The regulation of phenotypic plasticity of eyespots in the butterfly Bicyclus anynana

We use an outcrossed stock and selected lines of Bicyclus anynana in combination with measurements and manipulations of ecdysteroid hormones in early pupae to examine the regulation of eyespot size in adult butterflies. The eyespots on the ventral wing surfaces express adaptive phenotypic plasticity in response to the dry-wet seasonal environments of the butterflies. Larvae reared at low or high temperatures produce adults with small or large ventral eyespots, respectively. Our experiments examine the role of ecdysteroids in mediating this phenotypic plasticity. Higher titers of ecdysteroids shortly after pupation yield eclolarger ventral wing eyespots. There is an uncoupling of the ventral eyespots and those on the dorsal forewing. The latter do not show phenotypic plasticity. They show very little response to rearing temperature, and variation in their size is not associated with differences in the dynamics of ecdysteroids in early pupae. A testable hypothesis in terms of the distribution of hormone receptors in the developmental "organizers" or foci of the eyespots is proposed to account for how some eyespots express plasticity while others do not.     

Adaptive plasticity in wing melanisation of a montane butterfly across a Himalayan elevational gradient

1. Traits that are significant to the thermal ecology of temperate or montane species are expected to prominently co-vary with the thermal environment experienced by an organism. The Himalayan Pieris canidia butterfly exhibits considerable variation in wing melanisation. We investigated: (i) whether variation in wing melanisation and (ii) activity period of this montane butterfly was influenced by the seasonally and elevationally changing thermal landscape.
2. We discovered that wing melanisation varied across elevation, seasons, sex, and wing surfaces, with the variation strongly structured in space and time: colder seasons and higher elevations produced more melanic individuals. Notably, melanisation did not vary uniformly across all wing surfaces: (i) melanisation of the ventral hindwing co-varied much more prominently with elevation, but (ii) melanisation on all other surfaces varied with seasonal changes in the thermal environment.
3. Observed wing surface-specific patterns indicated thermoregulatory function for this variation in melanisation. Such wing surface-specific responses to seasonal and elevational variation in temperature have rarely been reported in montane insects.
4. Moreover, daily and seasonal thermal cycles were found to strongly influence activity periods of this species, suggesting the potential limits to wing melanisation plasticity.
5. Overall, these results showed that the seasonal and elevational gradients in temperature influence the thermal phenotype as well as activity periods of this Himalayan butterfly. It will be critical to study the phenotypic evolution of such montane insects in response to the ongoing climate change, which is already showing significant signs in this iconic mountain range.

     

Colour pattern homology and evolution in Vanessa butterflies (Nymphalidae: Nymphalini): eyespot characters

Ocelli are serially repeated colour patterns on the wings of many butterflies. Eyespots are elaborate ocelli that function in predator avoidance and deterrence as well as in mate choice. A phylogenetic approach was used to study ocelli and eyespot evolution in Vanessa butterflies, a genus exhibiting diverse phenotypes among these serial homologs. Forty‐four morphological characters based on eyespot number, arrangement, shape and the number of elements in each eyespot were defined and scored. Ocelli from eight wing cells on the dorsal and ventral surfaces of the forewing and hindwing were evaluated. The evolution of these characters was traced over a phylogeny of Vanessa based on 7750 DNA base pairs from 10 genes. Our reconstruction predicts that the ancestral Vanessa had 5 serially arranged ocelli on all four wing surfaces. The ancestral state on the dorsal forewing and ventral hindwing was ocelli arranged in two heterogeneous groups. On the dorsal hindwing, the ancestral state was either homogenous or ocelli arranged in two heterogeneous groups. On the ventral forewing, we determined that the ancestral state was organized into three heterogeneous groups. In Vanessa, almost all ocelli are individuated and capable of independent evolution relative to other colour patterns except for the ocelli in cells ?1 and 0 on the dorsal and ventral forewings, which appear to be constrained to evolve in parallel. The genus Vanessa is a good model system for the study of serial homology and the interaction of selective forces with developmental architecture to produce diversity in butterfly colour patterns.     

Phenotypic variation in adult morphology and pupal colour within and among families of the speckled wood butterfly Pararge aegeria

Abstract .1. Larvae from eggs of fifteen wild-caught speckled wood females were reared individually under common conditions (LD 18:6 h and 17 °C) in the laboratory.
2. Pupal colour (green or brown) and the following adult morphological variables that are known or assumed to be related to behavioural variation (thermoregulation, flight, crypsis) were studied: size, relative thorax mass, area of yellow patches on the dorsal forewing, size of submarginal eyespots on the dorsal hindwing, colour of the dorsal thorax, dorsal basal wing, and dorsal distal wing.
3. The results of the breeding experiment indicate significant differences in adult morphological traits among families, sexes, and pupal colour types.
4. All adult morphological variables (except spot size and thorax colouration) differed significantly among families, suggesting genetic variation underlying the phenotypic variation. Heritabilities for these features were intermediate (0.38) to high (> 1).
5. Apart from known aspects (e.g. size), novel aspects of the sexual dimorphism were found: females had paler thoraxes than males, which relates to higher abundance of fur on the thorax and hence to thermoregulatory differences.
6. Green pupae produced larger individuals with a larger relative thorax mass than brown pupae. Green pupae produced adults with a paler basal wing colour in females, but not in males. These relationships are novel and suggest a trade-off between juvenile and adult investment.     

Ultrastructural variation tune wing coloration of a moth Asota caricae Fabricius, 1775

Butterflies and moths develop highly ordered coloration in their wing for signal transmission. We have investigated the ultrastructural arrangement of wing coloration of a moth Asota caricae, applying light, optical polarized, and scanning electron microscopy, and spectrophotometry. The forewing of the moth is brown in color with a white spot at the center. The hindwing is golden yellow in color with many black patches in it. The ventral part of the forewing and dorsal hindwing share the similar color pattern. The ventral part of the hindwing has dull coloration in comparison to the dorsal one although the pattern remains same. The spectrometry analysis reveals various patterns of absorbance and reflectance spectra for various colors. The peak observed for various colors remain same although the intensity of peak changes. Bright colors possess highly ordered structures whereas irregular structures are found in dull colored scales. The color variation observed due to dorsal and ventral part of the wing is due to the minute difference observed in terms of ultrastructural arrangement revealed by scanning electron microscope. The color pattern of A. caricae is due to variation of microstructures present within the scale.     

Thermoregulatory significance of wing melanization in Pieris butterflies (Lepidoptera: Pieridae): physics,posture, and pattern

Summary Pieris butterflies use a novel behavioral posture for thermoregulation called reflectance basking, in which the wings are used as solar reflectors to reflect radiation to the body. As a means of exploring the thermoregulatory significance of wing melanization patterns, I examine the relation of basking posture and wing color pattern to body temperature. A mathematical model of the reflectance process predicts certain combinations of dorsal wing melanization pattern and basking posture that maximize body temperature. Laboratory experiments and field observations show that this model correctly predicts qualitative differences in the relation of body temperature to basking posture based on differences in the extent of dorsal melanization on the wing margins, both between species and between sexes within species of Pieris. This is the first demonstration in insects that coloration of the entire wing surface can affect thermoregulation. Model and experimental results suggest that, in certain wing regions, increased melanization can reduce body temperature in Pieris; this effect of melanization is exactly the opposite of that found in other Pierid butterflies that use their wings as solar absorbers. These results are discussed in terms of the evolution of wing melanization pattern and thermoregulatory behavior in butterflies.     

Sexual Size Dimorphism in the Color Pattern Elements of Two Mimetic Heliconius Butterflies

Sexual dichromatism and sexual dimorphism of body size are reasonably well studied in butterflies. Sexual size dimorphism of color pattern elements, however, is much less explored. The object of this study is Heliconius, a genus of butterflies well known for the coevolution between mate color preferences and mimicry. Given the sexual role of wing coloration, we investigated the existence of sexual size dimorphism in the wing color elements of a mimetic pair—Heliconius erato phyllis Fabricius and Heliconius besckei Ménétriés—and analyzed the allometric patterns of these traits. Correlation between size of elements in the dorsal and ventral wing surfaces were also estimated. In both species, three out of four elements were larger in males, but the non-dimorphic element was not the same. With regard to the allometric patterns, our most important finding was that smaller males of one species have proportionally larger yellow bars. This is the first study specifically concerning quantitative sexual dimorphism in the coloration of this well-known genus of butterflies and it opens new prospects to investigate sex-related natural selection and sexual selection of color pattern elements.     

Inter- and intra-sexual variation in immune defence in the cabbage white butterfly, Pieris rapae L. (Lepidoptera: Pieridae)

Abstract.  1. Immune defence imposes fitness costs as well as benefits, so organisms are expected to optimise, not maximise, their immune responses. This should result in variation in immune responses under varying environmental conditions.
2. Males and females are expected to exhibit different immune responses because life-history differences between the sexes affect optimal immune response. These life-history differences should usually result in a greater female, than male, immune defence. In this study, intra- and inter-sexual variation in one component of immune defence, the encapsulation response, in cabbage white butterflies ( Pieris rapae L.), was examined.
3. Encapsulation decreased with increasing age and in response to reduced diet quality.
4. Contrary to predictions, males generally had greater immune responses than females, although this pattern varied with age.
5. These patterns of inter- and intra-sexual variation in encapsulation may result from resource-based trade-offs with components of reproductive effort and/or because of sexual dimorphism in melanin-based wing patterns.     

Why Small Is Beautiful: Wing Colour Is Free from Thermoregulatory Constraint in the Small Lycaenid Butterfly,Polyommatus icarus

We examined the roles of wing melanisation, weight, and basking posture in thermoregulation in Polyommatus Icarus, a phenotypically variable and protandrous member of the diverse Polyommatinae (Lycaenidae). Under controlled experimental conditions, approximating to marginal environmental conditions for activity in the field (= infrequent flight, long duration basking periods), warming rates are maximised with fully open wings and maximum body temperatures are dependent on weight. Variation in wing melanisation within and between sexes has no effect on warming rates; males and females which differ in melanisation had similar warming rates. Posture also affected cooling rates, consistent with cooling being dependent on convective heat loss. We hypothesise that for this small sized butterfly, melanisation has little or no effect on thermoregulation. This may be a factor contributing to the diversity of wing colours in the Polyommatinae. Because of the importance of size for thermoregulation in this small butterfly, requirements for attaining a suitable size to confer thermal stability in adults may also be a factor influencing larval feeding rates, development time and patterns of voltinism. Our findings indicate that commonly accepted views of the importance of melanisation, posture and size to thermoregulation, developed using medium and large sized butterflies, are not necessarily applicable to small sized butterflies.     

Genotype‐environment interactions rule the response of a widespread butterfly to temperature variation

Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. This issue is of special interest in the current era of rapidly changing climatic conditions. Here, we investigate clinal variation and plastic responses in life history, morphology and physiology in the butterfly Pieris napi along a pan‐European gradient by exposing butterflies raised in captivity to different temperatures. We found clinal variation in body size, growth rates and concomitant development time, wing aspect ratio, wing melanization and heat tolerance. Individuals from warmer environments were more heat‐tolerant and had less melanised wings and a shorter development, but still they were larger than individuals from cooler environments. These findings suggest selection for rapid growth in the warmth and for wing melanization in the cold, and thus fine‐tuned genetic adaptation to local climates. Irrespective of the origin of butterflies, the effects of higher developmental temperature were largely as expected, speeding up development; reducing body size, potential metabolic activity and wing melanization; while increasing heat tolerance. At least in part, these patterns likely reflect adaptive phenotypic plasticity. In summary, our study revealed pronounced plastic and genetic responses, which may indicate high adaptive capacities in our study organism. Whether this may help such species, though, to deal with current climate change needs further investigation, as clinal patterns have typically evolved over long periods.     

Females show greater changes in wing colour with latitude than males in the green‐veined white butterfly,Pieris napi (Lepidoptera: Pieridae)

In butterflies, wing colour may simultaneously be under sexual selection in the context of mating selection and natural selection in the context of thermoregulation. In the present study, we collected mated females of the green‐veined white butterfly (Pieris napi) from locations spanning 960 km of latitude across Fennoscandia, and investigated sex‐specific latitudinal wing colour variation in their offspring raised under identical conditions. We measured wing colour characteristics, including reflectance at wavelengths 300–700 nm and the degree of wing melanization. At all latitudes, females reflected more light in the short wavelengths (< 400 nm) and less in the long wavelengths (> 450 nm), and they were more melanized than males. However, female wing colour varied more with latitude than that of males. Among females, long wavelength reflectance decreased, whereas short wavelength reflectance and melanization increased, towards the north. By contrast, among males, latitudinal variation was found only in the ventral hindwing melanization. These results are consistent with the idea that the balance between natural and sexual selection acting on wing colour changes with latitude differently in males than females. The dark wing colour of females in the north may be a thermoregulatory adaptation, although males may be constrained from evolving the dark dorsal wing colour favoured by natural selection because of constant sexual selection across latitudes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Expression of chemosensory proteins in hairs on wings of Locusta migratoria (Orthoptera: Acrididae)

The hairs on the wings of Locusta migratoria were observed and mapped using light microscopy, as well as by scanning and transmission electron microscopy. Based on their ultrastructure, we can distinguish four main types of hairs on the wings of adult L. migratoria , viz, short, medium and long hairs, and sensilla chaetica. The long hairs are located only on the ventral surface of the hindwing, whereas the other three types are present both on the dorsal and ventral surfaces of forewing and hindwing in both sexes. Medium hairs and sensilla chaetica are significantly more abundant on the dorsal surface of forewings in both females and males, than on the ventral surface, whereas the opposite was observed for short hairs (P < 0.01). No significant difference between males and females was observed in the density of any type of hairs (P > 0.1). Several dendritic branches, enveloped by a dendrite sheath, are situated in the lymph cavity of sensilla chaetica. Instead, no dendritic structure was observed in the cavity of the other three types of hairs. Immunocytochemical localization of chemosensory proteins (CSPs) was performed on ultrathin sections of hairs on wings. The antiserum against chemosensory proteins from L. migratoria (LmigCSP-II) strongly labelled sensilla chaetica, with gold granules only found in the outer sensillum lymph. In addition, the epidermal cell membrane of the wing was stained by the antiserum against LmigCSP-II. The other three types of hairs were never labelled. The results indicate that the wings might involve in contact chemoreception process.     

FEMALE CHOICE AND SEXUAL SELECTION OF MALE WING MELANIN PATTERN IN PIERIS OCCIDENTALIS (LEPIDOPTERA)

Using field experiments, I examined the role of 13 melanin-pattern elements in mate choice by female Pieris occidentalis butterflies. Males that mated successfully differed significantly from unsuccessful males in the extent of marginal forewing melanization but not on the basis of the entire forewing pattern. Deletion of the marginal forewing melanin characters significantly reduced the mating success of manipulated males relative to controls under field conditions. Female choice in P. occidentalis may act to maximize divergence of male color pattern from that of its close relative Pieris protodice. Sexual selection for increased melanization of the marginal dorsal forewing is generated by the strong preference of females for males with such wings and may be constrained by correlations between forewing melanin characters that have different functional roles.     

Wing colors of Chrysozephyrus butterflies (Lepidoptera; Lycaenidae): ultraviolet reflection by males

Wing colors of the four species of Chrysozephyrus butterflies were analyzed by a spectrophotometer. As the dorsal wing surface of males showed a strong reflectance when the specimen was tilted, measurements were made by the tilting method. The dorsal wing surface of males which appears green to the human eye reflected UV (315-350 nm) as well as green light (530-550 nm). The reflectance rate of UV to visible green light varied among species with a higher rate for C. hisamatsusanus and C. ataxus, and a lower rate for C. smaragdinus and C. brillantinus. The peak wavelength and the peak height did not shift when the specimen was exposed to direct sunlight at least for 16 hr. Artificial removal of scales by scratching the wing surface decreased reflectance. Blue marks on the forewings of C. brillantinus, C. hisamatsusanus and C. ataxus females reflected UV to visible light of short wavelength, and orange marks on the dorsal surface of the forewing and the ventral surface of the hindwing of C. samaragdinus females showed a higher reflectance at longer wavelengths.     

Thermal characterization of butterfly wings—1. Absorption in relation to different color, surface structure and basking type

1. 1. Spectral integral reflectance, transmittance and the resulting absorption of intact and descaled butterfly wings of the black-winged Pachliopta aristolochiae (Papilionidae), the white-winged Pieris brassicae (Pieridae), and the yellow-winged Gonepteryx rhamni (Pieridae) were determined between 350 and 800 nm.

2. 2. Whereas in the black forewing of the dorsal basking Pachliopta almost all incident light is absorbed nearly independent of the wavelength and thus converted into heat, the white forewing of the body basking Pieris absorbs less than 20% in the visible range of the spectrum.

3. 3. The yellow hindwing of the lateral basking Gonepteryx absorbs to a higher degree than the Pierid wing, but—due to the sparsely arranged scales—transmittance is clearly increased (40–50% between 525 and 800 nm).

4. 4. The varying thermal characteristics of the different wings with reference to the color and arrangement of the scales and the different basking strategies of the butterflies are discussed.

Assessing the Role of Wing Spots in Intraspecific Communication in the Cabbage White Butterfly (<Emphasis Type="Italic">Pieris rapae</Emphasis> L.) Using a Simple Device to Increase Butterfly Responses

Butterflies are regularly used as model systems for understanding the role of coloration in communication because of their highly variable and conspicuous phenotypes. Most research showing a role for color in communication has focused on aspects of brightness or hue of entire wings or large color patches. However, evidence is accumulating that butterflies sometimes use smaller wing pattern elements in communication. Here we provide evidence that both male and female cabbage white butterflies (Pieris rapae L.) discriminate among conspecifics on the basis of the number of small but conspicuous black wing spots of the dorsal forewing. Male butterflies were more interactive with model butterflies with two spots, which resemble female butterflies, than with model butterflies with only one spot, which resemble male butterflies. Female butterflies showed the opposite response, being more interactive with male-like (one-spot) models than with female-like (two-spot) models. Some of our experiments were conducted with an electronic device designed to create a realistic and controlled fluttering behavior of the models. We describe the design and function of this device and provide evidence that it increased butterfly responses compared to a non-fluttering model. This device could prove useful for others addressing questions of communication in butterflies or other flying insects.     

Sexual dimorphism and intraspecific variation in wing size and shape of Tongeia fischeri (Lepidoptera: Lycaenidae)

Wing morphological variations are described here for the lycaenid butterfly Tongeia fischeri. A landmark‐based geometric morphometric approach based on wing venation of 197 male and 187 female butterflies collected in Japan was used to quantify wing size and shape variations between sexes and among populations. Sexual dimorphism in wing size and shape was detected. Females had significantly larger wings than males, while males showed a relatively elongated forewing with a longer apex and narrower wing tornus in comparison to females. Intraspecific variations in wing morphology among populations were revealed for the wing shape, but not wing size. Distinct wing shape differences were found in the vein intersections area around the distal part of the discal cell where median veins originated in the forewing and around the origin of the CU1 vein in the hindwing. In addition, phenotypic relationships inferred from wing shape variations grouped T. fischeri populations into three groups, reflecting the subspecies classification of the species. The spatial variability and phenotypic relationships between conspecific populations of T. fischeri detected here are generally in agreement with the previous molecular study based on mitochondrial and nuclear sequences, suggesting the presence of a phylogenetic signal in the wing shape of T. fischeri, and thus having taxonomic implications.     

Quantitative genetics of butterfly wing color patterns

Developmental processes exert their influence on the evolution of complex morphologies through the genetic correlations they engender between traits. Butterfly wing color patterns provide a model system to examine this connection between development and evolution. In butterflies, the nymphalid groundplan is a framework used to decompose complex wing patterns into their component pattern elements. The first goal of this work has been to determine whether the components of the nymphalid groundplan are the products of independent developmental processes. To test this hypothesis, the genetic correlation matrices for two species of butterflies, Precis coenia and Precis evarete, were estimated for 27 wing pattern characters. The second purpose was to test the hypothesis that the differentiation of serial homologs lowers their genetic correlations. The “eyespots” found serially repeated across the fore- and hindwing and on the dorsal and ventral wing surfaces provided an opportunity to test this hypothesis. The genetic correlation matrices of both species were very similar. The pattern of genetic correlation measured between the different types of pattern elements and between the homologous repeats of a pattern element supported the first hypothesis of developmental independence among the elements of the groundplan. The correlation pattern among the differentiated serial homologs was similarly found to support the second hypothesis: pairs of eyespots that had differentiated had lower genetic correlations than pairs that were similar in morphology. The implications of this study are twofold: First, the apparent developmental independence among the distinct elements of wing pattern has facilitated the vast diversification in morphology found in butterflies. Second, the lower genetic correlations betweendifferentiated homologs demonstrates that developmental constraints can in fact be broken. The extent to which genetic correlations readily change, however, remains unknown. © 1994 Wiley-Liss, Inc.     

EVOLUTION AND COADAPTATION OF THERMOREGULATORY BEHAVIOR AND WING PIGMENTATION PATTERN IN PIERID BUTTERFLIES

This paper addresses the question of how the relationship between morphological structure and functional performance differs in related groups of organisms. I describe the relationship between a suite of phenotypic characters (behavioral posture and the pattern of wing pigmentation) and one function of these characters (thermoregulatory performance) for two groups of butterflies in the family Pieridae, focusing on how behavior and wing pattern interact to affect specific aspects of thermoregulation. Using both natural and experimentally created variation in wing-melanization patterns, I develop and test a series of predictions about the relations among thermoregulatory posture, melanization pattern, body temperature, and flight activity. Results show that increased melanization in different wing regions has positive, negative, or neutral effects in increasing body temperature of Pieris butterflies. The angle of the wings used during basking alters the relative importance of different modes of heat transfer and thereby determines the contribution of different dorsal wing regions to thermoregulation. Experimentally increased dorsal melanization can either increase or decrease the onset of flight activity and can directly alter thermoregulatory posture. For Pieris, dorsal melanization affects basking and flight, while ventral melanization primarily affects overheating. These results are used to generate a functional map relating melanization pattern to thermoregulatory performance in Pieris. Reflectance-basking posture, white background color, and melanization pattern represent coadapted characters in Pieris that interact to determine thermoregulatory performance. The differences in thermoregulatory posture and background color between pierid butterflies in the subfamilies Pierinae and Coliadinae have led to a reorganization and partial reversal of the thermoregulatory effects of melanization pattern. I suggest that this change in the physical mechanism of thermoregulatory adaption in pierids has qualitatively altered the nature of selection on wing-melanization pattern.     

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.