Mechanisms of black and white stripe pattern formation in the cuticles of insect larvae

Molecular mechanisms that produce pigment patterns in the insect cuticle were studied. Larvae of the armyworm Pseudaletia separata have stripe patterns that run longitudinally along the body axis. The pattern in the cuticle became clear by being emphasized by the increasing contrast between the black and white colors of the lines after the last larval molt. We demonstrated that dopa decarboxylase (DDC) mRNA as well as protein are expressed specifically in the epidermal cells under the black stripes. The pigmentation on the stripes was clearly diminished by injection of a DDC inhibitor (m-hydroxybenzylhydrazine) to penultimate instar larvae for 1 day before molting, suggesting that DDC contributes to the production of melanin. Further, electron microscopic observation showed that the epidermal cells under the gap cuticle region (white stripe) between the black stripes contain many uric acid granules, which gives a white color. Our findings suggest that the spatially regulated expression of DDC in the epidermal cells produces the black stripes while abundant granules of uric acid in the cells generate the white stripes in the cuticle. Based on these results, we concluded that this heterogeneity in the epidermal cells forms cuticular stripe patterns in the armyworm larvae.     

Caterpillar color patterns are determined by a two‐phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.     

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.     

A homolog of the human Hermansky–Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ² , and oa ^v ), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

Mosaic formation by developmental loss of a chromosomal fragment in a “mottled striped” mosaic strain of the silkworm,Bombyx mori

A separable chromosomal fragment of about 2.5 Mb, which carries the larval body marking gene striped (p ^S), is present in a recessive background in the kind of genetic mosaic called mottled striped in the silkworm, Bombyx mori. The somatic loss of this chromosomal fragment during cell division gives rise to white patches (variegated pigmentation) in the dorsal black stripes of the fifth instar larva. Each larger white patch in the black p ^S stripe represents the clonal expansion of an epidermal cell that lost the fragment carrying p ^S during an early developmental stage. To gain information on the developmental history of the larval epidermal cells, we have analysed a variety of mosaic individuals showing extreme mottling patterns. Based on several common features observed in mosaic patterns, we constructed a schematic model for migration of epidermal cells, which implies that several polyclonal founding cells on each lateral side of a segment move and expand toward the dorsal mid-line. To determine the timing of loss of the fragment in half-stripe mosaics, which are completely lacking the mottled black stripe on one half of the larval body, we examined several tissues from either body side for the chromosomal fragment. Pulsed field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis showed that testes and silk glands from each side of the half-stripe mosaics (two and five individuals, respectively) contained the chromosomal fragment carrying the p ^S allele, independent of the epidermal phenotypes of the respective body half. This result suggests that loss of the chromosomal fragment leading to external half-stripe mosaics might occur, not at an early stage of development such as the first nuclear division, but rather after the progenies of epidermis and internal tissues examined here diverged from each other developmentally.     

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.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3(oe) mutant

The w-3(oe) silkworm mutant has white eyes and eggs due to the absence of ommochrome pigments in the eye pigment cells and serosa cells. The mutant is also characterized by translucent larval skin resulting from a deficiency in the transportation of uric acid, which acts as a white pigment in larval epidermal cells. A silkworm homolog of the fruitfly white gene, Bmwh3, a member of ATP-binding cassette transporter superfamily, was mapped on the w-3 locus. The w-3(oe) mutant has a single-base deletion in exon 2 and a premature stop codon at the 5' end of exon 3. These results show that w-3 is equivalent to Bmwh3 and is responsible for the transportation of ommochrome precursors and uric acid into pigment granules and urate granules, respectively.     

Pigment pattern formation in the quail mutant of the silkworm, Bombyx mori: parallel increase of pteridine biosynthesis and pigmentation of melanin and ommochromes

The larval pigment pattern in the silkworm, Bombyx mori, is formed by melanin, ommochromes and pteridines. During development all these pigments are synthesized autonomously, and possibly also with mutual interaction between them, to yield unique pigment patterns. In order to find the key trigger for such pigment pattern formation, developmental changes in pteridine biosynthesis were studied using the quail mutant (q/q), which has darker larval marks formed by melanin and an abundance of ommochromes in the integument. In the current study, emphasis has been placed on the analysis of GTP-cyclohydrolase I (GTP-CH I), which is a key enzyme for the biosynthesis of pteridines, during the development of the silkworm. Results of Northern blotting showed that in the quail mutant strong signals of GTP-CH I mRNA appeared around each period of ecdysis, while no such signals appeared in the background strain (+q/q) used. Also, both GTP-CH I activities and pteridine content were higher in the quail mutant compared with the background strain. These results strongly suggest that pteridine biosynthesis is closely linked to the formation of melanin and ommochromes. It is also suggested here that in the silkworm a recessive gene (q) may be involved in the regulation of its pigment pattern formation.     

Ultraviolet Radiation Induces Dose‐Dependent Pigment Dispersion in Crustacean Chromatophores

Pigment dispersion in chromatophores as a response to UV radiation was investigated in two species of crustaceans, the crab Chasmagnathus granulata and the shrimp Palaemonetes argentinus. Eyestalkless crabs and shrimps maintained on either a black or a white background were irradiated with different UV bands. In eyestalkless crabs the significant minimal effective dose inducing pigment dispersion was 0.42 J/cm² for UVA and 2.15 J/cm² for UVB. Maximal response was achieved with 10.0 J/cm² UVA and 8.6 J/cm² UVB. UVA was more effective than UVB in inducing pigment dispersion. Soon after UV exposure, melanophores once again reached the initial stage of pigment aggregation after 45 min. Aggregated erythrophores of shrimps adapted to a white background showed significant pigment dispersion with 2.5 J/cm² UVA and 0.29 J/cm² UVC. Dispersed erythrophores of shrimps adapted to a black background did not show any significant response to UVA, UVB or UVC radiation. UVB did not induce any significant pigment dispersion in shrimps adapted to either a white or a black background. As opposed to the tanning response, which only protects against future UV exposure, the pigment dispersion response could be an important agent protecting against the harmful effects of UV radiation exposure.     

Fine structural analysis of the thoracic longitudinal stripes of Zaprionus vittiger (Diptera)

The structure of the longitudinal zebra stripes on the thorax of adult Zaprionus vittiger has been investigated by light-, polarization-, transmission electron-, and scanning electron microscopy. Each stripe consists of a central white stripe of about 50 μm width and two lateral dark brown stripes about 30 μm wide. Three different types of trichomes occur: Very long bent trichomes of the grooved-type, long bent trichomes of the crested-type, and short straight trichomes. The central white stripe contains neither bristle organs nor short straight trichomes but carries many long bent trichomes most of which are of the grooved type, contain two cavities and polarize the light in the polarization microscope. The dark brown stripes carry bristle organs and many trichomes of the short and straight-type. Bent trichomes of the crested-type are found on the whole zebra stripe at about equal frequencies; they contain no cavities and do not polarize the light. The cuticle of the dark stripes is underlain by pigment cells. It is suggested that the pigment granules in the epidermal cells cause the dark color of the dark brown stripes, whereas the form and structure of the bent grooved type trichomes cause the white color of the central stripe.     

The scanning electron microscopic study of the infection and conidial development of Aspergillus tamarii Kita on its host, the silkworm, Bombyx mori Linn.

Development of Aspergillosis on the integument of the silkworm, Bombyx mori Linn., was examined by scanning electron microscopy. Aspergillosis is a fungal disease caused by an insect mycopathogen Aspergillus tamarii Kita, which infects the silkworms in countries where sericulture (the rearing of silkworms)is prevalent. The present study showed the course of infection and the conidial development of A. tamarii on the integument of B. mori. Five different strains (KA, NB₁₈, NB₄ D₂, NB₇ and PM) of B. mori were inoculated on their body surface with ca. 1 × 10⁶ conidia/ml. Among the five breeds tested, the conidial germination was greatest on the larval surface of KA breed, and least on PM. Most of the conidia germinated on the cuticle approximately 8–12 hours after inoculation, forming a suctorial appressorium within 24 hours. The hyphae reached the hemocoel, where they grew and multiplied extensively, forming a mycelial complex and causing death of the host larva in about 5–6 days. The death of the host was followed by growth of the fungus through mesodermal and epidermal tissues, leading to larval mummification about 6–7 days post-inoculation. Extensive aerial outgrowths of the fungus followed, mostly through the intersegmental regions of larvae. Abundant branched conidiophores developed, forming a confluent yellow brown mat over the entire host body 7 days after inoculation. Each conidiophore had an apical vesicle bearing numerous phialides from which conidia were developed in long chains.     

The female-killing chromosome of the silkworm, Bombyx mori, was generated by translocation between the Z and W chromosomes

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p ^Sa + ^p W + ^od )Fem, derived from the translocation-carrying W chromosome (p ^Sa + ^p W + ^od ), is inert as femaleness determinant. Moreover, this Df(p ^Sa + ^p W + ^od )Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p ^Sa + ^p W + ^od )Fem chromosome are produced. Initially, to investigate whether the Df(p ^Sa + ^p W + ^od )Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(p ^Sa + ^p W + ^od )Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(p ^Sa + ^p W + ^od )Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(p ^Sa + ^p W + ^od )Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(p ^Sa + ^p W + ^od )Fem chromosome is viable. Therefore, we concluded that the Df(p ^Sa + ^p W + ^od )Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(p ^Sa + ^p W + ^od )Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(p ^Sa + ^p W + ^od )Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, p ^Sa + ^p W + ^od .     

Observations on the development of unusual melanization of leopard frog ventral skin

The ontogeny of ventral pigmentation of two species of leopard frog, Rana pipiens and R. chiricahuensis, was examined by light microscopy and transmission electron microscopy to reveal how the unusual melanistic ventral pigmentation of R. chiricahuensis is achieved at the cellular level. Ventral skin of R. pipiens is always white. Ventral skin of adult R. chiricahuensis is white when frogs are background-adapted to a white substrate, but ventral skin becomes nearly as dark colored as the dorsal skin when frogs darken in response to a black background. Skin samples from tadpoles of both species, newly metamorphosed frogs, and adult frogs were analyzed for chromatophore composition and distribution. Ventral skin of R. pipiens larvae, newly metamorphosed frogs, and adults and of R. chiricahuensis larvae was white due to abundant iridophores and no melanophores. Melanophore density in the ventral integument of R. chiricahuensis was 9.1 ± 2.8/mm² in newly metamorphosed frogs and 87.0 ± 4.8/mm² in adult frogs. Pigment within ventral melanophores migrated during physiological color change during background adaptation. © 1993 Wiley-Liss, Inc.     

Histology of melanic flank and opercular color pattern elements in the firemouth cichlid,Thorichthys meeki

Dark melanic color pattern elements, such as bars, stripes, and spots, are common in the skin of fishes, and result from the differential distribution and activity of melanin‐containing chromatophores (melanophores). We determined the histological basis of two melanic color pattern elements in the integument of the Firemouth Cichlid, Thorichthys meeki. Vertical bars on the flanks were formed by three layers of dermal melanophores, whereas opercular spots were formed by four layers (two lateral and two medial) in the integument surrounding the opercular bones. Pretreatment of opercular tissue with potassium and sodium salts effectively concentrated or dispersed intracellular melanosomes. Regional differences in epidermal structure, scale distribution, and connective tissues were also identified. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.