Effects of altered catecholamine metabolism on pigmentation and physical properties of sclerotized regions in the silkworm melanism mutant

Catecholamine metabolism plays an important role in the determination of insect body color and cuticle sclerotization. To date, limited research has focused on these processes in silkworm. In the current study, we analyzed the interactions between catecholamines and melanin genes and their effects on the pigmentation patterns and physical properties of sclerotized regions in silkworm, using the melanic mutant melanism (mln) silkworm strain as a model. Injection of β-alanine into mln mutant silkworm induced a change in catecholamine metabolism and turned its body color yellow. Further investigation of the catecholamine content and expression levels of the corresponding melanin genes from different developmental stages of Dazao-mln (mutant) and Dazao (wild-type) silkworm revealed that at the larval and adult stages, the expression patterns of melanin genes precipitated dopamine accumulation corresponding to functional loss of Bm-iAANAT, a repressive effect of excess NBAD on ebony, and upregulation of tan in the Dazao-mln strain. During the early pupal stage, dopamine did not accumulate in Dazao-mln, since upregulation of ebony and black genes led to conversion of high amounts of dopamine into NBAD, resulting in deep yellow cuticles. Scanning electron microscope analysis of a cross-section of adult dorsal plates from both wild-type and mutant silkworm disclosed the formation of different layers in Dazao-mln owing to lack of NADA, compared to even and dense layers in Dazao. Analysis of the mechanical properties of the anterior wings revealed higher storage modulus and lower loss tangent in Dazao-mln, which was closely associated with the altered catecholamine metabolism in the mutant strain. Based on these findings, we conclude that catecholamine metabolism is crucial for the color pattern and physical properties of cuticles in silkworm. Our results should provide a significant contribution to Lepidoptera cuticle tanning research.     

Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405 nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing.     

Evidence of selection at melanin synthesis pathway loci during silkworm domestication

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) more than 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. In the present study, we examined within- and between-species nucleotide diversity for eight silkworm melanin synthesis pathway genes, which play a key role in cuticular pigmentation of insects. Our results showed that the genetic diversity of B. mori was significantly lower than that of B. mandarina and 40.7% of the genetic diversity of wild silkworm was lost in domesticated silkworm. We also examined whether position effect exists among melanin synthesis pathway genes in B. mandarina and B. mori. We found that the upstream genes have significantly lower levels of genetic diversity than the downstream genes, supporting a functional constraint hypothesis (FCH) of metabolic pathway, that is, upstream enzymes are under greater selective constraint than downstream enzymes because upstream enzymes participate in biosynthesis of a number of metabolites. We also investigated whether some of the melanin synthesis pathway genes experienced selection during domestication. Neutrality test, coalescent simulation, as well as network and phylogenetic analyses showed that tyrosine hydroxylase (TH) gene was a domestication locus. Sequence analysis further suggested that a putative expression enhancer (Abd-B-binding site) in the intron of TH gene might be disrupted during domestication. TH is the rate-limiting enzyme of melanin synthesis pathway in insects. Real-time polymerase chain reaction assay did show that the relative expression levels of TH gene in B. mori were significantly lower than that in B. mandarina at three different developmental stages, which is consistent with light body color of domesticated silkworm relative to wild silkworm. Therefore, we speculated that expression change of TH gene may contribute to the body color transition from B. mandarina to B. mori. Our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.     

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.     

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.     

Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation

Melanism, the overall darkening of the body, is a widespread form of animal adaptation to particular environments, and includes bookcase examples of evolution by natural selection, such as industrial melanism in the peppered moth. The major components of the melanin biosynthesis pathway have been characterized in model insects, but little is known about the genetic basis of life-stage specific melanism such as cases described in some lepidopteran species. Here, we investigate two melanic mutations of Bicyclus anynana butterflies, called Chocolate and melanine, that exclusively affect pigmentation of the larval and adult stages, respectively. Our analysis of Mendelian segregation patterns reveals that the larval and adult melanic phenotypes are due to alleles at different, independently segregating loci. Our linkage mapping analysis excludes the pigmentation candidate gene black as the melanine locus, and implicates a gene encoding a putative pyridoxal phosphate-dependant cysteine sulfinic acid decarboxylase as the Chocolate locus. We show variation in coding sequence and in expression levels for this candidate larval melanism locus. This is the first study that suggests a biological function for this gene in insects. Our findings open up exciting opportunities to study the role of this locus in the evolution of adaptive variation in pigmentation, and the uncoupling of regulation of pigment biosynthesis across developmental stages with different ecologies and pressures on body coloration.     

云南乌骨绵羊乌质性状与TYR基因多态性的相关分析

乌骨乌肉是乌骨绵羊的主要乌质性状。本文测定了乌骨绵羊、兰坪本地羊和罗姆尼羊血液TYR活性并分析了乌骨绵羊与乌骨鸡组织器官黑色素结构,结果表明：乌骨绵羊TYR活性显著高于兰坪本地绵羊和罗姆尼羊（P<0.05）;乌骨绵羊黑色素与乌骨鸡黑色素的IR光谱基本一致,黑色物质主要是真黑色素。首次克隆了绵羊TYR基因第一外显子长667 bp序列,并检测了乌骨绵羊和非乌骨绵羊TYR基因多态性。结果发现,检测的乌骨绵羊TYR基因第一外显子有两个变异位点,分别位于第64和154号编码氨基酸上,但都属于同义突变。通过对64号位置设计酶切位点检测TYR基因多态性,结果发现,该突变与乌质性状有关基因紧密连锁,TYR基因上可能存在乌质性状相关功能突变位点。另外,TYR基因多态性与毛色的表型相关极显著（P<0.01）,该基因可能也是毛色功能基因。     

昆虫着色及体表黑色斑纹和斑点形成机制研究进展

昆虫在生长发育的过程中,会不断受到捕食者的攻击,为逃避被捕食在长期的适应进化中展现出各种适应性的形态特征,体色和斑纹的适应性变化是其中重要的防御策略。昆虫多样的着色模式常用于释放警告信号或者模仿宿主植物,避免被其他动物捕食并且加速逃避学习,而且在寻求伴侣、适应地理、调节体温和抵抗紫外线等方面发挥重要的生物功能,是昆虫学研究的热点之一。鳞翅目昆虫具有分布广、种类多的特点,大量的斑点和斑纹模式常见于鳞翅目昆虫中,其生物学功能比其他动物更明显。近年来研究发现色素色和结构色是昆虫主要的着色模式,眼色素、黑色素以及喋啶类色素是影响昆虫着色最重要的色素;而昆虫的寄主、环境因素、激素显著影响昆虫多样性着色模式的形成。利用定位克隆、经典遗传连锁图谱、RNA干涉、基因组编辑、高通量测序等技术分离鉴定出了多个调控鳞翅目昆虫着色的关键基因。研究表明, TH, DDC, yellow, laccase2, ebony, AA-NAT, tan和GTPCHI是昆虫色素合成信号通路中的关键基因,而多效性基因spz3, apt-like和wnt1以及20E诱导的转录因子E75A和spalt通过影响鳞翅目昆虫黑色素合成信号通路的活性从而调控黑色素的合成与沉积。本文对昆虫体色和斑纹多样性的形成和影响因素,昆虫着色类型及物质基础,以及黑色斑点和斑纹形成和调节机制方面的研究进展作了整理和总结,以期为今后着色基因的利用以及害虫防治提供理论参考。     

对家蚕第18连锁群隐性基因elp、ch-2和mln测交系的分子定位分析

家蚕长形卵(elp)、第二隐性赤蚁(ch-2)、暗化型(mln)均为第18染色体上的隐性突变, 在经典连锁图谱上的顺序和遗传距离已经排定。文章采用正常卵、正常黑蚁及正常白蛾品种P50与包含此3个隐性突变的三隐性测交系W18组配正反交群体, F1回交W18后获得回交群体(P50×W18)♀×W18♂ 和W18♀×(P50×W18)♂, 分别记作BC1F和BC1M, 利用已构建的家蚕SSR分子连锁图谱和根据家蚕基因组精细图设计的STS标记, 对这3个突变基因elp、ch-2、mln进行了分子定位研究, 并根据家蚕基因组精细图, 将第18连锁群的经典遗传图、分子连锁图和基因组物理图进行了对应。整合后的图谱遗传距离为94.2 cM, 突变基因和分子标记的排列顺序分别与形态标记连锁图和基因组精细图相一致, 研究结果对家蚕第18 染色体上其他突变的定位与克隆有重要的借鉴作用。     

Multiple roles for laccase2 in butterfly wing pigmentation,scale development,and cuticle tanning

Lepidopteran wing scales play important roles in a number of functions including color patterning and thermoregulation. Despite the importance of wing scales, however, we still have a limited understanding of the genetic mechanisms that underlie scale patterning, development, and coloration. Here, we explore the function of the phenoloxidase‐encoding gene laccase2 in wing and scale development in the nymphalid butterfly Vanessa cardui. Somatic deletion mosaics of laccase2 generated by CRISPR/Cas9 genome editing presented several distinct mutant phenotypes. Consistent with the work in other nonlepidopteran insect groups, we observed reductions in melanin pigmentation and defects in cuticle formation. We were also surprised, however, to see distinct effects on scale development including complete loss of wing scales. This study highlights laccase2 as a gene that plays multiple roles in wing and scale development and provides new insight into the evolution of lepidopteran wing coloration.     

Characterization of the effect of Melanocortin 1 Receptor,a member of the hair color genetic locus,in alpaca (Lama pacos) fleece color differentiation

Little is known about the inheritance and influence of the fleece color gene Melanocortin 1 Receptor (MC1R). Melanocortin 1 Receptor (MC1R) is a well-known gene responsible for red versus black fleece pigmentation and is hypothesized to be a candidate gene for variation in alpaca coloration patterns. Inheritance of red versus black pigmentation in the context of genetic mutation is well understood in many domesticated mammals. We characterized the MC1R gene in a population of multi-colored alpacas in order to better understand its effect on coat color in the alpaca. Our characterization of the alpaca MC1R gene revealed 11 mutations. Of these one is a 4 bp deletion, four are silent mutations and six are single nucleotide polymorphisms (SNPs) that alter the amino acid sequence (T28V, M87V, S126G, T128I, S196F, R301C). No mutation correlated completely with fleece color in alpacas at the MC1R locus. This may be due to the epistatic relationship of MC1R with other coat color genes especially agouti signaling protein (ASIP).     

Genetic and environmental components of variation in eumelanin and phaeomelanin sex-traits in the barn owl

Knowledge of the mechanism underlying the expression of melanin-based sex-traits may help us to understand their signalling function. Potential sources of inter-individual variation are the total amount of melanins produced but also how biochemical precursors are allocated into the eumelanin and phaeomelanin pigments responsible for black and reddish-brown colours, respectively. In the barn owl (Tyto alba), a eumelanin trait (referred to as 'plumage spottiness') signals immunocompetence towards an artificially administrated antigen and parasite resistance in females, whereas a phaeomelanin trait ('plumage coloration') signals investment in reproduction in males. This raises the question whether plumage coloration and spottiness are expressed independent of each other. To investigate this question, we have studied the genetics of these two plumage traits. Crossfostering experiments showed that, for each trait, phenotypic variation has a strong genetic component, whereas no environmental component could be detected. Plumage coloration is autosomally inherited, as suggested by the similar paternal-to-maternal contribution to offspring coloration. In contrast, plumage spottiness may be sex-linked inherited (in birds, females are heterogametic). That proposition arises from the observation that sons resembled their mother more than their father and that daughters resembled only their father. Despite plumage coloration and spottiness signalling different qualities, these two traits are not inherited independent of each other, darker birds being spottier. This suggests that the extent to which coloration and spottiness are expressed depends on the total amount of melanin produced (with more melanin leading to a both darker and spottier plumage) rather than on differential allocation of melanin into plumage coloration and spottiness (in such a case, darker birds should have been less spotted). A gene controlling the production of melanin pigments may be located on sex-chromosomes, since the phenotypic correlation between coloration and spottiness was stronger in males than in females.     

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

Melanin‐Based Coloration in Juvenile Kestrels ( Falco tinnunculus) Covaries with Anti‐Predatory Personality Traits

Recent studies have shown that melanin‐based coloration is associated with the ability to cope with stressful environments, potentially explaining why coloration covaries with anti‐predator behaviours, boldness and docility. To investigate whether these relationships are consistent across species, we performed a study in the European kestrel ( Falco tinnunculus). Similar to our results found previously in the barn owl ( Tyto alba), nestling kestrels displaying a larger sub‐terminal black tail band stayed on their back longer (tonic immobility test) and breathed at a lower rate than individuals with a smaller black band when handled. However, in contrast to barn owls, nestling kestrels with a larger black tail band were more aggressive and more agitated. Our results strengthen the hypothesis that melanin coloration is related to stress response and in turn to the reaction to predators, a very important personality trait (i.e. boldness).     

Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.     

Color reversion of the albino medaka fish associated with spontaneous somatic excision of the Tol‐1 transposable element from the tyrosinase gene

The medaka fish albino mutant, i¹ is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol‐1. Recently, mosaic black‐pigmented i¹ medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol‐1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.