In nature, organisms display extreme variation for multiple aspects of pigmentation phenotype; this is commonly observed both within and among populations, as well as among taxa. Among populations across environmental gradients (at the macro-physiological level), pigmentation has been found to be associated with several fitness components such as mating ability, disease resistance, UV tolerance, thermal adaptation, and drought tolerance. From the molecular perspective, a number of genes and causal genetic changes underlying pigmentation phenotype have been identified in multiple taxa. The established connections between molecular variation, physiology, and fitness make this trait ideally suited for addressing fundamental questions at the crossroads of ecology, evolution and physiology.

Division of Biological and Life Sciences, School of Arts and Sciences, Ahmedabad University

Tokyo Metropolitan University

Frontiers in Physiology

Integrative Physiology

Melanism:macrophysiology to molecules

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Drosophila Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.

Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly Drosophila melanogaster also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, ebony and tan, which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in D. melanogaster females. More specifically, we report that ebony loss-of-function mutants have a CHC profile that is biased toward long (&gt;25C) chain CHCs, whereas tan loss-of-function mutants have a CHC profile that is biased toward short (&lt;25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in ebony mutants, making the CHC profiles similar to those seen in tan mutants. These observations suggest that genetic variation affecting ebony and/or tan activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Drosophila Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of ebony and tan expression in newly eclosed adults in a manner consistent with the ebony and tan mutant phenotypes. These data suggest that the pleiotropic effects of ebony and tan might contribute to covariation of pigmentation and CHC profiles in Drosophila.

Pleiotropic Effects of ebony and tan on Pigmentation and Cuticular Hydrocarbon Composition in Drosophila melanogaster

The effects of ultraviolet radiation (UV) on the animal body have been reported in many studies, and melanin has emerged as a protective mechanism. In smaller insects such as Drosophila, replicated patterns of geographical variation in pigmentation have been observed on multiple continents. Such patterns are particularly pronounced on the Indian subcontinent where several species show a parallel cline in pigmentation traits. However, the potential role of UV exposure in generating the observed patterns of pigmentation variation has not been addressed. Here, we examine the association between UV intensity and body pigmentation in D. melanogaster natural populations collected along the latitudinal gradient of the Indian subcontinent. A strong negative relationship was observed between UV intensity and body pigmentation. This analysis clearly indicates that, in the sampled populations, pigmentation variation is independent of UV exposure and related selection pressures. Patterns of pigmentation in natural populations from the Indian subcontinent are better predicted by latitude itself and temperature-related climatic variables.

The effects of ultraviolet radiation (UV) on the animal body have been reported in many studies, and melanin has emerged as a protective mechanism. In smaller insects such as Drosophila, replicated patterns of geographical variation in pigmentation have been observed on multiple continents. Such patterns are particularly pronounced on the Indian subcontinent where several species show a parallel cline in pigmentation traits. However, the potential role of UV exposure in generating the observed patterns of pigmentation variation has not been addressed. Here, we examine the association between UV intensity and body pigmentation in D. melanogaster natural populations collected along the latitudinal gradient of the Indian subcontinent. A strong negative relationship was observed between UV intensity and body pigmentation. This analysis clearly indicates that, in the sampled populations, pigmentation variation is independent of UV exposure and related selection pressures. Patterns of pigmentation in natural populations from the Indian subcontinent are better predicted by latitude itself and temperature-related climatic variables.

Latitudinal Pigmentation Variation Contradicts Ultraviolet Radiation Exposure: A Case Study in Tropical Indian Drosophila melanogaster

The molecular underpinnings of pigmentation diversity in Drosophila have recently emerged as a model for understanding how the evolution of different cis-regulatory variants results in common adaptive phenotypes within species. We compared sequence variation in a 5′ regulatory region harboring a modular enhancer containing a ∼0.7-kb core element contributing to abdominal melanisation in African, and a ∼0.5-kb core element contributing to thoracic pigmentation in D. melanogaster from Japan, to tropical and temperate populations from eastern Australia previously shown to be divergent in thoracic pigmentation and ebony expression. The Australian populations exhibited strong association with the core enhancer polymorphism cluster in complete association with Dark and Light phenotypes from Iriomote, Japan. Moreover, the Iriomote Light and Dark core enhancer haplotypes are common to the Australian populations in the direction predicted by pigmentation phenotype. We also confirmed the Japanese patterns of linkage disequilibrium and association of the tropical inversion In(3R)Payne with the Light enhancer haplotype in the Australian tropical light population. A worldwide survey of the ∼0.5-kb ebony control region SNPs and haplotypes in a subset of the Drosophila Genome Nexus (DGN) populations suggest origins in the sub-Saharan ancestral region surrounding Zambia and subsequent invasion following colonization out of Africa. A previous study demonstrated complex within and between population genetic architecture for abdominal pigmentation which is also correlated with thoracic pigmentation in melanized DGN sub-Saharan populations; however, the ∼0.5-kb ebony control region was not associated and both haplotypes are common even in the most intensely pigmented D. melanogaster from high altitude Ethiopia. In the Australian populations, the strong phenotypic association with the enhancer SNPs and haplotypes that at least partly regulates ebony expression in the Iriomote population, our previous work demonstrating opposing clines for thoracic pigmentation and ebony expression, where the expression cline parallels the In(3R)Payne cline, and the concerted evolution of pigmentation intensity and ebony expression under rapid experimental evolution, all point to a common adaptive evolutionary pathway in distinct populations.

The molecular underpinnings of pigmentation diversity in Drosophila have recently emerged as a model for understanding how the evolution of different cis-regulatory variants results in common adaptive phenotypes within species. We compared sequence variation in a 5′ regulatory region harboring a modular enhancer containing a ∼0.7-kb core element contributing to abdominal melanisation in African, and a ∼0.5-kb core element contributing to thoracic pigmentation in D. melanogaster from Japan, to tropical and temperate populations from eastern Australia previously shown to be divergent in thoracic pigmentation and ebony expression. The Australian populations exhibited strong association with the core enhancer polymorphism cluster in complete association with Dark and Light phenotypes from Iriomote, Japan. Moreover, the Iriomote Light and Dark core enhancer haplotypes are common to the Australian populations in the direction predicted by pigmentation phenotype. We also confirmed the Japanese patterns of linkage disequilibrium and association of the tropical inversion In(3R)Payne with the Light enhancer haplotype in the Australian tropical light population. A worldwide survey of the ∼0.5-kb ebony control region SNPs and haplotypes in a subset of the Drosophila Genome Nexus (DGN) populations suggest origins in the sub-Saharan ancestral region surrounding Zambia and subsequent invasion following colonization out of Africa. A previous study demonstrated complex within and between population genetic architecture for abdominal pigmentation which is also correlated with thoracic pigmentation in melanized DGN sub-Saharan populations; however, the ∼0.5-kb ebony control region was not associated and both haplotypes are common even in the most intensely pigmented D. melanogaster from high altitude Ethiopia. In the Australian populations, the strong phenotypic association with the enhancer SNPs and haplotypes that at least partly regulates ebony expression in the Iriomote population, our previous work demonstrating opposing clines for thoracic pigmentation and ebony expression, where the expression cline parallels the In(3R)Payne cline, and the concerted evolution of pigmentation intensity and ebony expression under rapid experimental evolution, all point to a common adaptive evolutionary pathway in distinct populations.

Enhancing Ebony? Common Associations With a cis-Regulatory Haplotype for Drosophila melanogaster Thoracic Pigmentation in a Japanese Population and Australian Populations

The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.

The main function of small heat shock proteins (sHSPs) as molecular chaperones is to protect proteins from denaturation under adverse conditions. Molecular and physiological data were used to examine the sHSPs underlying cold-hardiness in Harmonia axyridis. Complementary DNA sequences were obtained for six H. axyridis sHSPs based on its transcriptome, and the expression of the genes coding for these sHSPs was evaluated by quantitative real-time PCR (qRT-PCR) in several developmental stages, under short-term cooling or heating conditions, and in black and yellow females of experimental and overwintering populations under low-temperature storage. In addition, we measured water content and the super cooling and freezing points (SCP and FP, respectively) of H. axyridis individuals from experimental and overwintering populations. The average water content was not significantly different between adults of both populations, but the SCP and FP of the overwintering population were significantly lower than that of the experimental population. Overall, the six sHSPs genes showed different expression patterns among developmental stages. In the short-term cooling treatment, Hsp16.25 and Hsp21.00 expressions first increased and then decreased, while Hsp10.87 and Hsp21.56 expressions increased during the entire process. Under short-term heating, the expressions of Hsp21.00, Hsp21.62, Hsp10.87, and Hsp16.25 showed an increasing trend, whereas Hsp36.77 first decreased and then increased. Under low-temperature storage conditions, the expression of Hsp36.77 decreased, while the expressions of Hsp21.00 and Hsp21.62 were higher than that of the control group in the experimental population. The expression of Hsp36.77 first increased and then decreased, whereas Hsp21.56 expression was always higher than that of the control group in the overwintering population. Thus, differences in sHSPs gene expression were correlated with the H. axyridis forms, suggesting that the mechanism of cold resistance might differ among them. Although, Hsp36.77, Hsp16.25, Hsp21.00, and Hsp21.62 regulated cold- hardiness, the only significant differences between overwintering and experimental populations were found for Hsp16.25 and Hsp21.00.

Molecular Cloning and Induced Expression of Six Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis (Coleoptera: Coccinellidae)

Melanization reflects not only body color variation but also environmental plasticity. It is a strategy that helps insects adapt to environmental change. Different color morphs may have distinct life history traits, e.g., development time, growth rate, and body weight. The green slender planthopper Saccharosydne procerus (Matsumura) is the main pest of water bamboo (Zizania latifolia). This insect has two color morphs. The present study explored the influence of photoperiod and its interaction with temperature in nymph stage on adult melanism. Additionally, the longevity, fecundity, mating rate, and hatching rate of S. procerus were examined to determine whether the fitness of the insect was influenced by melanism under different temperature and photoperiod. The results showed that a greater number of melanic morphs occurred if the photoperiod was extremely long. A two-factor ANOVA showed that temperature and photoperiod both have a significant influence on melanism. The percentages of variation explained by these factors were 45.53 and 48.71%, respectively. Moreover, melanic morphs had greater advantages than non-melanic morphs under an environmental regime of high temperatures and a long photoperiod, whereas non-melanic morphs were better adapted to cold temperatures and a short photoperiod. These results cannot be explained by the thermal melanism hypothesis. Thus, it may be unavailable to seek to explain melanism in terms of only one hypothesis.

Melanization reflects not only body color variation but also environmental plasticity. It is a strategy that helps insects adapt to environmental change. Different color morphs may have distinct life history traits, e.g., development time, growth rate, and body weight. The green slender planthopper Saccharosydne procerus (Matsumura) is the main pest of water bamboo (Zizania latifolia). This insect has two color morphs. The present study explored the influence of photoperiod and its interaction with temperature in nymph stage on adult melanism. Additionally, the longevity, fecundity, mating rate, and hatching rate of S. procerus were examined to determine whether the fitness of the insect was influenced by melanism under different temperature and photoperiod. The results showed that a greater number of melanic morphs occurred if the photoperiod was extremely long. A two-factor ANOVA showed that temperature and photoperiod both have a significant influence on melanism. The percentages of variation explained by these factors were 45.53 and 48.71%, respectively. Moreover, melanic morphs had greater advantages than non-melanic morphs under an environmental regime of high temperatures and a long photoperiod, whereas non-melanic morphs were better adapted to cold temperatures and a short photoperiod. These results cannot be explained by the thermal melanism hypothesis. Thus, it may be unavailable to seek to explain melanism in terms of only one hypothesis.

The Environmental Plasticity of Diverse Body Color Caused by Extremely Long Photoperiods and High Temperature in Saccharosydne procerus (Homoptera: Delphacidae)