Electronische Mikroskopievon zweier Arten
reflektierender Chromatophoren (Iridophoren und Leucophoren)
in the Guppy, Lebistes reticulatus Peters
Ikuo K. Takeuchi*
Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony,
Kasugai, Japan

Summary

Reflecting chromatophores in the integument of the guppy, Lebistes
reticulatus Peters, are of two distinct types, iridophores and leucophores.
The iridophores are smaller and fixed, producing a metallic iridescent color.
The cytoplasmic organelles involved in the coloration of iridophores are
the reflecting platelets, as in the iridophores of other fish and amphibian
species on which earlier reports have been made. Spherical granules of pleiomorphic
internal structure, quite variable in size but generally 0.2 gm to
1.0 pm in diameter, are also numerous in the iridophores. The nature of
these granules remains unknown.
The leucophores are larger, and highly dendritic; their pigment granules
are migratory and they exhibit a dull whitish color. Pigment granules of
the leucophores are spherical in form, varying from 0.5-0.8 lam in
diameter, with a double membrane enclosing the internal fibrous materials.
Melamine-treatment of the fish caused degenerative changes in the pigment
granules and also the other cytoplasmic organelles of the leucophores,
whereas the other kinds ofchromatophores, including the iridiophores,
remained intact. Some problems in general characterization and classification
between these two types of chromatophores were discussed.
Key words: Chromatophores - Iridophore - Leucophore - Guppy -
Electron microscopy.

Introduction
The body color of fishes is produced by an interaction of several chromatophores
such as black chromatophores (melanophores), yellow chromatophores (xantho-
Send offprint requests to: Doctor Ikuo K. Takeuchi, Department of Embryology, Institute for
Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan
* The author wishes to thank Mr. Yoshiro Yamazaki for his assistance in operating the electron
microscope, and Dr. Takao Kajishima (Biological Institute, Nagoya University) for his encouragements
phores), red chromatophores (erythrophores), and reflecting chromatophores.
Reflecting chromatophores have iridescent or whitish appearances by virtue
of the reflecting capacity of their pigments, commonly present in integument,
peritoneum and eye. A wide variety of names, such as antaugonophores, guanophores,
iridocytes, iridophores, iridoleucophores, leucophores, etc., have been
used for these reflecting chromatophores. The historical problem of the nomenclature
of these chromatophores has been discussed elsewhere (Fries, 1958).
Bagnara (1966) classifies chromatophores in lower vertebrates on the basis of
the color that they produce and on the appearance of the pigment; he designates,
all reflecting chromatophores as iridophores.
More recently, Hama (1967, 1970) has distinguished two types of reflecting
chromatophores in the medaka, Oryzias latipes," one contains fixed crystalline
platelets, manifesting a scintillating iridescence, and the other has white reflecting
granules capable of migration. A striking difference between these two types
was indicated. If larvae or adults of the medaka were reared in melaminesaturated
water, the white granule-containing chromatophores underwent destruction
within one or two days, while the others having crystalline platelets
remained intact. Hama has named the iridescent, melamine-resistant chromatophores
iridophores and the whitish ones leucophores.
In the course of studying the ultrastructural features of chromatophores
in the integument of the guppy, we noticed that it also has two types of reflecting
chromatophores as in the medaka. The purpose of this paper is to describe
the fine structure of these reflecting chromatophores.
Materials and Methods
Male guppies having whitish tailfins because of their numerous, scattered reflecting chromatophores,
were obtained from a commercial hatchery and reared in filtered tap water. Some of them were
reared in melamine (2,4,6-triamino-s-triazine)-saturated water for one or two days. Tailfins were
cut off in a Ringer's solution, observed with dark-field microscopy, and dissected into small pieces
in a chilled Dalton's chrome-osmium solution (Dalton, 1955). Fixation was in the same solution
for two hours at 4 ~ C. Epon-embedding was according to Coulter (1967). Sections were obtained
using a Porter-Blum MT-2 ultramicrotome with a glass knife, double-stained with uranyl acetate
and lead nitrate, and examined with a Hitachi HU 11-DS or a JOEL JEM 100B electron microscope.
Results
Light Microscopy
With dark-field microscopy two distinct types of reflecting chromatophores
were observed to be numerously distributed in the guppy tailfin. The iridophores
are smaller, having a metallic iridescent color, and the leucophores are larger,
highly dendritic, and exhibited a dull whitish color (Fig. la). The leucophores
gradually contracted 20 or 30 rain after the tailfin was cut off, while the iridophores
appeared unchanged (Fig. lb). In the tailfin taken from the fish reared
in melamine-saturated water for two days, the leucophores had completely disappeared
but the iridophores remained intact (Fig. Ic).

Reflecting Chromatophores in the Guppy 19
Fig. la-e. Dark-field micrographs of tailfin of the male guppy in a Ringer's solution, a Normal
guppy. Small iridophores (IP) and large, dendritic leucophores (LP) are noticed. Contracted melanophores
(MP) are also found, b Thirty minutes after the tailfin was removed. Leucophores are
contracted (LP), while the iridophores remain unchanged (IP). c Two days after rearing the fish
in melamine-saturated water. Note the complete disappearance of leucophores. IP iridophore, x 210
Electron Microscopy
Iridophores. The cytoplasm of iridophores of the guppy contain a large number
of laminated cisternae or "reflecting platelets" (Bagnara and Ferris, 1971)
(Fig. 2). Each of these platelets is surrounded by its own limiting membrane
(Fig. 3). Their lengths vary from 0.7 gm to 0.2 lain, and their diameters are
approximately 35 lam (Figs. 2, 3). The platelets are found in several groups.
Within each group several platelets, sometimes up to 17 or 18, are stacked
up with an interval of about 150 lain. Angles among groups are variable (Fig. 2).
Many of the platelets are split longitudinally, exhibiting empty spaces (Fig. 3).
Electron microscopy reveals that these longitudinal splittings are initially narrow,
but the electron beam rapidly enlarged them in various degrees, markedly affecting
their appearance (Fig. 7). Therefore, carbon-coated sections were used for
detailed observation of these platelets. The longitudinal splitting extended either
the entire length of the platelets, or was interrupted at irregular intervals. In
the center of the platelets that were not split, very thin lamellar materials
extended over the entire axis. In some platelets in where only partial longitudinal
splitting had occurred, the interconnection between these lamellae and the splitting
was clearly noticed (Fig. 3).
The other conspicuous cytoplasmic organelles in the iridophores are spherical
or oval granules of quite variable sizes, generally 0.2 lam to 1.0 ~tm in diameter.
They incorporate pleiomorphic internal structures ; in particular, grainy, filamentous,
membranous, and amorphous materials in various degrees (Fig. 4). Many
of the granules are apparently limited with a single membrane (Fig. 4), but
in many others, a double membrane encloses the contents. The empty intervening
space between these two membranes varies (Fig. 5).
Golgi apparatus, composed of saccules and small vesicles, occurs in the
perinuclear region of the iridophore. Some small vesicles near the Golgi apparatus
have internal structures resembling those of pleiomorphic granules, suggesting
that Golgi apparatus might be involved in the production of these granules
(Fig. 6). Rod-shaped mitochondria and tubular or vesicular endoplasmic reticula
are scattered throughout the cytoplasm (Fig. 6), but both organelles are sparsely
distributed in the intervening spaces of a stack of reflecting platelets (Fig. 3).
Numerous ribosomal particles are distributed freely or are attached to the endoplasmic
reticulum (Fig. 6). At the plasma membrane numerous pinocytotic vesicles
have been noted (Fig. 4). The nucleus of the iridophore is frequently in
highly convoluted form; the nucleoplasm is filled with granular and fibrous
elements. Few nucleoli or chromatin-like aggregates of granular elements are
found (Fig. 4).
Leucophores. The dendritic leucophores are very large, having large and convoluted
nuclei and cytoplasms with a large number of characterictic pigment
granules (Fig. 7). Nuclei usually lie at the periphery of the cell. Granular and
fibrous elements are distributed throughout the nucleoplasm, with a few aggregates
of granular elements suggestive of nucleoli or chromatin (Fig. 7).
The spherical or ellipsoidal pigment granules of the leucophores, measuring
about 0.5-0.8 l.tm in diameter, are enclosed by a double membrane with a
rather broad intervening space; the outer membrane strongly resembles the
delineating membranes of other cytoplasmic organelles such as mitochondria
and endoplasmic reticulum, but the inner is reminiscent of lamellae, appearing
to originate from the fibrous materials so numerous in the internal parts of
these granules (Fig. 8).
Other cytoplasmiv organelles in the leucophores include numerous pinocytotic
vesicles at the plasma membrane, many granular and agranular endoplasmic
reticula, moderate numbers of mitochondria having well-developed cristae, and
sporadic Golgi apparatus composed of saccules and small vesicles (Figs. 7, 8).
Two types of small particles are numerous; one type is frequently attached
to the endoplasmic reticulum and considered to be ribosomal, while the other,
with a diameter somewhat larger than that of ribosomes, is considered to consist
of glycogen particles (Fig. 8).
Melamine-induced Degeneration of Pigment Granules in the Leucophores
For the identification of leucophores, the guppies reared for one day in melaminesaturated
water were sacrificed and pigment granules of each chromatophores
were examined. Only the pigment granules in the chromatophore described above
as leucophores exhibited-conspicuous degenerative changes. Outer limiting
membranes of the granules were torn off and frequently disappeared. Inner
lamellae and internal fibrous materials were coagulated into small, thick membranous
materials (Fig. 9).
mitochondria
were swollen and ruptured, endoplasmic reticula fragmented into small
vesicles. Golgi apparatus became unrecognizable, and the nucleus exhibited a
fibrous appearance in its inner components. These cytoplasmic changes were
very similar to those observed in the process of degeneration of xanthophores
in some mutants of the goldfish, and will be discussed in another paper (Takeuchi
and Kajishima, in preparation).

Discussion
Many authors (e.g. Fries, 1958) have recognized two different types of reflecting
chromatophores in some teleostean species; one type is characterized as whitish
and granular with extensive effector movements of pigment granules, whereas
the other type is fixed and strongly iridescent by virtue of small crystal-like
pigment bodies. Fries (1958) considered these two types to be variants of one
kind of chromatophore characterized by light-reflecting particles or crystals,
and proposed the name "antaugonophores" for all reflecting chromatophores.
Bagnara (1966) designated these as en bloc iridophores.
This study has revealed two types of reflecting chromatophores in the guppy
differing from each other in ultrastructural features. This observation, along
with Hama's observation described in the introduction of this paper, suggests
a justification to draw a distinction between the iridophore-type and leucophoretype
chromatophores.
It is somewhat surprising that electron microscopic studies hitherto published
on reflecting chromatophores of many lower vertebrate species were, with few
exceptions, concerned exclusively with iridophore-type chromatophores characterized
by numerous platelet structures in their cytoplasm. These platelets have
been considered to be stores of crystalline materials, mainly guanine-crystals,
and have been called reflecting platelets (Bagnara and Ferris, 1971) or crystal
sacs (Best and Nicol, t967; Arnott and Nicol, 1970). Many authors have demonstrated
that these crystalline materials are lost from the sections during treatment
of section with staining solution of lead nitrate which is alkaline and capable
of dissolving guanine-crystals, leaving behind longitudinal splitting in the
platelets (Harris and Hunt, 1973 ; Kawaguti et al., 1965; Kawaguti and Kamishima,
1966a; Kawaguti and Takeuchi, 1968; Setoguti, 1967; Setoguti et al., 1969;
Rohrich and Porter, 1972). Thin lamellar materials in the reflecting platelets
have also reported in iridophores of the clupeoid fish, Harengula zunasi, although
the authors have described them as "dark lines" (Kawaguti and Kamishima,
1966b). These materials may play a role in accumulating crystalline materials
in the platelets.
Cytoplasmic organelles having spherical granules with pleiomorphic inner
components found in guppy iridophores have not been described previously.
The physiological role of these granules remains unknown although the large
number in the cytoplasm suggests that they play a role in the coloration of
guppy iridophores.
Very little is known about the fine structure of leucophore-type chromatophores
in lower vertebrates. The only report seems to be our preliminary one
dealing with leucophores of the larval medaka, Oryzias latipes (Kamei-Takeuchi
et al., 1968). Three structurally distinct types of pigment granules, suggestive
of several developmental forms of the same pigment granules, were described,
and one of them is very similar to the pigment granules in guppy leucophores.
In some species of invertebrates such as crustaceans and insects, some authors
reported white-colored chromatophores containing pigment granules of very
similar structures to those in the guppy (Eloffson and Kauri, 1971; Robinson
and Charlton, 1973; Veron et al., 1974), but the relationship of these chromatophores
to the vertebrate leucophores is still unknown.
Reflecting chromatophores in integuments of some reptilian species have
been called leucophores (see Fries, 1958), but electron microscopic observationes
have revealed that they are actually iridophore-type chromatophores, although
their crystals are in the forms of small tablet-like bodies rather than of platelets
(Rohlich, 1974). This indicates that the distinction between two types of reflecting
chromatophores is, in some cases, far from a simple problem. Melamine-treatment
has been shown to be a simple and effective method for this distinction
at least in the medaka and the guppy, but it remains unknown whether all
leucophore-type chromatophores in other lower vertebrate species are sensitive
to melamine. Electron microscopy seems to be an accurate and indispensable
method for classification. But it is possible that in other species leucophore-type
chromatophores contain pigment granules that are morphologically different
from those of the guppy leucophores. Recent studies, for example, have revealed
that the fine structure of pigment granules in the same group of chromatophores
is sometimes very different among species (Taylor and Bagnara, 1969; Bagnara
etal., 1973; Takeuchi, 1975a, b). Greater knowledge on the morphological,
physiological as well as biochemical aspects of not only the leucophore-type
chromatophores but also those of the iridophore-type is necessary, before
conceptual generalizations can be made.

Zusammenfassung.
Die reflektierenden Chromatophoren im Integument des Guppys, Lebistes
reticulatus Peters, sind von zwei verschiedenen Arten, Iridophoren und Leucophoren.

Die Iridophoren sind kleiner und fester und erzeugen eine irisierende Metallic-Farbe.

Die zytoplasmatischen Organellen, die an der Färbung der Iridophoren beteiligt sind, heißen reflektierende Platelets wie bei den Iridophoren anderer Fisch-und Amphibienarten, auf die in früheren Berichten hingewiesen wurde.
Sphärisches Granulate mit pleiomorphischer
innerer Struktur, recht unterschiedlich in Größe, aber im allgemeinen mit einem Durchmesser von 0,2 gm bis
1,0 gm, sind auch in den Iridophoren zahlreich.

Die Art diese Granulate ist unbekannt.
Die Leucophoren sind größer, und sehr dendritisch; ihre Pigment-Granulate
wandern und sie weisen eine trübe weißliche Farbe auf. Die Pigment Granulen
der Leucophoren sind von kugelförmiger Art, mit einem Durchmesser von 0,5-0,8 lam, mit einer doppelten Membran, welche die inneren Faserstoffe umschließt.

Eine Melamin-Behandlung des Fisches verursacht degenerative Veränderungen in dem Pigment - Granulat und auch der anderen zytoplasmatischen Organellen der Leucophoren, während die anderen Chromatophoren Arten, einschließlich der Iridiophores, nach wie vor intakt bleiben.

Einige Probleme bei der allgemeinen Charakterisierung und Klassifizierung zwischen diesen beiden Chromatophoren Arten wurden erörtert.

Einführung
Die Körperfarbe der Fische wird durch ein Zusammenwirken mehrerer Chromatophoren produziert, wie schwarze Chromatophoren (Melanophoren), gelb Chromatophoren (Xanthophoren), rote Chromatophoren (Erythrophoren) und reflektierende Chromatophoren.
Durch die spiegelnde Kapazität ihrer Pigmente besitzen reflektierende Chromatophoren ein irisierendes oder weißliches Erscheinungsbild und befinden sich meist in der Haut, im Peritoneum (Bauchfell) und im Auge.
Für diese reflektieren Chromatophoren werden eine Vielzahl von Namen, wie Antaugonophoren, Guanophoren, Iridocyten, Iridophoren, Iridoleucophoren, Leucophoren, etc. verwendet.
Das historische Problem der Nomenklatur dieser Chromatophoren wurde an anderer Stelle (Fries, 1958) diskutiert.
Bagnara (1966) stuft Chromatophoren bei niederen Wirbeltieren auf der Grundlage der Farbe, die sie herstellen und auf das Aussehen des Pigments ein, er benennt,
alle reflektierenden Chromatophoren als Iridophoren.

In jüngerer Zeit hat Hama (1967, 1970) zwei Arten von reflektierenden
Chromatophoren beim Medaka, Oryzias latipes, " unterschieden, eine enthält feste kristalline Plättchen (platelets), welche sich in einem funkelnden Schillern manifestieren, und die andere hat weiße, reflektierende und bewegliche
Granulate.
Ein auffallender Unterschied zwischen diesen beiden Arten
wurde nicht mitgeteilt. Wenn Larven oder adulte Medaka in melamin-gesättigtem Wasser aufgezogen wurden, wurde die das weiße Granulat enthaltende Chromatophore innerhalb von ein oder zwei Tagen zerstört, während die andere mit den kristalline Plättchen intakt blieb.
Hama benannte die schillernden, Melamin-resistenten Chromatophoren
Iridophoren und die weißlichen Leukophoren.


Diskussion

Viele Autoren (z. B. Fries, 1958) haben zwei verschiedene Arten von reflektierenden Chromatophoren bei einigen Knochenfisch Arten angeführt; ein Typ ist als weißlich und granular (körnig) charakterisiert worden, mit umfangreichen Effektor Bewegungen der Pigmentkörnchen, während
der andere Typ fest ist und stark irisiert aufgrund der kleinen kristall-ähnlichen
Pigment-Körpern. Fries (1958) betrachtet diese beiden Typen als Varianten von einer Chromatophorenart, welche durch lichtreflektierende Partikel oder Kristalle gekennzeichnet ist,
und schlug den Namen "Antaugonophoren" für alle reflektierenden Chromatophoren vor.
Bagnara (1966) bezeichnet diese "en bloc" als Iridophoren.
Diese Studie hat zwei Arten von reflektierenden Chromatophoren beim Guppy ergeben, die sich von einander in ultrastrukturellen Eigenschaften unterscheiden.
Diese Beobachtung, zusammen mit Hamas Beobachtung in der Einleitung zu dieser Arbeit Papier, ergibt eine Rechtfertigung, um eine Unterscheidung zwischen den iridophoren-Typ und dem leukophoren Typ Chromatophoren zu treffen.

Es ist etwas überraschend, dass bisher nur elektronenmikroskopische Untersuchungen von reflektierenden Chromatophoren vieler unterer Wirbeltierarten veröffentlicht
wurden, mit wenigen Ausnahmen, die sich ausschließlich mit dem iridophoren-Typ von Chromatophoren befassten, der durch zahlreiche Blättchen (platelets) Strukturen
in seinem Zytoplasma charakterisiert ist. Diese Plättchen wurden als Speicher
von kristallinen Materialien betrachtet, hauptsächlich von Guanin-Kristallen,
und worden reflektierende Plättchen (Bagnara und Ferris, 1971) oder Kristall
Säckchen (Best und Nicol, t967; Arnott und Nicol, 1970) genannt.
Viele Autoren haben gezeigt,
dass diese kristallinen Materialien aus den Sektionen, durch die Behandlung mit der Färbelösung Bleinitrat, die alkalisch und in der Lage ist,
Guanin-Kristallen aufzulösen und eine Längsspaltung der Blättchen bewirkt, verloren gingen
(Harris und Hunt, 1973; Kawaguti et al., 1965; Kawaguti und Kamishima,
1966a; Kawaguti und Takeuchi, 1968; Setoguti, 1967; Setoguti et al., 1969;
Röhrich und Porter, 1972).
Dünne lamellenartige Materialien in den reflektierenden Plättchen wurden auch
in den Iridophoren von Clupeiformes ( Heringe etc.), Harengula zunasi, berichtet, obwohl
die Autoren sie als "dunkle Linien" (Kawaguti und Kamishima beschrieben haben,
1966b).
Diese Materialien können eine Rolle bei der Anhäufung von kristallinen Materialien
in den Blättchen (Platelets) spielen.

Beim Guppy wurden zytoplasmatische Organellen mit kugelförmigen Körnchen mit pleomorphen inneren Komponenten gefunden, Iridophoren wurden bisher nicht beschrieben.
Die physiologische Rolle dieser Granula bleibt unbekannt, obwohl die große
Zahl im Zytoplasma vermuten läßt, dass sie bei der Färbung von
Guppy Iridophoren eine Rolle spielen.
Sehr wenig ist über die Feinstruktur des leucophoren-Typ Chromatophoren bei niederen Wirbeltieren bekannt
Der einzige Bericht scheint vorläufig unserer zu sein sein, der sich
mit Leucophoren des larvalen Medaka, Oryzias latipes (Kamei-Takeuchi
et al., 1968) befasst.
Drei strukturell unterschiedliche Arten von Pigmentkörnchen, suggestiv (hinweisend)
für mehrere Entwicklungs-Formen der gleichen Pigmentkörnchen, wurden beschrieben,
und einer von ihnen ist den Pigmentkörnchen beiden Guppy Leucophoren sehr ähnlich.
.
Bei einigen Arten wirbelloser Tiere wie Krebsen und Insekten, berichten einige Autoren über weiß-farbige Chromatophoren mit Pigmentkörnchen, sehr
ähnlich den Strukturen wie beim Guppy (Eloffson und Kauri, 1971; Robinson
und Charlton, 1973; Veron et al., 1974), aber die Beziehung dieser Chromatophoren
mit den Wirbeltieren Leucophoren ist noch unbekannt.

Reflektierende Chromatophoren in den Integumenten (Häuten) einiger Reptilien Arten wurden als Leucophoren bezeichnet(vgl. Fries, 1958), aber elektronenmikroskopische Observationen
haben gezeigt, dass sie tatsächlich vom iridophoren-Typ Chromatophoren sind, obwohl
ihre Kristalle eher eine kleine Pillen-Form statt der Plättchenform haben
(Röhlich, 1974).
Dies deutet darauf hin, dass die Unterscheidung zwischen beiden Arten von reflektierenden
Chromatophoren, in manchen Fällen, weit entfernt von einem einfachen Problem ist.
Es hat sich gezeigt, dass Melamin-Behandlung eine einfache und effektive Methode für die Unterscheidung zumindest beim Medaka und beim Guppy ist, aber es bleibt unbekannt, ob alle
leucophoren-Typ Chromatophoren bei anderen niederen Wirbeltieren empfindlich auf
Melamin sind. Elektronenmikroskopie scheint ein genaues und unverzichtbares
Verfahren zur Einstufung zu sein.
Aber es ist möglich, dass andere Spezies leucophoren-Typ
Chromatophoren Pigmentkörnchen enthalten, die morphologisch unterschiedlich
von denen der Guppy Leucophoren sind.
Aktuelle Studien haben zum Beispiel ergeben,
dass die Feinstruktur der Pigmentkörnchen in der gleichen Gruppe von Chromatophoren
manchmal sehr unterschiedlich zwischen den Arten ist (Taylor und Bagnara, 1969; Bagnara
et al., 1973; Takeuchi, 1975a, b).
Ein größerer Kenntnisstand über die morphologischen,
physiologischen sowie biochemischen Aspekte nicht nur der leucophoren-Typ
Chromatophoren, sondern auch jene des iridophore-Typs sind erforderlich, bevor
begriffliche Verallgemeinerungen vorgenommen werden können.