Levels Of Melanization Research Articles

Size-related changes and chemical basis of melanin-based body coloration in the amphisbaenian Trogonophis wiegmanni

Abstract Melanin is a fundamental pigment in animal coloration as it is involved in many different adaptive functions such as signaling or thermoregulation. Two forms of melanin are known to produce different colors (eumelanin: black; pheomelanin: yellow or brown). Here, we explored whether there were intersexual differences and/or size-related changes in melanization of the amphisbaenian Trogonophis wiegmanni, a fossorial species with a characteristic black and yellow color pattern of scales. We found a clear effect of body length, but not of sex, on the levels of melanization, which was also dependent on the body region; the proportion of melanin-dependent blackish coloration increased with body length in the dorsum, but decreased with body length in the ventral belly area. We also studied the chemical basis of this coloration using Raman spectroscopy and found that eumelanin was present in both black and yellow scales, but pheomelanin was not found in yellow scales, suggesting that this yellow coloration is caused by the reduction of eumelanin or dispersion of melanosomes and/or perhaps by other pigments.

Immune Response of Drosophila suzukii Larvae to Infection with the Nematobacterial Complex Steinernema carpocapsae-Xenorhabdus nematophila.

Entomopathogenic nematodes have been proposed as biological agents for the control of Drosophila suzukii, an invasive pest of small-stone and soft-skinned fruits. Larvae of the fly are susceptible to Steinernema carpocapsae infection but the reaction of immune defenses of the host are unknown. To determine the immune response, larvae were infected with S. carpocapsae and Xenorhabdus nematophila to evaluate the effector mechanisms of both humoral and cellular processes. The symbiont bacteria presented an inhibitory effect on the phenoloxidase cascade with a low level of melanization. Besides, X. nematophila activated the synthesis of putative antimicrobial peptides on the hemolymph of infected larvae. However, those peptides presented a lower antimicrobial activity compared to hemolymph from larvae infected with non-symbiont bacteria. Xenorhabdus nematophila avoided also the phagocytosis response of hemocytes. During in vitro and in vivo assays, S. carpocapsae was not encapsulated by cells, unless the cuticle was damaged with a lipase-treatment. Hemocyte counts confirmed differentiation of lamellocytes in the early phase of infection despite the unrecognition of the nematodes. Both X. nematophila and S. carpocapsae avoided the cellular defenses of D. suzukii larvae and depressed the humoral response. These results confirmed the potential of entomopathogenic nematodes to control D. suzukii.

Molecular and Potential Regulatory Mechanisms of Melanin Synthesis in Harmonia axyridis.

Melanization is a common phenomenon in insects, and melanin synthesis is a conserved physiological process that occurs in epidermal cells. Moreover, a comprehensive understanding of the mechanisms of melanin synthesis influencing insect pigmentation are well-suited for investigating phenotype variation. The Asian multi-colored (Harlequin) ladybird beetle, Harmonia axyridis, exhibits intraspecific polymorphism based on relative levels of melanization. However, the specific characteristics of melanin synthesis in H. axyridis remains elusive. In this study, we performed gene-silencing analysis of the pivotal inverting enzyme, tyrosine hydroxylase (TH), and DOPA decarboxylase (DDC) in the tyrosine metabolism pathway to investigate the molecular and regulatory mechanism of melanin synthesis in H. axyridis. Using RNAi of TH and DDC genes in fourth instar larvae, we demonstrated that dopamine melanin was the primary contributor to the overall body melanization of H. axyridis. Furthermore, our study provides the first conclusive evidence that dopamine serves as a melanin precursor for synthesis in the early pupal stage. According to transcription factor Pannier, which is essential for the formation of melanic color on the elytra in H. axyridis, we further demonstrated that suppression of HaPnr can significantly decrease expression levels of HaTH and HaDDC. These results in their entirety lead to the conclusion that transcription factor Pannier can regulate dopamine melanin synthesis in the dorsal elytral epidermis of H. axyridis.

Melanization, α-melanocyte stimulating hormone and steroid hormones in male western fence lizards from nine populations

Hormones can mediate suites of correlated traits. Melanocortins regulate melanin synthesis and elements of the melanocortin system can directly, and indirectly, affect a number of other traits, such as stress reactivity. Trait correlations within the melanocortin system have been studied mainly in birds and mammals but less so in reptiles. We examined adult male western fence lizards (Sceloporus occidentalis) and if melanization was correlated with plasma levels of three hormones, including peptide hormone α-melanocyte stimulating hormone (α-MSH), testosterone and corticosterone, and ectoparasite loads. This lizard is darker at higher elevations in California, and we compared five high-elevation and four low-elevation populations during comparable periods of the breeding season at each site. We first validated use of an α-MSH assay kit with lizard plasma. Since Anolis carolinensis is one of the few species with published values for α-MSH plasma levels, we assayed both Anolis and Sceloporus plasma and compared hormone values to those we generated for Anolis to the publish values. We also evaluated effects of different methods of storing spiked plasma pools on resulting α-MSH concentrations. Plasma levels of α-MSH did not differ significantly, but some populations differed significantly in mean corticosterone and mean testosterone. Combining all individuals from the nine populations, we found that individual variation in α-MSH was not associated with individual variation in melanization, but levels of α-MSH were positively associated with plasma testosterone and negatively associated with corticosterone. The lack of association between individual levels of melanization and expression of most other traits differs from a growing number of within-population studies of melanization, and we discuss what differences in physiological mechanisms could produce different hypothetical patterns. Circulating levels of -MSH are only one element of the melanocortin system; in situ synthesis of α-MSH by the skin and the diversity of melanocortin receptors could also contribute to variation in traits mediated by the melanocortin system and should be examined.

The physiology of melanin deposition in health and disease

Eumelanin is the major pigment responsible for human skin color. This black/brown pigment is localized in membrane-bound organelles (melanosomes) found in specialized cells (melanocytes) in the basal layer of the epidermis. This review highlights the steps involved in melanogenesis in the epidermis and the disorders in skin pigmentation that occur when specific steps critical for this process are defective. Melanosomes, which contain tyrosinase, a major enzyme involved in melanin synthesis, develop through a series of steps in the melanocyte. They are donated from the melanocyte dendrites to the surrounding keratinocytes in the epidermis. In the keratinocytes, the melanosomes are found singly or packaged into groups, and as the keratinocytes move upward in the epidermis, the melanosomes start to degrade. This sequence of events is critical for melanin pigmentation in the skin and can be influenced by genetic, hormonal, and environmental factors, which all play a role in levels of melanization of the epidermis. The effects these factors have on skin pigmentation can be due to different underlying mechanisms involved in the melanization process leading to either hypo- or hyperpigmentary disorders. These disorders highlight the importance of mechanistic studies on the specific steps involved in the melanization process.

Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq)

The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein–DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.

Regulated expression of cyclic AMP‐dependent protein kinase A reveals an influence on cell size and the secretion of virulence factors in Cryptococcus neoformans

Cyclic AMP-dependent protein kinase A (PKA) regulates elaboration of the virulence factors melanin and polysaccharide capsule in Cryptococcus neoformans. A mutation in PKA1 encoding the catalytic subunit is known to reduce virulence in mice while a defect in PKR1 encoding the regulatory subunit enhances disease. Here, we constructed strains with galactose-inducible and glucose-repressible versions of PKA1 and PKR1 by inserting the GAL7 promoter upstream of the genes. As expected, no capsule was found in dextrose-containing media for the P(GAL7):PKA1 strain, whereas a large capsule was formed on cells grown in galactose. Along with capsule thickness, high PKA activity also influenced cell size, ploidy and vacuole enlargement, as observed in previous reports of giant/titan cell formation. We employed the regulated strains to test the hypothesis that PKA influences secretion and found that elevated PKA expression positively regulates extracellular protease activity and negatively regulates urease secretion. Furthermore, proper PKA regulation and activity were required for wild-type levels of melanization and laccase activity, as well as correct localization of the enzyme. The latter phenotype is consistent with the discovery that PKA regulates the organization of intracellular membrane compartments. Overall, these results indicate that PKA influences secretion pathways directly related to virulence factor elaboration.

Infection with the wMel and wMelPop strains of Wolbachia leads to higher levels of melanization in the hemolymph of Drosophila melanogaster, Drosophila simulans and Aedes aegypti

Introduction of the life-shortening strain of Wolbachia pipientis, wMelPop, into the key dengue vector, Aedes aegypti, and the anti-pathogen effects in Wolbachia-infected hosts highlights the need for more research into its interactions with its original host, Drosophila melanogaster, and the novel mosquito host. The visual difference in darkness between the eggs of wMelPop Wolbachia-infected and uninfected mosquito hosts after egg deposition led to further investigation into melanization levels of the insects. Both D. melanogaster and A. aegypti infected with wMelPop showed increased levels of melanization, especially in females. This result was also seen in D. melanogaster and Drosophila simulans infected with the closely related wMel strain. D. simulans infected with other strains of Wolbachia did not display this difference. HPLC analysis of hemolymph from mosquitoes showed that this difference was not due to dopamine levels in the host as they were no different in wMelPop-infected and control mosquitoes before or after blood feeding.

The insect immune response and other putative defenses as effective predictors of parasitism

Parasitic wasps and flies (parasitoids) exert high mortality on caterpillars, and previous studies have demonstrated that most primary and secondary defenses do not protect caterpillars against parasitoids. We investigated the efficacy of tertiary defenses (i.e., immune responses) against parasitoids. Using a bead injection technique to measure the immune response and a 15-year database to measure parasitism, we compared the immune response for 16 species of caterpillars in nine different families. We found that caterpillar species with a strong immune response had the lowest incidence of parasitism, and when statistically compared to other defensive traits, the immune response was the best predictor of parasitism. Parasitoids either avoid attacking caterpillar species with a capacity for high levels of melanization or are killed once they have parasitized. In either case, the immune response is clearly one of the most effective defenses that caterpillars have against parasitism, and elucidating consistent predictors of variation in encapsulation could improve understanding of parasitism patterns in time and space and could enhance biological control efforts.

Different roles of natural and sexual selection on senescence of plumage colour in the barn swallow

Summary 1. Colour may show effects of senescence because the pigment or structures involved in production of colouration deteriorate with age. 2. We tested this hypothesis by investigating age-related changes in plumage colour for two feather tracts coloured by eumelanin or pheomelanin in a longitudinal study of a cohort of barn swallows Hirundo rustica that reached very old age (at least 5 years). 3. The level of melanization of the throat increased with age in both sexes, but particularly in females. In contrast, the black colour of the plumage of the back was unrelated to age in both sexes. 4. These age-dependent patterns of colouration of different feather tracts of male and female barn swallows suggest that effects of senescence are trait-specific depending on their importance in sexual signalling. The red throat colour based on pheomelanin is involved in sexual selection, with a strong effect in males, but not in females. In contrast, the black colour of the back based on eumelanin is unrelated to sexual selection, but is under natural selection due to intense abrasion of this feather tract. 5. These findings suggest that the relative importance of natural and sexual selection are important determinants of the pattern and rate of senescence of colour.

Seasonal Polymorphism in Elytral Coloration Pattern of Anomala flavipennis Burmeister (Coleoptera: Scarabaeidae) in Mexico

Elytral polychromatism in Anomala flavipennis Burmeister was detected in both spring and fall generations in northeastern Mexico from 2000 to 2002. Four distinctive coloration patterns were observed: (a) immaculated, (b) one-spotted, (c) two-spotted, and (d) striped. These categories were represented by 0, 1.4, 7.8, and 28.0% respective levels of melanization of elytral area. A significant interaction was detected between elytral pattern and generation, with melanized forms occurring more commonly during the spring and clearer forms during the fall. Weighted mean of elytral melanized area in the population ranged from 1.46% for fall to 7.80% for spring generations. Linear regression models suggested (R2 ≥ 0.93) an inverse association between temperature during pupation-adult ecdysis and elytral melanized area. The rationale and advantage for A. flavipennis responding to temperature by elytral melanization remain unknown, particularly because of the crepuscular-nocturnal habits of adults.

Parasitism of Lacanobia oleracea (Lepidoptera) by the ectoparasitoid, Eulophus pennicornis, is associated with a reduction in host haemolymph phenoloxidase activity

When haemolymph from fifth instar Lacanobia oleracea was incubated in vitro, rapid melanization occurred. Similar levels of melanization occurred in haemolymph from larvae that had been experimentally injected with venom from the ectoparasitic wasp, Eulophus pennicornis. In contrast, haemolymph from larvae parasitized by this wasp melanized more slowly and less extensively. Phenoloxidase assays indicated that enzyme activity was present in haemocyte lysate supernatants, serum and plasma from L. oleracea and that on day 5 post-parasitization, fractions prepared from parasitized larvae had significantly less phenoloxidase activity than similar fractions from untreated or experimentally envenomated larvae. In addition, no PO activity was detectable in wasp venom, and the venom had no effect on L. oleracea plasma phenoloxidase activity in vitro. These results indicate that parasitism of L. oleracea by E. pennicornis suppresses host haemolymph phenoloxidase activity and that this suppression is not induced by adult wasp venom. The results are discussed with reference to the survival advantages of suppressing the activity of this host enzyme, and to the possible source(s) of putative suppressive factors.

Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns

Background: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids.Results: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins.Conclusions: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.

Comparison of HPLC and Stereologic Image Analysis for the Quantitation of Eu- and Pheomelanins in Nevus Cells and Stimulated Melanoma Cells

The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.

Functional Analysis of Tyrosinase Isozymes of Cultured Malignant Melanoma Cells During the Recovery Period Following Interrupted Melanogenesis Induced by Glycosylation Inhibitors

Multiple forms of tyrosinase, T1, T2, T3, have been shown to differ with respect to carbohydrate moieties of these isozymes. We demonstrated that, in cultured B-16 melanoma cells, melanization can be completely interrupted by glycosylation inhibitors, such as glucosamine and tunicamycin, and that these inhibitors cause a selective loss of membrane-bound T3. It is further found that inhibition of melanization induced by glucosamine occurs even in the presence of protease inhibitors, such as phenylmethylsulfonyl fluoride and leupeptin, and that melanization inhibition is reversible upon removal of the inhibitor. In this report we have also examined the process of development and recovery of the tyrosinase isozymes in cells in which the interruption of melanogenesis has been released by the removal of these glycosylation inhibitors. The recovery process, which occurs during the period after interrupted melanogenesis and is a process of remelanization, has been biochemically followed. Tyrosinases obtained from the deoxycholate-solubilized large-granule fraction of these melanoma cells have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immediately after removal (0 h recovery) of the glycosylation inhibitor, loss of melanization and T3 is accompanied by T1 heterogeneity which is visualized as two electrophoretically distinct species, T1' and T1''. At this time, T1' and T1'' do not have a concanavalin A affinitive carbohydrate moiety but do possess in vitro dopa reactivity. When recovery of melanization begins visibly 24 h later, T3 is re-formed with disappearance of T1 heterogeneity. By 48 h, the previous normal level of melanization is almost attained. These results suggest that maturation of tyrosinase may occur via T1' and T1'' as precursors of T3, or possibly T1 through the addition of N-glycosydically linked oligosaccharide moieties which can be interrupted by glucosamine and tunicamycin.