinous also exhibit pupal color diphenism in response to environmental cues such as temperature and/or humidity as found in this report, although it has been thought that nondiapause pupae are represented by a monochromatic coloration of yellow types (Kato, 2000). Further study of B. alcinous should be conducted to clarify some of the above questions, and further comparative study between different butterfly families is necessary in order to reveal the diversity of pupal color diphenism (or polyphenism) in butterflies. Acknowledgements We thank Prof. K. Endo (Yamaguchi University), Dr. I. Kodama (Asa High School) and Mr. T. Uchiyama for encouragement throughout this work. References Clarke, C.A., Sheppard, P.M., 1972. Genetic and environmental factors influencing pupal colour in the swallowtail butterflies Battus philenor (L.) and Papilio polytes L. Journal of Entomology 46, 123–133. Hazel, W.N., West, D.A., 1983. The effect of larval photoperiod on pupal colour and diapause in swallowtail butterflies. Ecological Entomology 8, 37–42. Honda, K., 1979. Environmental factors affecting the pupal coloration in Papilio protenor demetrius Cr. (Lepidoptera: Papilionidae) I. Effect of chemical stimuli ˆ 47, 191–195. (odor). Kontyu Honda, K., 1981. Environmental factors affecting the pupal coloration in Papilio protenor demetrius Cr. (Lepidoptera: Papilionidae) II. Effect of physical stimuli. Applied Entomology and Zoology 16, 467–471. Ishii, M., 1977. Melanization of pupae caused by low temperature in the rice-plant skipper, Parnara guttata guttata Bremer et Grey (Lepidoptera, Hesperiidae). Kontyu´ 45, 271–275. Ishizaki, H., Kato, M., 1956. Environmental factors affecting the formation of orange pupae in Papilio xuthus. Memoirs of the College of Science, University of Kyoto, Series B 23, 11–18. Kato, Y., 2000. Interpopulational variation in pupal diapause of the butterfly Atrophaneura alcinous (Klug) (Lepidoptera, Papilionidae) in the Kanto District, eastern Japan. Transactions of the Lepidopterological Society of Japan 51, 233–242. Kato, Y., 2001. Seasonal occurrence of the Aristolochia-feeding butterfly Atrophaneura alcinous (Lepidoptera, Papilionidae): comparison between the lowland and mountain populations. Transactions of the Lepidopterological Society of Japan 52, 139–149.
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