The Genetic,Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly,Bicyclus anynana

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.     

Melanism in a larval Lepidoptera: repeatability and heritability of a dynamic trait

Abstract.  1. Although it is well established that the deposition of melanin pigment in the cuticle of larval Lepidoptera is influenced by both environmental and genetic factors, few studies have examined intra-individual regional variation in the degree of melanism or the ontogenetic dynamics of this trait. Here, heritable and density-dependent effects on within-individual and stage-specific variation in melanism were examined in caterpillars of the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval).
2. Using quantitative spectrometric methods, it is shown that cuticular melanism changes dramatically within larval stadia, showing the highest and lowest levels of melanism early (first day) and late (final day) in each larval stadium respectively. However, solitary-reared caterpillars were significantly paler than those reared gregariously at all stages of development and maintained greater levels of variation in melanism. This variation in melanism was repeatable and exhibited a significant heritable component (narrow sense heritability based on offspring–parent regressions: h ² = 0.18–0.30).
3. The degree of melanism was correlated negatively with larval body weight in solitary caterpillars, but not gregarious ones. Melanism also varied spatially, with the lateral longitudinal band being consistently darker than the dorsal or dorso-lateral bands. Crowd-rearing increased melanism in all regions of larval cuticle, but the extent of crowding-induced melanism was more pronounced in the dorsal and dorso-lateral bands than in the lateral one.
4. These results indicate that although cuticular melanism is a highly dynamic trait, ontogenetic changes in relative cuticular melanism are both predictable and repeatable within individuals and genotypes. This has implications for our understanding of the evolution of melanism and for applying artificial selection on the basis of colour.     

Genetic and phenotypic relationships between immune defense,melanism and life-history traits at different temperatures and sexes in Tenebrio molitor

Insect cuticle melanism is linked to a number of life-history traits, and a positive relationship is hypothesized between melanism and the strength of immune defense. In this study, the phenotypic and genetic relationships between cuticular melanization, innate immune defense, individual development time and body size were studied in the mealworm beetle (Tenebrio molitor) using three different temperatures with a half-sib breeding design. Both innate immune defense and cuticle darkness were higher in females than males, and a positive correlation between the traits was found at the lowest temperature. The effect of temperature on all the measured traits was strong, with encapsulation ability and development time decreasing and cuticle darkness increasing with a rise in temperature, and body size showing a curved response. The analysis showed a highly integrated system sensitive to environmental change involving physiological, morphological and life-history traits.     

An Opposite Pattern to the Conventional Thermal Hypothesis: Temperature-Dependent Variation in Coloration of Adults of Saccharosydne procerus (Homoptera: Delphacidae)

Melanism is a common polymorphism in many insect species that also influences immune function. According to the thermal melanin hypothesis, ectothermic individuals from cooler environments have darker cuticles and higher polyphenol oxidase (PO) levels, which represent a better immunocompetence. In this study, the links among environmental temperature, melanism, and PO activity of Saccharosydne procerus (Matsumura) were examined. Most S. procerus have a black spot on their forewings at high temperatures in the field and in the laboratory. In PO activity assay, a positive association between PO level and temperature was found. Our results showed that a diversification of melanism occurred under different temperatures and that melanism in S. procerus presented an opposite pattern to the one proposed by the thermal hypothesis.     

Immune and Stress Responses Covary with Melanin-Based Coloration in the Barn Swallow

Eumelanin and pheomelanin are the main endogenous pigments in animals and melanin-based coloration has multiple functions. Melanization is associated with major life-history traits, including immune and stress response, possibly because of pleiotropic effects of genes that control melanogenesis. The net effects on pheo- versus eumelanization and other life-history traits may depend on the antagonistic effects of the genes that trigger the biosynthesis of either melanin form. Covariation between melanin-based pigmentation and fitness traits enforced by pleiotropic genes has major evolutionary implications particularly for socio-sexual communication. However, evidence from non-model organisms in the wild is limited to very few species. Here, we tested the hypothesis that melanin-based coloration of barn swallow (Hirundo rustica) throat and belly feathers covaries with acquired immunity and activation of the hypothalamic–pituitary–adrenal (HPA) axis, as gauged by corticosterone plasma levels. Individuals of both sexes with darker brownish belly feathers had weaker humoral immune response, while darker males had higher circulating corticosterone levels only when parental workload was experimentally reduced. Because color of belly feathers depends on both eu- and pheomelanin, and its darkness decreases with an increase in the concentration of eu- relative to pheomelanin, these results are consistent with our expectation that relatively more eu- than pheomelanized individuals have better immune response and smaller activation of the HPA-axis. Covariation of immune and stress response arose for belly but not throat feather color, suggesting that any function of color as a signal of individual quality or of alternative life-history strategies depends on plumage region.     

Intraspecific variation in wing and pupal melanization in copper butterflies (Lepidoptera: Lycaenidae)

Melanin is a widespread pigment causing variation in skin darkness, with darker phenotypes typically reaching higher equilibrium temperatures than lighter ones. Therefore, selection is expected to favour darker phenotypes in colder environments. In the present study, we show intraspecific variation in pupal (and wing) melanization along an altitudinal gradient in two species of copper butterflies. Both, pupal and wing melanization increased with increasing altitude. Consistent with the thermal melanism hypothesis, darker (high-altitude) pupae reached higher equilibrium temperatures than paler (low-altitude) ones. However, as temperature differences were rather small despite pronounced differences in melanization, we cannot rule out that factors (e.g. ultraviolet protection, disease resistance) other than temperature comprise the principal selective agents. Mechanistically, variation in melanization might be related to variation in hormone titres, as demonstrated by low-altitude pupae showing higher ecdysteroid and juvenile hormone titres compared to high-altitude ones. Furthermore, we report sex differences in wing melanization, with males being darker than females, which is potentially related to a higher flight activity of males.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 301–312.     

Greater cuticular melanism is not associated with greater immunogenic response in adults of the polymorphic mountain stone weta, Hemideina maori

Abstract.  1. Greater immune function is associated with the high-density melanic phase of polyphenic insects, appearing to compensate for density-dependent increases in susceptibility to parasites and/or pathogens. Other types of discrete variation in cuticular colour occur in insects (which may or may not be associated with melanin pigmentation), but whether this variation is predictive of immune ability has not been investigated.
2. In the mountain stone weta Hemideina maori , a black morph and yellow banded morph occur. These morphs are not seasonally polyphenic and have discrete haplotype genetic markers. Black individuals are typically found at lower local densities than yellow individuals, contrary to relations between cuticular melanism and density seen in polyphenic insects.
3. Yellow males and females had greater melanotic encapsulation responses upon immune challenge than did black males and females, but these differences were not associated with differences in temperature selection between morphs. Morph differences in melanotic encapsulation responses were somewhat related to differences between morphs in haemocyte concentrations.
4. These results indicate that a common form of immune expression is not heightened with dark coloration in the mountain stone weta. Thus, earlier findings of greater immunity associated with darker cuticles in phase polyphenic insects cannot be extended to insects with other forms of discrete colour variation. These findings will help in elucidating causes and consequences of such colour polymorphism, which is widespread in several insect orders.     

Wood ant colouration as an ecological indicator for the level of disturbance in managed coniferous forests

The search for ecological indicators of population well-being in natural and managed ecosystems is a crucial aspect of effective biomonitoring, conservation and nature protection. In long-term monitoring programs environmental stress has a measurable effect on naturally selected traits, such as body shape or size. However, changes in colouration provide information about early warning responses. The red wood ant Formica aquilonia is ecologically and territorially dominant among wood ant species in European boreal coniferous forest, and possesses variable red-brownish melanin-based cuticular colouration. F. aquilonia gynes, which are unfertilized queens, exhibit colour variability on the head, propodeum and abdomen, yet only head colour features allow setting clearly visible and symmetrical (left/right) classes of morphs. We studied phenotypic colour variability, melanisation and fluctuating asymmetry of colour patterns in faces of F. aquilonia gynes from natural (forest interiors) and disturbed (clear-cut zones and forest edges) habitats in Finland. We defined five variable, clearly visible and symmetrical (left/right) colour morphs of gynes' faces. Individuals of totally dark morph were present only in disturbed habitats. General analyses of melanisation degree showed, that gynes from disturbed habitats were significantly darker compared to those from forest interiors. Individuals from the same nest tend to have a similar degree of melanisation. The mean darkness of face was highest among individuals from forest-edge nests and the lowest from forest interior nests. In all habitat types the darkness of face increased with an increase in head width. Increase of cuticular melanisation in F. aquilonia gynes in disturbed habitats could be explained with the theory of thermal melanism and stress-induced immune defence. Although some amount of fluctuating asymmetry among left/right symmetrical colour variations on faces of F. aquilonia gynes was found, it was affected neither by habitat type nor by head width. Melanisation degree of red wood ants have a potency to be used as ecological indicator for the level of disturbance in managed coniferous forests and fluctuating asymmetry of colour variations in ants might be studied furtherly in cases of more severe environmental concerns.     

Melanin-based coloration is a nondirectionally selected sex-specific signal of offspring development in the alpine swift

Two mutually exclusive hypotheses have been put forward to explain the evolution and adaptive function of melanin-based color traits. According to sexual selection theory melanism is a directionally selected signal of individual quality, whereas theory on the maintenance of genetic polymorphism proposes that alternative melanin-based variants achieve equal fitness. Alpine swift (Apus melba) males and females have a conspicuous patch of white feathers on the breast with their rachis varying continuously from white to black, and hence the breast varies from white to striated. If this trait is a sexually selected signal of quality, its expression should be condition dependent and the degree of melanism directionally selected. If variation in melanism is a polymorphism, its expression should be genetically determined and fitness of melanin-based variants equal. We experimentally tested these predictions by exchanging eggs or hatchlings between randomly chosen nests and by estimating survival and reproduction in relation to melanism. We found that breast melanism is heritable and that the environment and body condition do not significantly influence its expression. Between 5 and 50 days of age nestlings were heavier and their wings longer when breast feathers of their biological father were blacker, and they also fledged at a younger age. This shows that aspects of offspring quality covary positively with the degree of melanism. However, this did not result in directional selection because nestling survival and recruitment in the local breeding population were not associated with father breast melanism. Furthermore, adult survival, age at first reproduction and probability of skipping reproduction did not covary with the degree of melanism. Genetic variation in breast melanism is therefore maintained either because nonmelanic males achieve fitness similar to melanic males via a different route than producing fast-growing offspring, or because the advantage of producing fast-growing offspring is not sufficiently pronounced to result in directional selection.     

Cuticular colour change after imaginal eclosion is time-constrained: blacker beetles darken faster

Abstract Adult mealworm beetles, Tenebrio molitor L. (Coleoptera: Tenebrionidae), show density dependent polyphenism in adult cuticular colour that is correlated with prophylactic immunity ( Barnes & Siva-Jothy, 2000 ). The degree of cuticular colour is important in understanding the biology of a number of insect-related phenomena (e.g. industrial melanism, thermoregulation). In this paper, the extent of variation in cuticular colour in a large outbred population is documented; the rate of darkening under three different rearing protocols including experimental selection for cuticular colour is examined; and the time taken for cuticular colouring to be completed is determined. The results show that cuticular colouring after eclosion is time-constrained in T. molitor , regardless of whether beetles are reared in large outbred cultures, inbred cultures, or cultures subjected to selection for darkness or lightness. Blacker beetles always darkened faster. The potential reasons for the constraint and the potential costs of rapid darkening are discussed.     

Role of cuticular lipids and water-soluble compounds in tick susceptibility to Metarhizium infection

The arthropod cuticle acts as a physiochemical barrier protecting the organism from pathogens' entry. Entomopathogenic fungi actively penetrate the cuticles of arthropod hosts and are therefore directly affected by cuticle composition. Previously we have observed that Metarhizium spp. developing on resistant ticks ultimately die without penetrating tick's cuticle, suggesting that the cuticles of resistant ticks have antifungal compounds. In the present study, lipids and water-soluble cuticular components were extracted from engorged female tick cuticles, of one susceptible and one resistant tick species to Metarhizium spp. While conidia exposed to lipids from the susceptible tick, Rhipicephalus annulatus, germinated and differentiated into appressorium, conidia exposed to lipids from the resistant tick, Hyalomma excavatum, were inhibited. Soluble cuticular component extracts from both susceptible and resistant ticks stimulated conidial germination but not appressorium differentiation. A comparative analysis of the fatty acid profile in lipid extract of each tick exhibited similar compositions, but the relative abundance of C16:0, C18:0, C18:1ω9C and C20:0 was 2–5 times higher in the extracts from resistant ticks. All of these fatty acids inhibited conidial germination in vitro at 1% and 0.1% w/v concentration, but C20:0 stimulated appressorium differentiation at low concentration. This is the first report demonstrating a possible link between the presence of antifungal compounds in a specific concentration in tick cuticle and tick resistance to infection.     

Multivariate heredity of melanin-based coloration,body mass and immunity

The genetic covariation among different traits may cause the appearance of correlated response to selection on multivariate phenotypes. Genes responsible for the expression of melanin-based color traits are also involved in other important physiological functions such as immunity and metabolism by pleiotropy, suggesting the possibility of multivariate evolution. However, little is known about the relationship between melanin coloration and these functions at the additive genetic level in wild vertebrates. From a multivariate perspective, we simultaneously explored inheritance and selection of melanin coloration, body mass and phytohemagglutinin (PHA)-mediated immune response by using long-term data over an 18-year period collected in a wild population of the common kestrel Falco tinnunculus. Pedigree-based quantitative genetic analyses showed negative genetic covariance between melanin-based coloration and body mass in male adults and positive genetic covariance between body mass and PHA-mediated immune response in fledglings as predicted by pleiotropic effects of melanocortin receptor activity. Multiple selection analyses showed an increased fitness in male adults with intermediate phenotypic values for melanin color and body mass. In male fledglings, there was evidence for a disruptive selection on rump gray color, but a stabilizing selection on PHA-mediated immune response. Our results provide an insight into the evolution of multivariate traits genetically related with melanin-based coloration. The differences in multivariate inheritance and selection between male and female kestrels might have resulted in sexual dimorphism in size and color. When pleiotropic effects are present, coloration can evolve through a complex pathway involving correlated response to selection on multivariate traits.     

Cuticular Pores and Transcuticular Canals in Diverse Fruit Varieties

Dewaxed thin-sectioned and dewaxed isolated mature fruit cuticlesrevealed the unequivocal presence in situ of visibly discrete,ubiquitous, cuticular pores or orifices concomitant with anticlinally-orientedtranscuticular canals in 51 varieties of fruit among 20 plantfamilies. More than 66 per cent of the fruit cuticles have poresand/or canals. No correlation exists between either fruit sizeor pore size and cuticle thickness. Dewaxed cuticles rangedfrom 1.25–22.5 µm in thickness. Canal lengths aredirectly related to cuticle thickness. Cuticular occlusionsof the epidermal cells were found in 76 per cent of the fruitsexamined. Evidence is provided by light microscopy photomicrographs. Fruit cuticles, cuticle morphology, cuticular pores, transcuticular canals     

Needle browning and death in the flagged crown of Abies mariesii in the timberline ecotone of the alpine region in central Japan

In the flagged crown, which is asymmetric growth formed by severe stresses during winter in alpine regions, needles of evergreen conifers often became brown and died in early spring, but did not in a cushion-shaped crown. Needle browning and death is thought to occur by increasing transpiration due to a thinner cuticle or mechanical damage to the cuticle by wind-born snow and ice particles. To confirm whether the needle browning and death in the flagged crown of Abies mariesii Mast., in the alpine region of Japan conform with this concept, we assessed mechanical damage of the needle cuticle in a timberline ecotone and evaluated the effect of cuticle thickness on cuticular resistance. Mechanical damage on needle cuticles of A. mariesii was not observed. In the cushion-shaped crown, epicuticular wax covered the cuticle and plugged stomatal antechambers. In the flagged crown, epicuticular wax was mostly absent. Cuticular resistance in the flagged crown was lower than that in the cushion-shaped crown. However, the cuticle in the flagged crown was thicker than that in the cushion-shaped crown. The needle browning and death in the flagged crown of A. mariesii occurred even though needle cuticles were not mechanically damaged. The thicker cuticle of the flagged crown may play a role in other stresses. To estimate desiccation stress in relation to the cuticle, we need to elucidate not only cuticular resistance and cuticle thickness, but also cuticle quality and structure.     

Anatomical and ultrastructural study of the pharyngeal bulb in Protodrilus (Polychaeta,Archiannelida) II. The stomodeal epithelium and its cuticle

The epidermal and stomodeal cuticles of Protodrilus are described then compared. The thin epidermal cuticle, the thickness of which is about the same over all the body, is characterized both by the absence of fibrils in its deepest part and by the extension of epidermal microvilli above the cuticle. The stomodeal cuticle, the thickness of which is as variable as that of the epithelium, presents two layers of fibrils comparable to the collagen fibrils described in the cuticle of other Annelida, as well as a relatively diversified supramicrovillous coating. The anterior cuticular thickening or grating plate, is characterized by the length of the epithelial microvilli, the thickness of the cuticular matrix and the superficial cuticular zone with supramicrovillous denticles supported by an axis of fibrous bundles. In the stomodeal cuticle, the fibrillar material seems to give to the cuticle a best resistance to deformation during the pharyngeal bulb contraction, while an especially elaborated supramicrovillous coating is found in regions most exposed to friction. These features contrast with the relative simplicity of the epidermal cuticle.     

Ultrastructure and mineral distribution in the tergal cuticle of the terrestrial isopod Titanethes albus. Adaptations to a karst cave biotope

Composition and spatial distribution of organic and inorganic materials within the cuticle of isopods vary between species. These variations are related to the behaviour and habitat of the animal. The troglobiotic isopod Titanethes albus lives in the complete darkness of caves in the Slovenian Karst. This habitat provides constant temperature and saturated humidity throughout the year and inconsistent food supply. These conditions should have lead to functional adaptations of arthropod cuticles. However, studies on structure and composition of cave arthropod cuticles are rare and lacking for terrestrial isopods. We therefore analysed the tergite cuticle of T. albus using transmission and field-emission electron microscopy, confocal μ-Raman spectroscopic imaging, quantitative X-ray diffractometry, thermogravimetric analysis and atomic absorption spectroscopy. The ultrastructure of the epicuticle suggests a poor resistance against water loss. A weak interconnection between the organic and mineral phase within the endo- and exocuticle, a comparatively thin apical calcite layer, and almost lack of magnesium within the calcite crystal lattice suggest that the mechanical strength of the cuticle is low in the cave isopod. This may possibly be of advantage in maintaining high cuticle flexibility and reducing metabolic expenditures.     

Apple Fruit Cuticles and the Occurrence of Pores and Transcuticular Canals

Developmental studies were made on the dewaxed thin-sectionedapple fruit cuticles of 10 Malus sylvestris Mill, cultivarsfrom 4 weeks before anthesis through fruit maturation and harvest.Cuticular development appears to correlate well with the generalgrowth of the fruit. However, no correlation exists betweenfruit size and cuticle thickness. Cuticular pores were evidentas early as 1 week before anthesis and transcuticular canalsbecame evident by 1 week following anthesis. Dewaxed thin-sectioned,as well as isolated, mature fruit cuticles of 16 cultivars andfour crab-apples consistently revealed the distinct presenceof ubiquitous pores and canals. Evidence is provided by lightmicroscopy and scanning electron microscopy (SEM). Measurementswere taken of cuticle thicknesses during development and ofcuticular pore dimensions, and calculations were made of poreand canal numbers. Fruit size alone is not directly indicativeof total pore numbers per surface area. Canal lengths are directlyrelated to the developmental thickness of the cuticle. No correlationwas found between the thickness of the mature cuticle and eitherthe number of pores present or the pore diameters. Malus sylvestris Mill, apple fruit, morphology, cuticle, cuticular flanges, cuticular pores, transcuticular canals, ultrastructure     

Immune function responds to selection for cuticular colour in Tenebrio molitor

Cuticular colour in the mealworm beetle (Tenebrio molitor) is a quantitative trait, varying from tan to black. Population level variation in cuticular colour has been linked to pathogen resistance in this species and in several other insects: darker individuals are more resistant to pathogens. Given that cuticular colour has a heritable component, we have taken an experimental evolution approach: we selected 10 lines for black and 10 lines for tan adult cuticular phenotypes over at least six generations and measured the correlated responses to selection in a range of immune effector systems. Our results show that two immune parameters related to resistance (haemocyte density and pre-immune challenge activity of phenoloxidase (PO)) were significantly higher in selection lines of black beetles compared to tan lines. This may help to explain increased resistance to pathogens in darker individuals. Cuticular colour is dependent upon melanin production, which requires the enzyme PO that is present in its inactive form inside haemocytes. Thus, the observed correlated response to selection upon cuticular colour and immune variables probably results from these traits' shared dependence on melanin production.     

Low proton conductance of plant cuticles and its relevance to the Acid-growth theory

Evidence obtained on the relation between the pH of the medium and the growth of intact stem sections is compatible with the acid-growth theory only if the proton conductance of the cuticle is so low that the cuticle is an effective barrier to the entry or exit of protons from the tissue. By measuring the rate at which protons cross frozen-thawed epidermal strips of sunflower (Helianthus annuus L.) and soybean hypocotyls (Glycine max Morr.) and enzymically isolated cuticles of Berberis aquifolium Persh. and tomato (Lycopersicum esculentum Mill.) fruit, we have now demonstrated the low proton conductance of the cuticular layer. Unless the conductance is enhanced by abrasion of the cuticle or by removal of the cuticular waxes, proton movement into and out of a tissue across the cuticle will be significant only over long time periods.