Ultrastructural and functional study of the liver pigment cells from Rana esculenta L.

Summary A study of the liver pigment cells of Rana esculenta L. has been performed on both liver in toto and cells in culture. Ultrastructural and cytochemical analyses showed a close relationship between this visceral pigment cell system and the cells of hepatic macrophage lineage. Like the latter, the liver pigment cells present phagocytic activity, in the sinusoids and in vitro, and give a positive response to tests for peroxidase and lipase. The liver pigment cells are isolated, together with the Kupffer cells, from the sinusoidal cell fraction of the liver. In culture, they maintain their melanogenetic ability, demonstrated by the presence of dopaoxidase activity in the soluble, membranous, and melanosome fractions. Analysis of the cultures showed that as culture time increased, so did melanosome dopaoxidase activity, the number of pigmented fields, and the level of pigmentation of the cells. The values of dopaoxidase activity of the pigment cells in culture show the same seasonal oscillations as the system in toto, indicating that the cells maintain an internal clock, at least in the first 72 h of culture. There is evidence that the pigment cells are macrophages which can express a melanogenetic function. Our results and other experimental data provide a basis for hypothesizing that the pigment cells in Rana esculenta L. liver may derive from, or have a common origin with, the Kupffer cells.     

A microsatellite‐based method for genotyping diploid and triploid water frogs of the Rana esculenta hybrid complex

Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR‐amplified (polymerase chain reaction‐amplified) genome‐specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.     

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.     

A comparative study of physiological and structural changes at the myoneural junction in two species of frog after transection of the motor nerve

Summary Structural and functional behaviour of motor end-plates after transection of the motor nerve has been studied in two species of frog: Rana esculenta and Rana temporaria. The physiological results show that in both species there is a transient cessation of spontaneous activity followed by a resumption of miniature end-plate potentials (min. e.p.p.s.) after denervation. The characteristics of these potentials (frequency, distribution of amplitudes, time-course) are similar in the two species. However, some differences have been observed: Firstly, the period of silence lasts for 2–4 days in the case of Rana temporaria whereas it is prolonged to about 15 days in Rana esculenta. Secondly, the resumption of min. e.p.p.s. is gradual and after the 10th day of denervation remains constant in Rana temporaria. It is inconstant independent of the period of denervation in Rana esculenta. The morphological results show that the Schwann cell is constantly in contact with the post-synaptic membrane after about 6 days of denervation in both species. It is suggested that either the Schwann cell is capable of functioning for a limited period of time in Rana esculenta or is activated to produce min. e.p.p.s. only in certain cases.     

Elektronenmikroskopische Untersuchungen am Stirnorgan von Anuren

The ultrastructure of the frontal organ (pineal end-vesicle, Stirnorgan) of Rana temporaria L. and Rana esculenta L. is similar to the submicroscopic organization of the retina and other photosensitive organs. There are five different cell types in the frontal organ: sensory (receptor) cells, ependymal cells, ganglion cells, glial cells and epithelial cells. The ependymal cells may be secretory. There is no evidence for a typical pigment epithelium. The sensory cells have inner and outer segments. The inner segments contain numerous mitochondria, a Golgi complex, filaments, lipid droplets, two centrioles and a fibrillar apparatus (within the connecting piece). The mitochondria are very abundant in the Ellipsoid and Ersatzellipsoid areas (Holmgren) of the inner segment. The outer segment consists of about 60 to 110 discs formed by infoldings of the cell membrane. Most of the sensory cells are cone-like, but there are some elements with rod-like structures. Plexiform areas of the frontal organ contain terminations of the receptor cells, and processes of the nerve cells and glia cells. Synaptic structures have been determined within these areas. Non-medullated and medullated nerve fibers with adjacent glial satellites are observed in the pineal nerve (Nervus pinealis). The anatomical findings are described in detail and discussed in respect to the physiological results of Dodt and Heerd (1962) in Rana temporaria and Rana esculenta.

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Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Über Kanälchenzellen und dunkle Zellen im Nephron von Anuren

Zusammenfassung Die elektronenmikroskopische und enzymcytochemische Untersuchung der Nieren von Rana cancrivora und R. esculenta, Hyla arborea und H. regilla, Bufo carens, B. mauretanicus und B. viridis ergibt, daß im Epithel ihrer Verbindungsstücke verschiedene Zelltypen vorkommen.Die Verbindungsstücke von Rana cancrivora und R. esculenta zeichnen sich — abgesehen von dem Vorhandensein heller Zellen — durch den Besitz sog. Kanälchenzellen aus, die nach Form und Struktur an die Belegzellen der Magendrüsen erinnern. Es handelt sich um sehr mitochondrienreiche, elektronendicht erscheinende Elemente, die durch teilweise verzweigte intrazelluläre Kanälchen mit der Lichtung des Verbindungsstückes kommunizieren. Aus der Oberfläche dieser Canaliculi ragen kurze Cytoplasmafortsätze in das Lumen. An der Basis der Kanälchenzellen bilden zahlreiche Cytoplasmafortsätze ein Labyrinth, das mit den seitlichen Interzellularräumen in Verbindung steht. Die Kanälchenzellen geben eine starke Reaktion auf Bernsteinsäuredehydrogenase und NADH-Diaphorase, die dem Reichtum der Zellen an Mitochondrien entspricht. Unterschiede in der Zahl der Kanälchenzellen bei dem marinen Frosch Rana cancrivora und bei R. esculenta scheinen nicht zu bestehen.Im Verbindungsstück der untersuchten Hyla- und Bufoarten fallen dunkle, d.h. elektronendichte Zellen auf, die zwar viele Mitochondrien enthalten und ein basales Labyrinth bilden, jedoch nicht kanalisiert sind. Auch diese dunklen Zellen geben eine intensive Reaktion auf Bernstein- säuredehydrogenase und NADH-Diaphorase.Die Kanälchenzellen weisen Zeichen einer sekretorischen Aktivität (Bildung von Mukosubstanzen) auf. Die Frage, ob sie außerdem resorptiv tätig sind und ob die dunklen Zellen sowohl sezernieren als auch Ionen aktiv transportieren, kann mit den angewandten Methoden nicht beantwortet werden. Unbekannt ist ferner, ob die Unterschiede im Aufbau der Verbindungsstücke bei den Rana-Arten auf der einen und den Hyla- und sowie Bufo-Arten auf der anderen Seite zu Verschiedenheiten der Harnbildung bei diesen Formen in Beziehung stehen.

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On canaliculi cells and dark cells in the nephron of anurans

Summary The electronmicroscopical and enzymecytochemical investigation of the kidneys of Rana cancrivora, R. esculenta, Hyla arborea, H. regilla, Bufo carens, B. mauretanicus and B. viridis reveals that the epithelium of the connecting tubule (Verbindungsstück) consists of different cell typs.The connecting tubules of Rana cancrivora and R. esculenta are characterized — apart from the occurrence of light cells — by the differentiation of so-called canaliculi cells, the shape and structure of which resemble those of the parietal cells of the gastric glands. These electron dense elements are rich in mitochondria and communicate by partly ramified intracellular canaliculi with the lumen of the connecting tubule. The plasma membrane bordering the canaliculi forms short microvilli-like processes. At the basis of the canaliculated cells numerous cytoplasmic processes form a labyrinth which is in connection with the lateral intercellular space. The canaliculi cells exhibit a strong activity of succinic dehydrogenase and NADH-diaphorase, which corresponds to the abundance of mitochondria in these cells. Differences in the number of canaliculi cells in the marine frog Rana cancrivora and the freshwater species Rana esculenta apparently do not exist.The connecting tubules of the Hyla and Bufo species investigated contain strikingly dark, i.e. electron dense cells, which contain numerous mitochondria, and which possess a basal labyrinth. Canaliculi, however, are lacking. These dark cells, too, give an intensive reaction for succinic dehydrogenase and NADH-diaphorase.The canaliculi cells show features of a secretory activity (production of mucous substances). The question, whether in addition they have a resorptive function and whether the dark cells are secretory and actively ion-transporting elements, cannot be answered by means of the methods applied. Further, it remains unknown whether the differences in the structure of the connecting tubule of the Rana species on the one hand and the Hyla-and Bufo species on the other, are related to differences in the mechanisms of urine production.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

<Emphasis Type="Italic">Phellinus</Emphasis> extracts inhibit migration and matrix metalloproteinase secretion in porcine coronary artery endothelial cells

Phellinus linteus is a fungus which is found primarily in tropical regions of the Americas, Africa, and Asia.P. linteus has been used in traditional medical practice for the treatment of arthritis, liver damage and cancer. Angiogenesis is a process that involves migration, proliferation and cell differentiation, as well as the formation of new capillary structures. The anti-angiogenic activities evidenced by natural compounds may actually be a critical effect for the inhibition of angiogenesis-dependent disease by these agents via the blockage of vascular development. This study assessed the effects of water extracts fromP. linteus (Phellinus extracts) on primary cultured porcine coronary artery endothelial cells (PCAECs).Phellinus extracts induced no changes in DNA synthesis or cell numbers, but inhibited the migration of PCAECs.Phellinus extracts also induced a reduction in the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results show that, in endothelial cells,Phellinus extracts may inhibit angiogenesis by reducing levels of MMP-2 and MMP-9 secretion.     

On the biology of Amphibia in the ecosystem of Lake Glubokoe

In Lake Glubokoe, the Amphibia are represented by five species. Two numerous species, Rana temporaria L. and Bufo bufo L., spawn in the lake and appear to be a significant component of its ecosystem. Thus, in autumn R. temporaria is a common food for predatory fish (pike, perch). During their wintering R. temporaria participate in the consumption of oxygen from the water. They may be a source of dangerous infections. Their tadpoles and those of B. bufo feed in the lake and annually remove substances from it. Adult R. temporaria in autumn, and the young of the year of R. temporaria and of B. bufo influence the population numbers of small littoral invertebrates. Several dozen pairs of Rana terrestris Andrzejw are observed to spawn regularly. Rana esculenta L. and Rana ridibunda Pallas are scarce. The influence of introducing a nature preserve on the numbers of Amphibia in Lake Glubokoe is discussed.     

Saisonbedingte Veränderungen in der Morphologie der Leydigzellen von Rana esculenta

Zusammenfassung Saisonabhängige Veränderungen in der Morphologie der Leydigzellen von Rana esculenta wurden über den Zeitraum eines Jahres in Licht- und Elektronenmikroskop untersucht.In den Monaten November bis Februar kommen im Zytoplasma der Leydigzellen kleine Mitochondrien des tubulären Typs, Vesikel des glatten endoplasmatischen Retikulum und freie Ribosomen vor, Fettvakuolen treten nur vereinzelt auf, der Golgikomplex ist gering entwickelt. In der Zeit von März bis Juni nimmt die Zahl der Zellorganellen zu. Es erscheinen in Gruppen angeordnete Lysosomen und außer den kleinen Mitochondrien Riesenmitochondrien, des Golgifeld ist größer, die Fettvakuolen sind vermehrt. Sowohl die kleinen als auch die Riesenmitochondrien enthalten Kristalle, die aus osmiophilen globulären Einschlußkörpern hervorzugehen scheinen. In den Monaten Juni/Juli ist fast der ganze Zelleib einer Leydigzelle von Fettvakuolen ausgefüllt. Die Zahl der Riesenmitochondrien und Lysosomen verringert sich, das Golgifeld ist wieder unauffällig.Eine Beziehung zwischen der Vermehrung und Entfaltung der Zellorganellen und der in der Literatur angegebenen Phase der gesteigerten Steroidproduktion konnte festgestellt werden. Das Auftreten der Riesenmitochondrien und Lysosomen und ihre Bedeutung für die saisonbedingte Entwicklung und Involution der Leydigzellen wird diskutiert.

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Seasonal changes in the morphology of leydig cells in Rana esculenta

Summary Seasonal changes in the structure of the testicular interstitial cells in Rana esculenta were investigated.Throughout November–February, small mitochondria of the tubular type, visicles of the smooth endoplasmic reticulum and free ribosomes predominate and the cytoplasm contains few lipid droplets and a poorly developed Golgi apparatus. During the period from March to June the cell organelles increase in number. There are numerous aggregated lysosomes and conspicuous accumulations of lipid droplets. Besides small mitochondria increasing numbers of giant mitochondria occur and the Golgi complex is enlarged. The small and giant mitochondria contain crystals which seem to originate from osmiophilic globular inclusion bodies. During June and July the whole cytoplasm may be filled with lipid droplets. Concomitantly there is a decrease in the number of lysosomes and giant mitochondria and the Golgi apparatus is in a state of involution.Possible correlations between the development of the cell organelles and the period of increased steroid production are pointed out. The appearance of giant mitochondria and lysosomes and their importance for seasonal Leydig cell development and involution are discussed.

Auszugsweise vorgetragen auf der 68. Versammlung der Anatomischen Gesellschaft in Lausanne, April 1973. — Herrn Prof. Dr. Dr. E. Horstmann danke ich für die Anregung zu dieser Arbeit und Herrn Prof. Dr. A. F. Holstein für die Durchsicht des Manuskriptes.     

ASYMMETRIC COMPETITION IN MIXED POPULATIONS OF TADPOLES OF THE HYBRIDOGENETIC: RANA ESCULENTA COMPLEX

Hybridogenetic Rana esculenta tadpoles display tolerance to extreme environmental conditions and fit criteria of the “general-purpose” genotype. A trade-off between generality and competitive ability is assumed to occur in asexual species, but the evidence remains unclear. The purpose of my experiment was to test the competitive ability of hemiclonal hybrid Rana esculenta tadpoles relative to the parental species Rana lessonae. Mixed and single genotype populations of R. esculenta and R. lessonae tadpoles were reared at three densities in artificial ponds. Survival of R. esculenta was higher than for R. lessonae tadpoles, but did not differ among densities. Body size at metamorphosis was the same between genotypes, but decreased with increasing density. Larval period was not affected by density, but R. esculenta tended to metamorphose earlier than R. lessonae. Percentage of individuals metamorphosing was higher for R. esculenta at both medium and high densities, but the same as R. lessonae at the low density. The difference in survival, body size, and larval period between tadpoles reared in single and mixed genotype populations was unaffected by genotype or density. The difference in the percentage of metamorphs, however, was strongly affected. The percentage of hybrids metamorphosing was 9% above the responses of single genotype populations at the highest density. Conversely, the percentage of R. lessonae metamorphosing was 12% below the responses of single genotype populations at the same density. Hybrid success in this experiment further supports the criterion of a “general-purpose” genotype without assumptions of reduced competitive ability.     

The role of frogs in the epidemiology of Weil's disease

Summary 1. A high percentage of frog sera (ofRana esculenta andRana fusca) reacts positively with respect to many pathogenic and apathogenic leptospira-strains; this reaction is highest (100%) with respect to the incomplete biotype ofL. icterohaemorrhagiae.2. Under natural conditions frogs do not excrete leptospirae with the urine and neither is this the case, when a single leptospira has somehow succeeded in penetrating into them. This penetration does not easily take place.3. By the inoculation of many virulent leptospirae it is possible to break the congenital immunity. Up till 3 days after the inoculation leptospirae can be detected in the blood, up till 7 days in the liver and kidneys. The frogs did not appear to fall ill.4. Frogs do not play a part in the epidemiology of Weil's disease.5. In normal frog sera precipitation of the agglutinins by means of ammonium sulphate and acetone alcohol could not be observed so definitely as in immune sera. Normal frog serum is thermostabile at 56–57°C., immune serum thermolabile at the same temperature. These differences are explained, in accordance withBordet, by the lesser strength of the antibodies in normal sera.     

Spleen and Liver Pigmented Macrophages of Rana Esculenta L. A New Melanogenic System?

The present study reports the results of a morpho‐functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin‐synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosine‐hydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p‐phenylenediamine‐pyrocatechol (PPD‐PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenol‐oxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue‐specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Annual cycle of activity of the neurocytes of the ventral lateral septum in the brainof Rana esculenta L.∗

Abstract

Sexually mature male and femaleRana esculenta L. were captured in their natural habitat in six phases of the annual cycle. Nuclear volumes of neurocytes in the ventral lateral septum (VLS) were found to fluctuate distinctly in the course of the year. In both sexes nuclear volumes of neurocytes were maximal in the phases preceding the breeding season (3rd decade of January, and 1st decadeof April), and minimal in the phaseat the beginning of hibernation (3rd decade of October). The results lead to the assumption that the VLS ofRana esculenta may have an active role in controlling gonadotropin release.     

Fluoreszenzmikroskopischer Nachweis biogener Monoamine in der Epiphysis cerebri von Rana esculenta und Rana pipiens

Zusammenfassung Die Epiphysis cerebri von Rana pipiens und Rana esculenta wurde fluoreszenzmikroskopisch auf das Vorhandensein bestimmter biogener Amine untersucht. Unter normalen Bedingungen lassen sich im Parenchym der Froschepiphyse keine Amine fluoreszenzmikroskopisch darstellen. Nach Vorbehandlung der Tiere mit dem Monoaminoxydase-Hemmer Nialamid ist aber eine intensive, durch Formaldehyd-Behandlung induzierte Gelbfluoreszenz in den Sinnes- und Stützzellen zu beobachten. Mikrospektrofluorometrische Messungen zeigen, daß das Fluorophor mit 5-Hydroxytryptamin identisch ist, obwohl das Vorkommen von anderen verwandten Indolen — z.B. 5-Hydroxytryptophan und Melatonin — nicht ausgeschlossen werden kann. Grünfluoreszierende adrenerge Nervenfasern sind im meningealen Hüllbindegewebe der Epiphyse zu erkennen; einige dieser Fasern scheinen auch in die Epiphyse einzudringen.Der Nachweis von 5-Hydroxytryptamin wird im Zusammenhang mit der Frage einer Melatoninsynthese diskutiert. Das 5-Hydroxytryptamin könnte außerdem in einer funktionellen Beziehung zu einem bisher noch unbekannten Protein- der Polypeptid-Hormon des Epiphysenparenchyms stehen.

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Fluorescence microscopic studies of biogenic amines in the pineal organ of Rana esculenta and Rana pipiens

Summary The pineal organ of Rana pipiens and Rana esculenta was studied by fluorescence microscopy for the histochemical demonstration of certain biogenic monoamines. Under normal conditions, no fluorogenic amines were visible in the organ. After pretreatment of the animal with a monoamine-oxidase inhibitor, nialamide, an intense yellow formaldehyde-induced fluorescence appeared both in the sensory cells and in the supporting cells. Microspectrofluorometric analysis indicated that the fluorophore is identical with 5-hydroxytryptamine; the presence of other closely related indoles, such as 5-hydroxytryptophan and melatonin, however, cannot be excluded. Fluorescent adrenergic nerves were found in the connective tissues surrounding the pineal organ; fluorescent fibers were observed also in the pineal parenchyma.The presence of 5-hydroxytryptamine in the anuran pineal organ is discussed with regard to the role that the amine plays in melatonin synthesis and with regard to a possible functional relation to some as yet unidentified protein- or polypeptid-hormone within the pineal parenchyma.

Finanzierung eines Studienaufenthaltes an der Universität Lund durch die Deutsche Forschungsgemeinschaft (Forschungsvorhaben — Ok 1/15 — von Prof. Dr. A. Oksche u.a.).     

Formation of desmosomes and other contact specializations in cultured skin of the frog (Rana esculenta)

Summary Small trypsinized explants from ventral skin of frogs (Rana esculenta) were maintained in culture for 4 days during which a newly formed epithelium differentiated along the cut edges of the dermis. During the first 6 h adjacent cells produced numerous interdigitating lamellipodia. After 2 days, epithelial polarity was restored by the formation of zonulae occludentes and the epithelial cells were joined by a few small newly formed desmosomes and by numerous interdigitations. Bipartite junctional complexes consisting of a zonula occludens, followed by a series of typical desmosomes, and characteristic of adult frog epidermis were formed only after 4 days. When cultured in the presence of an inhibitor of protein synthesis (cycloheximide) the trypsinized epidermis no longer formed desmosomes. Therefore pools of one or more crucial desmosomal proteins must be very low or non-existent. However, cycloheximide did not prevent the formation of cell contact specializations, consisting of a highly developed system of complex lamellar interdigitations, between adjacent cells.     

Immunocytochemical localization of Vitamin A in the retina and pineal organ of the frog,Rana esculenta

Summary Vitamin A immunoreactive sites were studied in the retina and pincal organ of the frog,Rana esculenta, by the peroxidase antiperoxidase, avidin-biotinperoxidase and immunogold methods. Indark-adapted material, strong immunoreaction was found in the outer and inner segments of the photoreceptor cells of both retina and pineal organ, as well as in the pigment epithelium, retinal Müller cells and pineal ependymal cells. Inlight-adapted retina, cones and green (blue-sensitive) rods were immunopositive.At the electron microscopic level, immunogold particles were found on the membranes of the photoreceptor outer segments as well as on the membranes of the endoplasmic reticulum and mitochondria. Individual retinal photoreceptor cells exhibited strong immunoreaction in the distal portion of the inner segment, the ciliary connecting piece and the electron-dense material covering the outer segment. In the pigment epithelium, the immunolabeling varied in intensity in the basal and apical cytoplasm and phagocytosed outer segments.The immunocytochemical results indicate that retinoids (retinal, retinol and possibly retinoic acid) are present not only in the photoreceptor cells of the retina but also in those of the pineal organ. The light-dependent differences in the immunoreactivity of vitamin A underlines its essential role in the visual cycle of the photopigments. Our results suggest that the pineal ependyma plays a role comparable to that of the Müller cells and pigment epithelium of the retina with regard to the transport and storage of vitamin A. The presence of a retinoid in nuclei, mitochondria and cytoplasmic membranes suggests an additional role of vitamin A in other metabolic processes.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Hungarian OTKA grant Nr. 1619 to B.V., and a grant from the Pardee Foundation to G.H.W.     

Selection of a high and stable pigment-producing strain in cultured Euphorbia millii cells

Summary A high pigment-producing strain of cultured Euphorbia millii cells was isolated by clonal selection. The pigment obtained was red and consisted mainly of anthocyanin. The amount of this pigment obtained after 24 selections was seven times that found in the original cells. Statistical and cell-pedigree analyses proved that this cell strain has stable productivity for this red pigment.     

Food supply modifies the trade-off between past and future reproduction in a sexual parasite–host system (<Emphasis Type="Italic">Rana esculenta,Rana lessonae</Emphasis>)

Life history theory is concerned with the costs of survival, growth and reproduction under different ecological conditions and the allocation of resources to meet these costs. Typical approaches used to address these topics include manipulation of food resources, followed by measures of subsequent reproductive traits, and measures of the relationship between current and future reproductive investment. Rarely, however, do studies test for the interaction of past investment, present resource availability and future investment simultaneously. Here, we investigate this interaction in females of a sexual parasite–host system consisting of the hybridogenetic frog Rana esculenta (E) and one of its parental species Rana lessonae (L). We kept females from each of two groups (with or without previous reproduction) under two food treatments (low or high) and regularly recorded their growth as well as their body condition and hormone titres as measures of future reproductive condition. After keeping them in hibernation until the following spring, we exposed the females to males, recorded whether they spawned or not and related this response to their condition in the previous autumn. Past reproduction negatively affected growth during summer and condition during autumn which, in turn, reduced the following year’s reproductive output. These costs of previous reproduction were less pronounced under the high than under the low food treatment and lower in R. lessonae than in R. esculenta. Increasing food supply improved reproductive condition more in L than in E females. These species differences in reproductive costs and food requirements provide a mechanistic explanation for why E females skip annual reproduction almost twice as often as L females. Since R. esculenta is a sexual parasite that depends on R. lessonae for successful reproduction, these species-specific life history patterns not only affect individual fitness but also the spatial structure and temporal dynamics of mixed LE populations.