Franz Romer

Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro

Abstract Isolated prothoracic glands from Tenebrio larvae synthesize in vitro α-ecdysone, but not β-ecdysone from 4-14C-cholesterol. Isolated abdominal oenocytes from the larvae synthesize mainly β-ecdysone, but only little α-ecdysone. When prothoracic glands and oenocytes are cultured together, the α-ecdysone derived from the prothoracic glands is oxidized by the oenocytes to β-ecdysone. The newly synthesized hormones are not stored in the cells, but are secreted into the medium if sufficient amounts of non-labelled hormones are present. If no unlabelled hormones are added to the culture medium, the newly formed hormones are converted to a large extent into polar conjugates.

Structure and function of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas (Insecta, Heteroptera)

Summary1.Active prothoracic glands and oenocytes of last larval stage are both characteristized by well-developed smooth and rough endoplasmic reticulum (ER). Prothoracic glands also show plasma membrane infoldings, but not oenocytes which contain a large number of pleomorphic vesicles.2.The fine structure of embryonic oenocytes corresponds after blastokinesis with that of active larval and adult cells. Thus, an activity in the late embryo can be assumed. Embryonic prothoracic glands reveal no signs of activity: smooth and rough ER are absent. The subcellular structure resembles that of organ anlagen, i.e. not yet fully differentiated tissue. Hormone synthesis is not likely.3.Ecdysone titer was determined throughout embryonic development and in mature adults. Although prothoracic glands break down during adult ecdysis, imagines contain in the Calliphora-bioassay active factors: females 0.9 CU/g and males 0.5 CU/g. As sites of synthesis the oenocytes are suggested.4.A relatively high ecdysone titer of 7 CU/g is measured in newly deposited eggs. The hormone is presumably of maternal origin. Subsequent to blastokinesis the hormone content increases dramatically up to about 180 CU/g, apparently due to endocrine function of the embryo. Oenocytes are proposed as the source of ecdysone during late embryonic development.5.The function of ecdysone during early and advanced embryogenesis, especially in view of “embryonic molts”, is discussed.

Cuticle: Formation, Moulting and Control

The relative rigidity of the arthropod exoskeleton makes it impossible for body size to increase continuously during the postembryonic development of these animals. Once they have hatched from the egg, they grow in steps, passing through a variable number of (larval) stages (Fig. 1 a). Apart from a few exceptions, there are between 3 and 10 such stages in the arachnids, 3–20 in the crustaceans, and 3–10 in the insects. In many cases a metamorphosis stage intervenes (some crustaceans; holometabolous insects) (Fig. 9b, c).

Die Prothorakaldrüsen der Larve vonTenebrio molitor L. (Tenebrionidae, Coleoptera) und ihre Veränderungen während eines Häutungszyklus

Summary1.The larval ecdysial glands ofTenebrio consist of two cords along the dorsal tracheal trunks and their ramifications in the prothorax.2.Glycogen, cholesterol and lipid droplets could be demonstrated histochemically in the glandular cells. The absorption of cholesterol by the ecdysial glands was demonstrated in tests with tritium labeled cholesterol dissolved in lipid droplets.3.By staining with methylene blue an innervation of the prothoracic glands by the suboesophageal and the prothoracic ganglion was demonstrated. Neurosecretory granules were found electronmicroscopically in axons which pass through the basement membrane and may indent the gland cells.4.Extracts from isolated ecdysial glands of prepupae or moulting larvae, extracted by the method of Karlson and Shaaya, showed hormone activity in theCalliphora test.5.Immediately after moulting the ecdysial glands have a distinct rough surfaced vesicular endoplasmic reticulum (RVER). Glycogen is deposited in vacuoles derived from vesicles. When the membranes of the vacuoles break down, lipid droplets migrate into the glycogen areas and seem to resorb a part of them. Thereafter, the lipid droplets are absorbed by the ER which is roughsurfaced or smooth. In connection with the ER there are extended Golgi areas near the nucleus or the basal infoldings, mainly during the second part of the moulting cycle.6.It is suggested that cholesterol dissolved in lipid droplets enters the gland cells and is then converted to ecdysone by enzymes localized in mitochondria and ER. The hormone may be extruded by Golgi vesicles.Zusammenfassung1.Die Ecdysialdrüsen der Larve vonTenebrio liegen in 2 Bändern entlang der dorsalen Tracheenäste und deren Verzweigungen im Prothorax.2.Mit histochemischen Methoden konnten in den Drüsenzellen Glykogen, Cholesterin und Lipidtröpfchen nachgewiesen werden. Versuche mit tritiummarkiertem Cholesterin zeigten, daß dieses mit den Lipidtröpfchen in die Zellen der Prothorakaldrüsen gelangt.3.Mit Hilfe der Methylenblaufärbung wurde eine Innervation der Prothorakaldrüsen nachgewiesen, die vom Unterschlund- und vom Prothoraxganglion ausgeht. Elektronenmikroskopisch konnten Neurosekretgranula in Axonen, die in die Basalmembran und z. T. durch die Drüsenzellen selbst ziehen, nachgewiesen werden.4.Aus isolierten Prothoraxdrüsen von Vorpuppen und Häutungstieren wurden nach der Methode von Karlson und Shaaya Extrakte hergestellt, die imCalliphoratest positiv waren.5.Kurz nach der Häutung zeigen die Prothorakaldrüsen ein sehr ausgeprägtes rauhes vesikuläres ER (RVER). In Vakuolen, die aus Vesikeln entstanden sind, wird Glykogen eingelagert. Während sich die Vakuolenmembranen auflösen, wandern in die Glykogenfelder Lipidvesikel ein, die wahrscheinlich einen Teil des Glykogens aufnehmen. Im Anschluß daran geht der Inhalt der Lipidvesikel ins ER, das teils ribosomenbesetzt, teils agranulär ist. In Verbindung zum ER stehen sehr aktive. Golgivesikel, die z.T. in der Nähe des Kerns, z.T. in der Nähe der basalen Zellmembraneinfaltungen liegen. Sie sind insbesondere in der 2. Hälfte des Häutungszyklus sehr ausgeprägt.6.Es wird vermutet, daß das Cholesterin mit den Lipidtropfen in die Prothorakaldrüsen gelangt und dort von Enzymen der Mitoehondrien und des ER in Ecdyson umgewandelt wird. Das Hormon dürfte dann durch die Golgibläschen ausgeschleust werden.1. Die Ecdysialdrusen der Larve vonTenebrio liegen in 2 Bandern entlang der dorsalen Tracheenaste und deren Verzweigungen im Prothorax. 2. Mit histochemischen Methoden konnten in den Drusenzellen Glykogen, Cholesterin und Lipidtropfchen nachgewiesen werden. Versuche mit tritiummarkiertem Cholesterin zeigten, das dieses mit den Lipidtropfchen in die Zellen der Prothorakaldrusen gelangt. 3. Mit Hilfe der Methylenblaufarbung wurde eine Innervation der Prothorakaldrusen nachgewiesen, die vom Unterschlund- und vom Prothoraxganglion ausgeht. Elektronenmikroskopisch konnten Neurosekretgranula in Axonen, die in die Basalmembran und z. T. durch die Drusenzellen selbst ziehen, nachgewiesen werden. 4. Aus isolierten Prothoraxdrusen von Vorpuppen und Hautungstieren wurden nach der Methode von Karlson und Shaaya Extrakte hergestellt, die imCalliphoratest positiv waren. 5. Kurz nach der Hautung zeigen die Prothorakaldrusen ein sehr ausgepragtes rauhes vesikulares ER (RVER). In Vakuolen, die aus Vesikeln entstanden sind, wird Glykogen eingelagert. Wahrend sich die Vakuolenmembranen auflosen, wandern in die Glykogenfelder Lipidvesikel ein, die wahrscheinlich einen Teil des Glykogens aufnehmen. Im Anschlus daran geht der Inhalt der Lipidvesikel ins ER, das teils ribosomenbesetzt, teils agranular ist. In Verbindung zum ER stehen sehr aktive. Golgivesikel, die z.T. in der Nahe des Kerns, z.T. in der Nahe der basalen Zellmembraneinfaltungen liegen. Sie sind insbesondere in der 2. Halfte des Hautungszyklus sehr ausgepragt. 6. Es wird vermutet, das das Cholesterin mit den Lipidtropfen in die Prothorakaldrusen gelangt und dort von Enzymen der Mitoehondrien und des ER in Ecdyson umgewandelt wird. Das Hormon durfte dann durch die Golgiblaschen ausgeschleust werden.

Morphogenesis of mechanoreceptor and epidermal cells of crickets during the last instar, and its relation to molting-hormone level

Summary(1) The fine structure of the cercal campaniform sensilla and epidermal cells of Gryllus bimaculatus Deg. (Saltatoria, Gryllidae) was examined, and the ecdysteroid level was monitored throughout the last larval instar. (2) The epidermal cells show changes in shape, cytoplasmic inclusions and differentiation of the apical cell membrane, coupled to the phases of buildup and breakdown of the (cercus) cuticle. (3) The imaginal epicuticle of the epidermal cells begins to form later (by about approximately 6 h) than that of the campaniform sensilla. (4) The campaniform sensilla were studied with respect to (a) the morphogenesis of the cuticular apparatus, (b) the inclusion of phenol oxidases in the cuticular apparatus, and (c) changes in the sensory apparatus preparatory to molting. (5) After apolysis the folding of the tormogen-cell wall into microvilli transiently disappears. Microvilli re-form shortly before imaginai ecdysis, and at the same time an outer receptor-lymph space develops. The role of the tormogen-cell “plaques” is discussed. (6) The levels of α- and β-ecdysone were determined separately by radioimmunoassay. (7) At the beginning of the instar the hormone level, especially that of β-ecdysone, falls. Prior to apolysis, the concentration of α-ecdysone rises, reaching an intermediate peak after apolysis is complete. The maximum hormone concentration (approximately 2,000 ng/g) is reached after the cuticulin layer is deposited, primarily due to the increase in β-ecdysone. While the proecdysial cuticle is forming, the hormone titer is reduced; at this time β-ecdysone is its chief component. (8) The identification of the ecdysteroids monitored by radioimmunoassay was confirmed by gas chromatography.

Ultrastructural changes of the oenocytes of Gryllus bimaculatus DEG (Saltatoria, Insecta) during the moulting cycle

Summary1.The oenocytes of Gryllus bimaculatus are characterized by an abundant smooth-surfaced ER (ATER). In spite of the great cell size the plasma membrane never shows extensive infoldings during the moulting cycle. In addition to mitochondria there are very large numbers of microbodies containing peroxidase but apparently not uricase. Within the second part of the instar the microbodies lie along the clefts which run through the whole cell.2.The following changes are observed in the course of a moulting cycle: Immediately after hatching the ATER is scarcely developed, some liposomes are located within areas of ATER disappearing some hours later. 20 hours after emergence glycogen deposits appear in two forms: large deposits reaching some μm in diameter, and in distinct rosettes dispersed between the tubules of the ATER. 30 hours post-moult profiles of RER appear, which disappear together with the large glycogen deposits one day later. At the same time ATER is increased and the clefts develop within areas of elongate granules smaller than ribosomes. The number of such clefts subsequently becomes reduced probably as a result of confluence. Towards the end of the moulting cycle numerous autophagosomes appear. These digest parts of the agranular reticulum, many microbodies, and to a lesser extent, mitochondria. Residual bodies are extruded during moult whereas the clefts remain.3.The ultrastructural features parallel those of steroid-producing cells in vertebrates. Besides this it is possible that oenocytes also engage in detoxication processes as shown for vertebrate liver.

Arachnid oenocytes: Ecdysone synthesis in the legs of harvestmen (Opilionidae)

SummaryCells measuring up to 130 μm have been found in the proximal segments of the femora of all four pairs of walking legs in various species of harvestmen (Phalangium opilio, Leiobonum limbatum, Opilio parietinus, and Opilio ravennae). These cells exhibit all the fine-structural characteristics of insect oenocytes, in particular the conspicuous agranular endoplasmic reticulum. Radioimmunoassay after in vitro incubation of these cells has demonstrated the synthesis of α- and β- ecdysone. These ecdysteroids have been found in the ovaries and tergites of the opisthosoma as well as in the oenocytes.

Metabolism of 3H-α-ecdysone in Gryllus bimaculatus during the eighth larval instar

Abstract During the 8th larval instar of Gryllus bimaculatus the metabolites of exogenous tritiumlabelled α-ecdysone were isolated from animals and from excrement, qualified by TLC and quantified in the scintillation counter. Besides β-ecdysone, there were 3 other compounds more polar than α-ecdysone (compounds I, II, and III), in addition to two less polar ones: compounds IV and V. The pattern of hormone changes characteristically during the period of development investigated. Thus on the 3rd day compound IV, on the 4th day α-ecdysone, and on the 5th day, at the moment of the hormone titre maximum, β-ecdysone dominates. Whether all ecdysone metabolites represent products of inactivation will be discussed in this article.

Der metabolismus von 3H-α-ecdyson bei larven von Tenebrio molitor

Abstract The metabolism of 3H-α-ecdysone has been investigated in larvae of Tenebrio molitor within a moulting cycle. The metabolization occurs by hydroxylation, dehydration, and conjugation. A total of 8 metabolites including α- and β-ecdysone were found. Peak I was cleaved enzymatically up to 56%. The hydrolized part consists of esters of sulfuric- and glucuronic acid with compounds II and III, also with α-ecdysone and 3-dehydroecdysterone to a lesser degree. The hydroxylation of α-ecdysone to β-ecdysone is a very rapid one. Velocity and intensity depends on the physiological stage of the larvae. On days with a large endogenous hormone titer they are maximal and on days with a low endogenous titer they are lower. The rate of conversion of α- to β-ecdysone shows a correlation with the endogenous titer and vice versa. At a low endogenous titer the rate of excretion is very strong, in larvae aged to 2 to 5 days and also 7 to 8 days, 8 h after injection 50 to 80% of injected activity was recovered in the faeces. At the beginning of the moulting cycle 3% and at the 6th day 30% (hormone titer maximum) of activity is excreted. The unexpected high rate of excretion at the first day is compensated by increasing metabolism and storage. The faeces mainly consist of conjugates and α- and β-ecdysone. A correlation between the appearance of these compounds and the physiological stage of the larvae could be found. On those days with low endogenous titer the quota of ecdysone (α and β) is higher.

Morphologie und ultrastruktur der larvalen oenocyten von Tenebrio molitor L. (Insecta, Coleoptera) bei larve, puppe und imago

Summary1.The oenocytes of the larval generation of Tenebrio stick to the lateral tracheal trunks in a narrow chord. They do not increase their number during post-embryonic development, and therefore become polyploid. They are still present in 8-day-old adults; thus a degeneration within metamorphosis does not occur.2.The following ultrastructural features are typical of oenocytes: a well developed ATER which allows only little room for the cytoplasma between the tubuli. Mitochondria are very numerous, but derived ones could not be observed. There are several free ribosomes sometimes adjoining endoplasmic reticulum to give rough surfaced profiles of ER. A most striking characteristic of the oenocytes of Tenebrio is the great amount of lipofuscin granules which reach their maximum 2 days after molting and are thereafter reduced. At least in the pupa an accumulation of granules take place. One way of degradation runs form scattering to a final exocytosis through the numerous enfoldings of the basal cell membrane. Distinct Golgi areas could not be found within the larval period, but were observed within the pupa and adult period, though very seldom. Acid phosphatases for the destruction of cell particles in autophagic vacuoles become organized in microbody-like structures directly in the tubuli of the ATER, and are transported to the vacuoles.3.In contrast to other steroid hormone-producing cells there is no synchronization evident within the molting cycle. Also lipid droplets and glycogen rosetts, as a rule characteristics of a series of activations, are not to be observed. The only sign of cell activation is the occurrence of rough surfaced ER-profiles following a reduction of volume. A striking parallel exists between the titer of molting hormones and the number of vacuolated cells induced by a hypoosmolar fixative.Zusammenfassung1.Die larvalen Oenocyten von Tenebrio liegen den lateralen Tracheerlängsstämmen in einem schmalen Bändchen auf. Sie vermehren ihre Zahl während der postembryonalen Entwicklung nicht, wachsen also durch Endopolyploidie. In 8 Tage alten Imagines sind sic immer noch vorhanden; sie werden also im Zuge der Metamorphose nicht abgebaut.2.In ihrer Ultrastruktur zeigen sie die für Oenocyten typischen Merkmale: ein sehr kräftig ausgebildetes ATER, das zwischen den Tubuli nur ganz wenig Platz für das Grundcytoplasma haft. Die Mitochondriendichte ist relativ hoch, spezialisierte Mitochondrien kommen nicht vor. Ribosomen treten hauptsächlich als freie Ribosomen auf, daneben auch mit dem ER verbunden zu kleinen Profilen mit rauhem ER. Hervorstechendes Merkmal der Oenocyten von Tenebrio ist die große Anzahl von Lipofuscingranula, die am 2. Tag nach der Häutung ihr Maximum erreichen und danach reduziert werden. Zum mindesten in der Puppe, bei der eine vorübergehende Akkumulation der Granula stattfindet, geht ein Weg des Abbaus über eine Zerstäubung mit anschließender Exocytose durch die zahlreich vorhandenen Einfaltungen der basalen Zellmembran. Ausgeprägte Golgiregionen wurden während der Larvalperiode nicht gefunden, jedoch vereinzelt bei der Puppe and bei der Imago. Die sauren Phosphatasen zum Abbau der zelleigenen Bestandteile im Rahmen der Autophagie werden, soweit beobachtet, direkt am ATER in microbody-ähnlichen Strukturen abgepackt and zu den autophagen Vakuolen transportiert.3.Bei einem Vergleich mit anderen Steroidhormon-bildenden Zellen fällt auf, daß a) keine klare Synchronisation innerhalb des Häutungszyklus festzustellen ist and b) die während der Aktivierungsfolge typischerweise vorhandenen Lipidtropfen and Glykogengranula nicht zu beobachten sind. Als einziges Zeichen einer Zellaktivierung ist das Auftreten von Profilen mit rauhem ER zu werden, das meist im Anschluß an eine vorausgegangene Reduktion des Volumens anknüpft. Eine deutliche Parallelität ist auch zwischen der in Homogenaten erschlossenen Häutungshormonmenge zur Zahl von vakuolisierten d.h. osmotisch aktiven Zellen nach hypoosmolarer Fixierung zu beobachten.