Intrinsic Pigment-Cell Stimulating Activity in the Catfish Integument

In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus. This species has a dark dorsal surface in marked contrast to an almost white midventral surface. Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev. Biol. 129:25). A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS). When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed. The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis. Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis. A similarly conditioned medium based upon Leibovitz's L-15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens. Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores. A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results. The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument. It is suggested that the light-colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF. Thus, the expression of melanophores is inhibited while that of iridophores is enhanced. In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.     

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.     

Melanization Stimulating Activity in the Skin of the Gilthead Porgy,Sparus auratus

The presence of a melanization-stimulating factor (MSF) was discovered in dorsal and/or ventral skin of Sparus auratus. Skin from this marine species was used to condition Steinberg's balanced salt solution (BSS), which was subsequently tested with the neural tube assay. BBS conditioned by dorsal and/or ventral skin of S. auratus at 25% and 50% concentrations had a profound stimulatory effect on the percentage of melanization of neural crest cells throughout the 3day assay period. In some cases 90% melanization occurred within the first 24 hr. Such stimulated cells showed a doubling of the number of dendrites per cell. assess the effects of MSF on other indices of melanization, dorsal and/or ventral skin was used to condition MEM used in the culture of B16-F10 murine melanoma cells. During the first 24 hr, B16-F10 murine melanoma cells responded to conditioned media by demonstrating a considerable increase in activities of tyrosine hydroxylase, dopa oxidase, and dopachrome tautomerase, but no effect was observed on melanin content. In contrast, melanin content increased after 48 hr of incubation, whereas the enzymatic activities were inhibited during this period. It seems that MSF activity, expressed in several ways, may be present generally among marine species.     

Melanization stimulating factors in the integument of the Mugil cephalus and Dicertranchus labrax

The pigment pattern expression resides in the chromatoblasts of the embryonic skin. The differentiation of these chromatoblasts is influenced by specific local factors such a melanization inhibiting factor (MIF) and a melanization-stimulating factor (MSF). We reveal the presence of these factors by means of a series of experiments on the skin of the marine species of fish Dicertranchus labrax and Mugil cephalus, each with different pigment pattern, the former having a light skin and the latter a darker one. Media conditioned by exposure to dorsal and/or ventral skin, stimulates the melanization of Xenopus laevis neural crest cells throughout a 3 day assay period. Similarly conditioned culture media tested on B16-F10 murine malignant melanocytes, revealed a considerable influence in enzymatic activities: dopachrome tautomerase (DCT), tyrosine hydroxylase and dopa oxidase. The use of media in a dose response basis suggests that the conditioned media may contain both melanophore stimulating and inhibiting factors. The results obtained may actually reflect the resultant activity of the two factors present.     

Control of melanoblast differentiation in amphibia by alpha-melanocyte stimulating hormone, a serum melanization factor, and a melanization inhibiting factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide:Dev. Biol., 129:25-36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of alpha-melanocyte stimulating hormone (alpha-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the "outgrowth" and "melanization" of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW less than 30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493-1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW greater than 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

Co-expression by keratinocytes of migration stimulating factor (MSF) and a functional inhibitor of its bioactivity (MSFI)

Migration stimulating factor (MSF) is a potent autocrine and paracrine factor expressed by fibroblasts and epithelial cells in foetal skin, tumours and healing wounds. In tissue culture, MSF bioactivity is present in the conditioned medium of foetal and tumour derived fibroblasts, but not in normal adult fibroblasts or keratinocytes. The conditioned medium of early passage keratinocytes or a keratinocyte line (HaCaT) effectively inhibited the motogenic activity of rhMSF. Fractionation of keratinocyte conditioned medium by size-exclusion chromatography revealed the presence of bioactive MSF as well as a functional inhibitor of MSF (MSFI) in fractions corresponding to approximately 70 kDa and 25 kDa, respectively. MSFI was purified and identified as neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2). Immunostaining confirmed that keratinocytes expressed both MSF and NGAL, whereas normal adult fibroblasts did not express either. Recombinant and cell-produced NGAL neutralised the motogenic activity of rhMSF. NGAL is known to bind MMP-9 and promote the activity of this protease. In contrast, there was no evidence of NGAL-MSF binding in keratinocyte conditioned medium. MSF displays a number of bioactivities of relevance to cancer progression and wound healing. Our findings indicate a novel function of NGAL and a possible mechanism for regulating MSF activity in tissues.     

THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO : Cellular and Subcellular Changes

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]⁺ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.     

Melanin-based plumage coloration and flight displays in plovers and allies

Plovers and their allies exhibit an impressive diversity of melanin-based plumage patterns ranging from non-melanized to completely melanized species. We use phylogenetic comparative methods to test whether melanization has evolved in relation to sexual selection for attracting mates, to selection for signalling territory defence, or to natural selection for camouflage. First, according to sexual-selection theory, melanized plumage has evolved to amplify the courtship displays of males. As predicted by this hypothesis, we found that males with aerial displays had more melanized plumage than males of ground-displaying species. In addition, sexual dimorphism in melanization was greater in species with display flights than in species with ground displays. Second, melanization may have evolved through social interactions to signal competitive ability in territory defence. We did not find evidence for this hypothesis, since breeding density was unrelated to the melanization of either sex. Finally, melanized plumage may camouflage the incubating parent. The latter hypothesis was not supported, since melanization was unrelated either to the darkness of nest substrate or the extent of vegetation cover. Taken together, our results are most consistent with the sexual-selection hypothesis, and suggest that melanized plumage has evolved to enhance the aerial displays of male plovers.     

An association of actin isoforms with the expression of motile response in pigmentation variants induced from goldfish erythrophoroma cells

Comparison of actin isoforms in unpigmented goldfish cells (a normal dermal fibroblast-like cell line, and an unpigmented erythrophoroma cell line capable of being induced to undergo melanization) and in normal and neoplastic melanized goldfish cells shows that the melanized phenotype is accompanied by the presence of multiple actin isoforms. In contrast, the unpigmented cells have only beta-actin. The possible significance of this to pigment organelle translocation is discussed.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Characterization of the intracellular melanization response in Anopheles quadrimaculatus against subperiodic Brugia malayi larvae.

Intracellular melanization, a defense or an immune response in the thoracic muscle cells, was investigated in a refractory strain of Anopheles quadrimaculatus infected with larvae of Brugia malayi. In mosquitoes fed on B. malayi-infected jirds, intracellular melanization against first-stage larvae (L1) was better expressed when fewer than 40 microfilariae reached the thoracic muscle cells than when more than 40 microfilariae reached the thoracic muscle cells. This result suggests that when large numbers of microfilariae invade the thoracic muscle cells, the immune response of the mosquito may become overloaded. Intracellular melanization response against L1 in the thoracic muscle cells also showed a significant decrease in older females (14-16-day-old) as compared to the younger ones (4-9-day-old). A comparison is made between intracellular and extracellular responses of mosquitoes to filarial larvae. It is significant that in both cases high rate of infection can reduce both the number and percentage of larvae melanized.     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.     

A ventrally localized inhibitor of melanization in Xenopus laevis skin

Melanophores normally differentiate in dorsal but not in ventral skin of Xenopus laevis. We have sought factors which might regulate this differentiation pattern, and we have obtained a putative melanization inhibiting factor (MIF) from ventral but not from dorsal skin. Preliminary studies reveal that MIF is destroyed by heat or trypsin treatment, indicating its protein composition, and has a molecular weight in the range of 300 kDa. The effects of MIF on the differentiation of neural crest derivatives to melanophores were examined in vitro in the presence of tyrosine and fetal calf serum (FCS). Tyrosine enhances melanophore differentiation in vitro at concentrations equivalent to those estimated in adult Xenopus blood plasma (20 microM). FCS also stimulates melanization, by way of materials other than the tyrosine contained in FCS. MIF strongly inhibits outgrowth and melanization of neural crest cells from neural tube explants. MIF also inhibits the differentiation of melanoblasts contained in cultured explants of ventral skin. Inhibition of melanization or melanophore differentiation by MIF occurs even in the presence of L-tyrosine and/or FCS. We suggest that MIF plays an important role in the establishment of dorso-ventral pigment patterns in amphibia.     

Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates

Sporothrix schenckii is known to produce DHN melanin on both conidial and yeast cells, however little information is available regarding the factors inducing fungal melanization. We evaluated whether culture conditions influenced melanization of 25 Brazilian S. schenckii strains and one control strain (ATCC 10212). Tested conditions included different media, pH, temperature, incubation time, glucose concentrations, and presence or absence of tricyclazole or L-DOPA. Melanization was reduced on Sabouraud compared to defined chemical medium. The majority of strains produced small amounts of melanin at 37 °C and none melanized at basic pH. Increased glucose concentrations did not inhibit melanization, rather increasing glucose enhanced pigment production in 27% of strains. Melanin synthesis was also enhanced by the addition of L-DOPA and its addition to medium with tricyclazole, an inhibitor of melanin synthesis, resulted in fungal melanization, including hyphal melanin production. Our results suggest that different S. schenckii strains have distinct control of melanization and that this fungus can use phenolic compounds to enhance melanization in vitro.     

Melanin in the dimorphic fungal pathogen Paracoccidioides brasiliensis: effects on phagocytosis, intracellular resistance and drug susceptibility

The fungal pathogen Paracoccidioides brasiliensis produces a melanin-like pigment in the presence of l-DOPA in vitro. We investigated whether melanization affected yeast uptake by alveolar and peritoneal macrophages, the intracellular resistance of fungal cells and their susceptibility to antifungal drugs. The interactions of melanized and nonmelanized P. brasiliensis with murine primary macrophages and J774.16 and MH-S macrophage-like cell lines were investigated. Melanized yeast cells were poorly phagocytosed by the cells even in the presence of complement. Melanization caused significant interference with the binding of cell wall components to lectin receptors on macrophages. Melanized cells were also more resistant than nonmelanized cells to the antifungal activity of murine macrophages. No difference in the susceptibilities of melanized and nonmelanized P. brasiliensis to antifungal drugs was observed using the minimum inhibitory concentration (MIC) method. However killing assays showed that melanization significantly reduced fungal susceptibility to amphotericin B and also protected against ketoconazole, fluconazole, itraconazole and sulfamethoxazole. The present results indicate that fungal melanin protects P. brasiliensis from phagocytosis and increases its resistance to antifungal drugs.     

A mutant affecting the crystal cells inDrosophila melanogaster

Summary Black cells (Bc, 2-80.6±) mutant larvae ofDrosophila melanogaster have pigmented cells in the hemolymph and lymph glands. In this report we present evidence that these melanized cells are a mutant form of the crystal cells, a type of larval hemocyte with characteristic paracrystalline inclusions.Bc larvae lack crystal cells. Furthermore, the distribution pattern of black cells inBc larvae parallels that of experimentally-blackened crystal cells in normal larvae (phenocopy).InBc/Bc zygotes black cells appear during mid embryonic development but inBc ⁺/Bc zygotes pigmented cells are not found until late in the first larval instar.Crystal cells are present in the heterozygous larvae until this time, and paracrystalline inclusions can be seen in some of the cells undergoing melanization in these larvae.The rate of phenol oxidase activity inBc ⁺/Bc larval cell-free extracts is less than half that ofBc ⁺/Bc ⁺extracts whereas enzyme activity is undetectable inBc/Bc larvae. We propose that theBc ⁺gene product is required for maintaining the integrity of the paracrystalline inclusions; inBc/Bc larvae either the product is absent or nonfunctional so an effective contact between substrate and enzyme results in melanization of the cells.Phenol oxidase itself is either destroyed or consumed in the melanization process accounting for the absence of enzyme activity inBc/Bc larvae. These studies confirm that the crystal cells store phenolic substrates and are the source of the hemolymph phenol oxidase activity in the larva ofD. melanogaster.     

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.     

Sexual Dichromatism of the Damselfly Calopteryx japonica Caused by a Melanin-Chitin Multilayer in the Male Wing Veins

Mature male Calopteryx japonica damselflies have dark-blue wings, due to darkly coloured wing membranes and blue reflecting veins. The membranes contain a high melanin concentration and the veins have a multilayer of melanin and chitin. Female and immature C. japonica damselflies have brown wings. We have determined the refractive index of melanin by comparing the differently pigmented wing membranes and applying Jamin-Lebedeff interference microscopy. Together with the previously measured refractive index of chitin the blue, structural colour of the male wing veins could be quantitatively explained by an optical multilayer model. The obtained melanin refractive index data will be useful in optical studies on melanized tissues, especially where melanin is concentrated in layers, thus causing iridescence.