Ultrastructure and migration of screening pigments in the retina of Pieris rapae L. (Lepidoptera,Pieridae)

Summary The retinal morphology of the butterfly, Pieris rapae L., was investigated using light and electron microscopy with special emphasis on the morphology and distribution of its screening pigments. Pigment migration in pigment and retinula cells was analysed after light-dark adaptation and after different selective chromatic adaptations. The primary pigment cells with white to yellow-green pigments symmetrically surround the cone process and the distal half of the crystalline cone, whilst the six secondary pigment cells, around each ommatidium, contain dark brown pigment granules. The nine retinula cells in one ommatidium can be categorised into four types. Receptor cells 1–4, which have microvilli in the distal half of the ommatidium only, contain numerous dark brown pigment granules. On the basis of the pigment content and morphology of their pigment granules, two distal groups of cells, cells 1, 2 and cells 3, 4 can be distinguished. The four diagonally arranged cells (5–8), with rhabdomeric structures and pigments in the proximal half of the cells, contain small red pigment granules of irregular shape. The ninth cell, which has only a small number of microvilli, lacks pigment. Chromatic adaptation experiments in which the location of retinula cell pigment granules was used as a criterium reveal two UV-receptors (cells 1 and 2), two green receptors (cells 3 and 4) and four cells (5–8) containing the red screening pigment, with a yellow-green sensitivity.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

FRACTIONATION OF THE EYE PIGMENTS OF DROSOPHILA MELANOGASTER

Eye pigments of normal and mutant types of D. melanogaster have been extracted with water and fractionated by chromatographic adsorption on powdered talc. Spectra of all the fractions obtainable in solution have been measured and the general chemical behavior of the pigments is described. Two chemically distinct groups of pigments are found, to be identified with the earlier designated red and brown components. The red component in the wild-type eye contains three well defined pigments, two of them capable of further subdivision so that the total number of fractions obtained is five. There is also present a brown component pigment which could not be treated quantitatively by the methods employed. All members of the wild-type red component are found in cinnabar eyes, unaccompanied by the brown component. Conversely, brown eyes contain a pigment indistinguishable from the wild-type brown component, virtually alone. In sepia eyes, one red component and two brown component pigments can be distinguished, all three pigments differing from those of wild-type eyes. Pigments apparently identical with those found in wild-type melanogaster eyes have also been found in D. virilis.     

Sub-cellular Localisation of the White/Scarlet ABC Transporter to Pigment Granule Membranes Within the Compound Eye of Drosophila Melanogaster

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lipofuscin in the corpus luteum of macaque ovaries

The present study examined the histochemistry of pigments in the corpus luteum of the ovaries of cynomolgus monkeys (Macaca fascicularis), Japanese monkeys (Macaca fuscata), and rhesus monkeys (Macaca mulatta). Yellowish brown pigments were found in the regressing corpus luteum cells. Histochemical studies revealed that these pigments consisted of lipofuscin, the so-called age pigment. The findings obtained suggest that accumulation of lipofuscin might be related to cellular aging of the corpus luteum.     

Biological and biochemical characterization of a red-eye mutant in Nilaparvata lugens （Hemiptera： Delphacidae）

A red-eye colony was established in our laboratory in brown planthopper （BPH）, Nilaparvata lugens （Stal）, a major rice pest in Asia. Except for the red-eye phenotype, no other differences were observed between the wild-type （brown eye） and the mutant-type （red eye） in external characters. Genetic analysis revealed that the red-eye phenotype was controlled by a single autosomal recessive allele. Biological studies found that egg produc- tion and egg viability in the red-eye mutant colony were not significantly different from those in the wild-type BPH. Biochemical analysis and electronic microscopy examination revealed that the red-eye mutants contained decreased levels of both xanthommatin （brown） and pteridine （red） and reduced number of pigment granules. Thus, the changes of amount and ratio of the two pigments is the biochemical basis of this red-eye mutation. Our results indicate that the red-eye mutant gene （red） might be involved in one common gene locus shared by the two pigments in pigment transportation, pigment granule formation or some other processes.     

Pigments associated with the flagellum ofEuglena gracilis

The pigments associated with the flagellum of the phytoflagellateEuglena gracllis were characterized by HPLC. The pigment pattern of the wild-type strain was compared with a set of white mutants which did not display phototaxis and photoaccumulation in response to blue light. Flagella of the wild type contained FMN and FAD. Two mutants which lacked the stigma but retained a small paraxonemal body (PAB) contained less flavins. The whiteEuglena mutant FB, which retained a residual stigma and also a PAB, and the white phytoflagellateAstasia longa, a close relative ofEuglena, had normal amounts of flagellar flavins. Cells and flagella ofEuglena wild type contained an unldentified pterin-like pigment, called Pt₁₆, which was substantially reduced inAstasia and theEuglena mutants. A third pigment, designated P₅₂₈ with major absorption at 528 nm and fluorescence emission at 550 nm was present mainly in flagella. The association of the three pigment types with flagella and their respective alterations in the white strains indicates their possible role in photoreception. Dedicated to Pill-Soon Song on the occasion of his 60th birthday.     

Melanin and novel melanin precursors from Aeromonas media

Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product.     

A solvent partitioning procedure for the separation of chlorophylls from their degradation products and carotenoid pigments

A simple liquid/liquid partitioning procedure was developed which employed aqueous acetonitrile and hexane, for the isolation of chlorophyll and pheophytin. This procedure separated these pigments from other interfering pigmented compounds. The efficacy of this solvent separation method was evaluated using commercially available chlorophylla, b, pheophytina, b, carotenoids, and algal pigment extracts. The recovery efficiencies of this solvent partitioning process for chlorophyll a and pheophytina have been shown to be 95–98% and 93–96%, respectively, furthermore, the chlorophylla fraction was practically free of any contaminating pigments. It appears that a more accurate assessment of chlorophylla and pheophytina can be accomplished employing liquid/liquid partitioning than with the present standard method.     

Leucine interference in the production of water-soluble red Monascus pigments

The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.Abbreviations mSG Monosodium glutamate - MOPS 3-(N-morpholine)propane sulfonic acid - DCW dry cell weight     

Pigmentation and autofluorescence of Cryptococcus species after growth on tryptophan and anthranilic acid media

Cryptococcus neoformans and Candida albicans produced a pink pigment from media containing tryptophan. Approximately 30% of the C. neoformans strains produced large amounts of the pink (purple after 6 days) pigment in the absence of light whereas 70% of the Cryptococcus neoformans strains, as well as C. laurentii, C. albidus, C. diffluens, and C. albicans also produced the pink pigment with light being required for significant early production (2–6 days). Significant production did occur for Cryptococcus but not Candida species in the dark after extended incubation (10–25 days). C. terreus produced brown pigments from tryptophan and C. luteolus produced a trace of a buff pigment. Most Candida species produced either pink or brown pigments but not both. In contrast, many Cryptococcus species producing the pink pigment simultaneously produced brown pigments. C. terreus, C. albidus, and C. diffluens produced brown pigments from anthranilic acid whereas C. neoformans, C. laurentii, C. luteolus, and the medically important Candida species did not produce significant amounts of pigments from anthranilic acid. Cryptococcus and Candida species were autofluorescent when tryptophan was a major nitrogen source whereas yeast cell autofluorescence was not observed when anthranilic acid was used. Pigmentation of some Cryptococcus species also the substrate.Operated for the U.S. Department of Energy under contract number EY-76-C-05-0033This article is based on work supported by the Division of Biomedical and Environmental Research.     

The dark brown integumentary pigment of a barnacle (Balanus eburneus)

Summary A histochemical analysis involving tinctorial and solubility tests was pursued in conjunction with electron microscopy for the purpose of identifying the dark brown epidermal pigment of a barnacle (Balanus eburneus) as melanin and/or ommochrome. Histochemically, comparisons were made with other brown pigments located in the subcarapal epidermis of another crustacean, the fiddler crab (Uca pugilator), the dorsal skin of the red-backed salamander (Plethodon cinereus), the liver and testis of a slider turtle (Chrysemys sp.) and the substantia nigra of human brain. The solubility properties of the pigment of the two crustacean invertebrates were in general similar to one another and markedly different from the pigment of the three vertebrates. Insolubility in appropriate solvents classified the vertebrate pigment as melanin. The invertebrate pigment, however, which was soluble in the ommochrome solvents, concentrated sulfuric and formic acids and 2-chloroethanol, remained insoluble in the ommochrome solvents, dilute aqueous and methanolic hydrochloric acid and dilute sulfuric acid. On the basis of these solubilities, an unequivocal classification of the invertebrate pigment as either melanin or ommochrome did not appear possible. The tinctorial and electron microscopic properties of the barnacle pigment were also equally ambiguous in regard to its specific classification.     

Production by Bacillis subtilis of brown sporulation-associated pigments

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.     

The pineal gland of the horse. Morphological and histochemical results. (With notes on the donkey and mule pineal)

The horse pineal gland has been investigated by morphological and histochemical methods. Particular care has been given to the cellular types, to the eventual presence of neurosecretory activity and to the nature of the pigments. Even in the horse pineal, it is possible to distinguish two populations of pinealocytes, morphologically but not histochemically distinct. A great number of pinealocytes are positive for the Masson- Hamperl reaction, and for Gomori- Bargmann 's chromic haematoxylin-phloxine and Gomori's paraldehyde-fuchsin. Along the connective septa, many brown- blackish pigmented cells were present; their pigment was positive for the Lillie and the Masson-Fontana reactions for the determination of melanin pigment. Another type of pigmented cells, carrying a brown yellowish pigment of lipofuscin nature was present, particularly in older animals, along the connective septa.     

Pigmentation and sporulation in selected Myxomycetes

Chemical, chromatographic and spectrometric methods are used to characterize plasmodial pigments and determine relationships between pigmentation and sporulation in selected Myxomycetes. In Physarum gyrosum (white) a single pigment is identified and characterized as a flavone. Physarum polycephalum (yellow) and Didymium iridis (brown) contain four and six components, respectively, in their plasmodial pigments which test negatively for flavones but show the presence of some type of phenolic compound. No detectable component is identified in the white plasmodium of Didymium squamulosum which proved to be independent of light for fruiting. The absorption spectra of all species that were light sensitive for fruiting showed common peaks in the 300–400-mμ region, among others. Pigment changes associated with light absorption are reported for some white, yellow and brown plasmodial types. In Physarum gyrosum a yellow pigment forms in light which did not show the characteristic flavones present in the white plasmodial stage. Changes in absorption spectra are reported for Physarum polycephalum, Didymium iridis and Didymium squamulosum as the plasmodial pigments change prior to fruiting. Results show a close relationship between the physiology of plasmodial pigmentation and sporulation in the Myxomycete species studied.     

Tissue specific effects of ommochrome pathway mutations in Drosophila melanogaster

The tissue-specific effects of 17 mutations affecting the synthesis of brown eye pigment (xanthommatin) have been investigated by combining them with chocolate and red cells, two mutations causing ectopic pigmentation of the Malpighian tubules and larval fat body (which normally only synthesize pigment precursors). The majority of mutations block the pigmentation of four organs; the normally pigmented eyes and ocelli, and ectopically pigmented tubules and fat body. They represent genes that would appear to be required for the normal operation of the pathway per se and are likely to encode structural proteins. Mutations at 5 loci affect pigmentation of a subset of organs: cd and po affect only the eyes and ocelli; kar affects the eyes, ocelli and fat body; car causes excretion of pigment from tubules; and z affects pigmentation of the eyes alone. Of these loci, only z has been shown to encode a regulatory protein and the role of the remaining four gene products is not clear. Two mutations affecting the red eye pigments (drosopterins), bw and mal, do not substantially perturb brown pigment synthesis in any of the four organs.     

Seasonal variation in cone sensitivity and short-wave absorbing visual pigments in the rudd Scardinius erythrophthalmus

Summary Microspectrophotometric and electroretinographic investigation of photoreceptor spectral sensitivity in the rudd Scardinius erythrophthalmus has revealed four spectral classes of cone with peak sensitivity in the ultra-violet, violet, green and red regions of the spectrum. These peak sensitivities were found to vary seasonally, and in response to artificial illumination, as a result of a change in the ratio of A1 to A2-based visual pigment in the cells. Short daylengths favoured the A2 pigment whereas long daylengths favoured the A1 analogue. Both the ultra-violet and violet-sensitive pigments have not previously been reported for the rudd. Evidence is presented supporting the hypothesis that the ultra-violet class of cells is not present in older fish.Abbreviation rvi response versus intensity (curve)     

Pigment biogenesis in freshwater shrimp ventral nerve chord chromatophores

Summary The possible biogenesis of two pigment granule types present in the monochromatic, brown chromatosomes enveloping the ventral nerve chord of the freshwater palaemonid shrimps Macrobrachium acanthurus, M. heterochirus and M. olfersii is examined by transmission electron microscopy in thin section and freeze fracture replicas. Prominent, membrane limited granules are suggested to have their origin in a complex, juxtanuclear, smooth endoplasmic reticulum labyrinth, continuous with the nuclear envelope. Amembranous, lipocarotenoid granules possibly derive from the external surface of the smooth endoplasmic reticulum. Nuclear envelope and SER membranes contain numerous 11 nm diameter intramembranous particles while pigment granule membranes exhibit fewer particles. A dictyosomal origin for the lipocarotenoid granules is discounted. Granulogenesis is suggested to be a continuous process in crustacean chromatophores.     

Growth responses and photosynthetic characteristics of wild and phycoerythrin-deficient strains of <Emphasis Type="Italic">Hypnea musciformis</Emphasis> (Rhodophyta)

The phycoerythrin-deficient strain (green phenotype) of Hypnea musciformis (Rhodophyta) originated from a green branch, which had arisen as a spontaneous mutation in a wild plant (brown phenotype) collected from the Brazilian coast. The present study describes the growth responses to irradiance, photoperiod and temperature variations, pigment contents, and photosynthetic characteristics of the brown and green strains of H. musciformis. The results showed that growth rates increased as a function of irradiance (up to 40 μmol photons m⁻² s⁻¹) but, with further increase in irradiance (from 40 to 120 μmol photons m⁻² s⁻¹), became light-saturated and remained almost unchanged. The highest growth rates of the brown and green strains were observed in temperatures of 20–25°C under long (14:10 h LD) and short (10:14 h LD) photoperiods. The brown strain had higher growth rates than the green strain in the short photoperiod, which could be related to the high concentrations of phycobiliproteins. Phycoerythrin was not detected in the green strain. The brown strain had higher concentrations of allophycocyanin and phycoerythrin in the short photoperiod while the green strain had higher concentrations of phycocyanin. The brown strain presented higher photosynthetic efficiency (α), and lower saturation parameter (I_k) and compensation irradiance (I_c) than the green strain. The brown strain exhibited the characteristics of shade-adapted plants, and its higher value of photosynthetic efficiency could be attributed to the higher phycoerythrin concentrations. Results of the present study indicate that both colour strains of H. musciformis could be selected for aquaculture, since growth rates were similar (although in different optimal light conditions), as the green strain seems to be adapted to higher light levels than the brown strain. Furthermore, these colour strains could be a useful experimental system to understand the regulation of biochemical processes of photosynthesis and metabolism of light-harvesting pigments in red algae.     

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.