Melanophores of Zacco temmincki (Teleostei) are light sensitive

The responses of melanophores of a cyprinid fish Zacco temmincki to changes in illumination were examined in isolated scale preparations of the adult fishes. Melanosomes in the melanophores aggregated in darkness and dispersed in light. These responses were invariably induced, even in denervated melanophores. These light responses, the dark-induced aggregation and the light-induced dispersion, were not affected by a number of alpha and beta adrenergic blocking agents. It was concluded that the melanophores of Zacco temmincki were themselves light sensitive and responded directly to light by melanosome translocations. The light responses were quantitatively assessed in relation to the intensity of illumination.     

Spectral sensitivity of melanophores of a freshwater teleost, Zacco temmincki

1. The melanophores of a freshwater teleost, Zacco temmincki, responded to changes in illumination: in darkness the melanophores induced a melanosome aggregation and when subjected to light they caused a melanosome dispersion. 2. Using monochromatic light, the spectral sensitivity of the melanophores was examined. 3. The melanophores showed a different sensitivity to light between 400 and 600 nm with a maximum at about 525 nm. 4. The action spectrum closely resembled a porphyropsin absorbance curve, suggesting a porphyropsin or similar photopigment is active in the melanophore light response of Zacco temmincki.     

Local light stimulation of melanophores of a teleost, Zacco temmincki

Responses of melanophores of the teleost, Zacco temmincki, to local light stimulation were examined in preparations of isolated scales. The melanophores induced the aggregation of melanosomes in darkness and their dispersion in light. Local illumination of a melanophore in the melanosome-dispersed state inhibited centripetal migration of melanosomes only in the stimulated area. Local illumination of a pigment-free branch of a melanophore with aggregated melanosomes generally brought about pigment dispersion into the stimulated area. However, when that area was at a significant distance from the edge of the central melanosome mass, the melanosomes never migrated into the irradiated area. Local illumination of the centrosphere of a cell inhibited the full aggregation of melanosomes in the dispersed and aggregated state. The degree of the inhibition depended on the size of the irradiated area. The results suggest that photoreceptive sites are distributed over the whole of a cell, and that the movements of melanosomes are regulated locally in a very precise manner.     

Basic properties of beta adrenergic receptors mediating melanosome dispersion of melanophores in a cyprinid fish, Zacco temmincki

In melanophores of a cyprinid fish, Zacco temmincki, receptor mechanisms of melanosome dispersion induced by catecholamines were examined. While possessing a melanosome-aggregating action in higher concentrations, isoproterenol and epinephrine in lower concentrations acted to disperse melanosomes. Norepinephrine, epinine and dopamine altered their action from melanosome aggregation to melanosome dispersion after alpha adrenergic blockade. The catecholamine-induced melanosome dispersion was inhibited by beta adrenergic blocking agents. Mediation of dispersion is regulated through beta adrenergic receptors. The beta adrenergic responses were unaffected by mersalyl, a sulfhydryl inhibitor. A prospective substance acting in dispersing melanosomes appears to be adrenaline, but not noradrenaline.     

Action of melanophore-stimulating hormone on melanophores of the cyprinid fish Zacco temmincki

1. Alpha MSH was extremely effective in inducing melanosome dispersion in both innervated and denervated melanophores in isolated scales of Zacco termmincki. 2. The sensitivity of the melanophores to MSH did not change after denervation. 3. The MSH action was blocked by mersalyl, a SH inhibitor, but not by any of alpha and beta adrenergic blockers. 4. Ca2+ was required for the MSH action, but not for melanosome dispersion itself, since the beta adrenergic response was normally produced in the absence of this ion. 5. Mg2+, Sr2+ and Ba2+ could not replace the Ca2+. 6. Mn2+ reversibly inhibited the MSH action.     

Muscarinic Cholinoceptors That Mediate Pigment Aggregation Are Present in the Melanophores of Cyprinids (Zacco spp.)

Like melanophores of many teleosts, those of the dark chub, Zacco temmincki, and the common minnow, Z. platypus (Cyprinidae, Cypriniformes) responded to norepinephrine (NE) by the aggregation of pigment. It was further found that some melanophores were responsive to acetylcholine (ACh) in the same way. The response to NE was blocked by an alpha-adrenergic blocker, phentolamine, whereas the response to ACh was not. By contrast, two muscarinic cholinoceptor antagonists, namely, atropine and scopolamine, effectively blocked the action of ACh. The pigment aggregation due to the liberated sympathetic neurotransmitter was blocked by phentolamine but not by cholinergic blockers. These results suggest that, although the melanophores of these species are controlled in an orthodox manner by the sympathetic nervous system, some of them possess extra muscarinic cholinoceptors that also mediate the aggregation of pigment. The present report is the first to describe the presence of cholinoceptors on the chromatophores in species of fish other than those that belong to the order Siluriformes. The evolutionary implications of these findings are discussed.     

Control of melanosome movement in intact and cultured melanophores in the bitterling, Acheilognathus lanceolatus

The melanophores in the dermis on scales in the bitterling, Acheilognathus lanceolatus were studies to obtain information about the control mechanism of aggregation and dispersion using intact, membrane-permeabilized and cultured cells. The cultured melanophores showed supersensitivity, namely, they responded to norepinephrine with much higher sensitivity than intact cells. The cultured melanophores failed to respond to high KCl. Melatonin aggregated and adenosine dispersed melanosomes within a cell. Digitonin permeabilized cells showed aggregation with Ca ions and dispersion by cyclic adenosine 3',5'-monophosphate (cAMP) in the presence of ATP. Movement of melanosomes was observed under the high magnification of light microscope and the tracks of each pigment granule were followed. The granules moved fast and linearly during aggregation, whereas they showed to-and-fro movement during dispersion.     

Dermal and epidermal chromatophores of the Antarctic teleost Trematomus bernacchii

The physiological response and ultrastructure of the pigment cells of Trematomus bernacchii, an Antarctic teleost that lives under the sea ice north of the Ross Ice Shelf, were studied. In the integument, two types of epidermal chromatophores, melanophores and xanthophores, were found; in the dermis, typically three types of chromatophores--melanophores, xanthophores, and iridophores--were observed. The occurrence of epidermal xanthophore is reported for the first time in fish. Dermal melanophores and xanthophores have well-developed arrays of cytoplasmic microtubules. They responded rapidly to epinephrine and teleost melanin-concentrating hormone (MCH) with pigment aggregation and to theophylline with pigment dispersion. Total darkness elicited pigment aggregation in the majority of dermal xanthophores of isolated scales, whereas melanophores remained dispersed under both light and dark conditions. Pigment organelles of epidermal and dermal xanthophores that translocate during the pigmentary responses are carotenoid droplets of relatively large size. Dermal iridophores containing large reflecting platelets appeared to be immobile.     

Possible involvement of nitric oxide in signaling pigment dispersion in teleostean melanophores

The possible involvement of nitric oxide (NO) in regulating the motile activities of teleostean melanophores was studied in the dark chub Zacco temmincki (Cyprinidae, Cypriniformes) and in the translucent glass catfish Kryptopterus bicirrhis (Siluridae, Siluriformes). NO donors, including (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexaneamide (NOR1), molsidomine (MSD), sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), had no pigment-aggregating action on melanophores, but actively dispersed melanosomes in those cells. Among those reagents, NOR 1, a spontaneous releaser of NO, was the most effective. Inhibitors for nitric oxide synthase (NOS), i.e. N omega-nitro-L-arginine methyl ester (L-NNA), N omega-nitro-L-arginine (L-NAME) and N omega-monomethyl-L-arginine (L-NMMA), showed melanosome-aggregating effects. A membrane-permeable analogue of cyclic guanosine-3',5'-monophosphate (8-Br-cGMP) was effective in dispersing melanosomes. The sum of these results suggests that NO plays an active role in the elaborate control of color changes in teleosts by dispersing pigment in melanophores via activation of soluble guanylyl cyclase to increase cytosolic levels of cGMP.     

Pigment migration in the dermal melanophores of amphibian larvae in the interphase and during mitosis

Functioning of the dermal melanophores was studied in the isolated skin of the Rana temporaria and R. esculenta tadpoles at stages 17-21 and 20-24 (after Kopsch). At all stages we studied melanophores exhibited reaction to light. From stage 18 on repeated alternation of pigment dispersion and aggregation was obtained using melanotropins and melatonin. When observing transition of the melanophores from interphase to mitosis, it was found that dividing dermal melanophores could be distinguished due to changes in their appearance shortly before the end of prophase.     

Purification of Black Moor Goldfish melanophores and responses to epinephrine

Summary Two key modifications of the previously reported method for isolation of goldfish xanthophores allowed the isolation and establishment of primary cultures of terminally differentiated melanophores from the Black Moor goldfish (Carassius auratus). First, pretreatment with 10⁻⁴ M epinephrine causing aggregation of the melanosomes and collapse of the dendrites, prevents damage to the melanophores during tissue dissociation and melanophore isolation. Second, maintenance of these cells in culture was successful only when the culture medium was supplemented with fish serum. The purified melanophores attached, flattened, and were maintained in culture for up to 3 mo. Although the morphology of the cultured melanophores is less dendritic than their in vivo counterparts, the melanophores translocate melanosomes in a normal manner except that they exhibit enhanced sensitivity to epinephrine. This epinephrine-induced pigment aggregation, as well as the redispersion of pigment after the removal of epinephrine, can occur in the presence of ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid and absence of Ca²⁺. This work was supported by grant AM13724 from the National Institutes of Health, Bethesda, MD.     

Relationship between intermediate host taxon and infection by nematodes of the genus Rhabdochona

We describe the intermediate and definitive hosts of the fish nematodes Rhabdochona coronacauda and R. denudata honshuensis and discuss the relationships between parasitism and the feeding habitats of their intermediate hosts. We found that the principal intermediate hosts of the two nematodes were filter-feeding mayflies of the genera Ephemera, Photamanthus and Isonychia. Ephemera strigata seemed to be the most important intermediate host of these nematodes. Adult R. coronacauda were found mainly in Hemibarbus longirostris and Rhinogobius flumineus, which are benthic fishes that feed on benthic aquatic insects, including E. strigata. For R. coronacauda, therefore, the feeding habits of the definitive hosts facilitate host alternation by this species. However, adult R. denudata honshuensis were found in cyprinids. In particular, Zacco temmincki was the principal natural definitive host in our study area. Since Z. temmincki is a swimming predator, E. strigata nymphs that burrow in the substrate are not the main prey of this species. This indicates that the transmission of R. denudata honshuensis hardly occurs from E. strigata nymph to Z. temmincki, suggesting another, unknown transmission route.     

Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.     

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 μM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 μM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca²⁺ ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 μM). Since it is known that medaka melanophores possess specific receptors for α-melanophore-stimulating hormone (α-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca²⁺ might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1–14) and MCH (5–17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 μM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.     

Alpha1 and alpha2 adrenoceptor mediated melanosome aggregatory responses in vitro in Oreochromis mossambica (Peters) melanophores

Effects of specific and non-specific adrenoceptor agonists and antagonists were examined on the isolated scale melanophores of O. mossambica in physiological Ringer solution. The responses were recorded as melanophore size index. It was observed that adrenaline, nor-adrenaline, phenylpropanolamine, clonidine and phenylepherine induced melanosome aggregation in a dose-dependent manner. Denervation of the fish melanophores increased the sensitivity of the melanophores to adrenaline but not to nor-adrenaline. Phentolamine (3.55 x 10(-5) M), prazosin (2.38 x 10(-5) M) and yohimbine (2.821 x 10(-5) M) significantly inhibited the aggregatory responses of the fish melanophores to adrenaline, nor-adrenaline, clonidine and phenylepherine. The blocking effect of yohimbine was significantly higher than that of prazosin. It is concluded that the effect of adrenaline is directly mediated through the receptors and alpha2 adrenoceptors are predominantly involved in the aggregatory responses of this fish melanophores, while alpha1 adrenoceptors presence has been indicated.     

Evidence for the presence of novel β-melatonin receptors along with classical α-melatonin receptors in the fish Rasbora daniconius (Ham.)

Abstract

The effects of melatonin (MT) were examined on the isolated scale melanophores from dorso-lateral (D-L) and band regions of a tropical fish Rasbora daniconius. Our study primarily aimed for further depiction of the signaling receptors involved in MT mediated pigment translocations in the fish. Melanophore Size Index (MSI) was employed as a recording parameter for the responses of melanophores to MT and various antagonists. MT has induced aggregation as well as dispersion in D-L region and aggregation in band region melanophores during summer season. During winter, MT-induced responses were only of aggregatory type in D-L region, while in the band region there was an increase in the sensitivity. The responses of the melanophores to MT were reversible. The aggregation of innervated melanophores induced by MT on the D-L and band regions was partially mediated through the neurotransmitters released under the influence of MT and partially by the specific MT receptors. Luzindole and K185 have completely blocked the aggregatory responses of D-L and band region melanophores. Aggregatory receptors may be of the conventional α-MT type. Dispersion of D-L and band region melanophores induced by MT in the presence of various antagonists and on denervated band region could be the result of activation of β-MT receptors of dispersive nature. Presence of α and β MT receptors is thus indicated in this fish melanophores.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Enhancement by fibronectin of spreading of isolated melanophores of the medaka,Oryzias latipes

Summary Cell spreading of isolated melanophores in medium containing fibronectin was observed in the wild type and two mutants of the medaka, Oryzias latipes. Isolated and cultured melanophores of the wild type and the mm mutant were different in appearance from those within scales but dendritic in shape and with fully dispersed pigment granules. Isolated melanophores of the cm mutant were stellate with dispersed pigment granules, whereas in scales the pigment granules are condensed. In the presence of fibronectin, spreading of cultured melanophores of wild type and cm mutant was observed. Spreading of melanophores from the mm mutant was observed only among dendritic melanophores, but not among condensed melanophores. The increase of spreading was inhibited by antibody against fibronectin. To test the involvement of cytoskeletal elements, colchicine, vinblastine or cytochalasin B were added to the culture medium; spreading did not increase, even in the presence of fibronectin. These results suggest that fibronectin-induced melanophore spreading is correlated with the state of pigment granule dispersal and that microtubules and microfilaments may play a role in the mechanism of spreading.Department of Zoology NJ-15, University of Washington, Seattle, Wa 98195, USA.     

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.