In vitro synthesis of peritrophic membranes of the blowfly, Calliphora erythrocephala.

Tyrode solution containing added glutamine and Leloup's medium 1 has been used as a basic medium for the in vitro culture of the so-called proventriculus of adult Calliphora erythrocephala to elucidate some of the factors controlling the synthesis of peritrophic membranes (PM) in vitro. The formation rate was chosen as a quantitative criterion for the evaluation of the modifications of the incubation media.After systematic variation of osmolarity, pH, and temperature optimal formation rates were obtained in media with an osmolarity of 320 to 360 mOsmol, a pH of 6·8, and an incubation temperature of 27°C. Under these conditions the average rate of formation was in the modified Tyrode solution 3·0±1·1 mm PM/hr, and in Leloup's medium 3·6±0·8 mm PM/hr. In the modified Tyrode solution the formation of PM was complete after 5 to 7 hr, whereas in Leloup's medium it continued up to 24 hr. The addition of β-ecdysone caused an increase of the formation rate of PM to 4·5 to 5·5 mm PM/hr.The results obtained led to the hypothesis that an osmotically regulated enzyme system could be the limiting factor of the formation rate of peritrophic membranes, i.e. a system which could regulate the internal osmolarity of the formative cells by the interconversion of a bulk polymer and its monomer which are needed for the synthesis of PM.     

The fine structure of peritrophic membranes of the blowfly, Calliphora erythrocephala, grown in vitro under different conditions.

The formation of peritrophic membranes (PM) in vitro was studied in a flow chamber in order to avoid the accumulation of metabolic substances during prolonged incubation. During the first 8 to 10 hr of incubation the production of PM was nearly constant—3·5 ± 1·4 mm PM/hr. After about 10 hr it decreased and stopped after about 35 hr. Between 1 and 8 hr after the beginning of incubation the width of the periodic crossband pattern reached or nearly reached the values found in PM which had formed in vivo; afterwards it decreased more and more. During the first 20 min of incubation a ‘disturbed zone’ of PM without any regular crossband pattern is formed.In the cardia of adult Calliphora erythrocephala there are three formation zones forming three PM of different fine structures. The fine structure of PM 1 to 3 formed in vitro during the first 6 to 8 hr of incubation in Leloup's medium 1, with an osmolarity of 340 mOsmol, a pH of 6·8, and a temperature of 27°C, does not differ from the PM grown in vivo. PM 1–3 grown in vitro in Tyrode's solution with added glutamine or in Leloup's medium with added β-ecdysone show a considerable increase in thickness and a disturbed formation of the electron dense layer of PM 1.     

The neurosecretory system of the adult Calliphora erythrocephala

Summary The histology of the corpus cardiacum (c. card.) and the hypocerebral ganglion of Calliphora has been described from sections mainly stained with paraldehyde-fuchsin (PAF) and counterstained with Halmi's mixture. Concurrently the nervous connections of these organs with the neurosecretory system and the stomatogastric nervous system were studied.Neurosecretory material from the medial neurosecretory cells of the brain (m.n.c.) could be traced through the cardiac-recurrent nerve, and passing through the c. card. it was seen to be abundantly present in the wall of the aorta and the two pairs of nerves leaving the c. card.-hypocerebral ganglion complex posteriorly, i.e. the aortic and the oesophageal nerves. However, in some old, fed flies a considerable amount of neurosecretory material was also observed in anastomosing branches of the cardiac-recurrent nerve inside the c. card. Thus storage of neurosecretory material originating in the m.n.c. may take place both in the aorta wall and in the c. card. This observation is relevant to the interpretation of previous experiments of E. Thomsen (1952).The c. card. cells proper (the c.n.c.) were not stained by the PAF, although they are known to be neurosecretory.This work was supported by grants from the Carlsberg Foundation. I am grateful to Professor C. Overgaard Nielsen for laboratory facilities.     

The neurosecretory system of the adult Calliphora erythrocephala

Summary Neurosecretory cells in the brain and suboesophageal ganglion of the adult female Calliphora erythrocephala have been studied in the light microscope. The paraldehydefuchsin stain (PAF) gave by far the clearest pictures.The medial neurosecretory cells of the protocerebrum (m.n.c.) show definite cyclic changes as to the size of the nuclei and the content of secretory material. The cytological changes depend on the age of the fly and the diet given and are correlated with ovarian development.The nuclear size is held to express the metabolic activity of the cells. Cells with large nuclei, as found in young sugar-flies (S1D) and meat-fed flies with developing ovaries (S4D/M2D), contain less secretory material which is released through the axons, while the m.n.c. of old sugar-flies (S6D) have small nuclei and are stuffed with secretory material which is stored in the perikarya.These results confirm those obtained by darkfield microscopy of living m.n.c. by Lea and E.Thomsen (1962 and unpublished).No really convincing evidence for the existence of more than one type of m.n.c. was found.Two small groups of lateral cells were observed. Possibly neurosecretory are further: 1-(2) cells at the base of each optic lobe, two groups of 2–3 cells on the caudal side of the brain, and 2 cells ventrally in the suboesophageal ganglion.Giant neurons of unknown function are situated very near the m.n.c. Their axons join those from the m.n.c., but end in the suboesophageal ganglion.The same region comprises a number of peculiar cells, each containing a large, fluid-filled vacuole (the vacuolated cells). Similar cells are associated with the possibly neurosecretory cells on the caudal side of the brain.My sincere thanks are due to my wife Dr. Ellen Thomsen, who made all the excisions of the brains and the measurements of the nuclei, to T.C.Normann for valuable assistance with the photographic work, and to Mrs. K. Bahnert for technical help with the gallocyanin method. The Carlsberg Foundation has supported the work with grants.     

The neurosecretory system of the adult Calliphora erythrocephala

Summary In continuation of previous light microscopical investigations using darkfield microscopy of the living cells and sections stained with paraldehyde-fuchsin, an electron microscopical study of the medial neurosecretory cells (m.n.c.) of virgin females of Calliphora has been performed. The neurosecretory material consists of elementary granules corresponding in quantity to the amount of secretory material found by the two other methods in flies of the same age and kept on the same diet. The majority of the cells (m.n.c. I) contain granules measuring c. 2000–3000 Å, while fewer cells (m.n.c. II) show a smaller granular diameter (c. 1000–1500 Å). Due to the Tyndall effect the elementary granules are visible when using darkfield microscopy.The granules were seen to be pinched off from the Golgi complexes. These are numerous and well-developed, except in the less active m.n.c. I of the six days old sugar-flies. The reticulum and mitochondria are described. Axoplasmic channels were observed in the m.n.c. I, probably corresponding to structures found by Wigglesworth (1959 and 1960) in other insect neurons with another technique.The fine structure of the giant neurons and the vacuolated cells has been studied, the observations supporting the conclusions of M. Thomsen in a light microscopical study (1965). Lacunae in the ramifying glia are interpreted as belonging to the glial lacunar system described by Wigglesworth (1960).Dedicated to the memory of Ernst Scharrer (1905–1965), pioneer in the study of neurosecretion.We are grateful to the Carlsberg Foundation and the State General Scientific Foundation for financial support.     

Salivary gland development in the blowfly,Calliphora erythrocephala

The salivary gland of adult Calliphora erythrocephala is a tubular structure composed of secretory, reabsorptive, and duct regions. Development of these structures has been followed during the six days of larval and ten days of pupal growth. Two small groups of imaginal cells located at the junction between larval gland and duct give rise to the adult gland. These presumptive adult cells divide during all larval stages and appear to be functional components of the larval gland. Shortly after pupation, the larval gland breaks down and the imaginal cells proliferate rapidly, forming sequentially the duct, reabsorptive and secretory regions. Proliferating regions of the developing gland are frequently encrusted with haemocytes. As it elongates the gland establishes intimate contacts first with the basement membrane of the degenerating larval gland, later with an epithelial layer surrounding the main dorsal tracheal trunks, and then with the gut. Cell division continues until about five days after pupation, bu t the gland is unable to secrete fluid in response to 5-hydroxytryptamine stimulation until two hours after the adult fly emerges. The Golgi complex appears to be involved in forming the highly folded membranes of the canaliculi in the secretory region. Presumptive adult salivary gland cells appear to increase in number logarithmically from the time of hatching of the larva until five days after pupation. This contrasts with the development of classical imaginal discs, in which cell division ceases prior to pupation.     

Rhythmische Erregungen in den optischen Zentren von Calliphora erythrocephala

Zusammenfassung Die rhythmischen Aktionspotentiale in den optischen Ganglien der Schmeißfliege (Calliphora erythrocephala) werden untersucht.Wird das Komplexauge von Calliphora belichtet, so können vom Ganglion opticum II schnelle, rhythmische Aktionspotentiale, 'Belichtungsrhythme , abgegriffen werden (Abb. 1). Sie treten im Bereich physiologischer Temperaturen und Lichtintensitäten stets und unabhängig von Schädigungen auf. Sie sind die einzige Form von Erregung, die zwischen dem retinalen Bereich und dem Cerebralganglion nachgewiesen werden kann. Die Belichtungsrhythmen zeigen gesetzmäßige Abhängigkeiten von den Reizgrößen. Es ist daher wahrscheinlich, daß sie in die Kausalkette der bei Belichtung des Auges ablaufenden zentralen Vorgänge eingeschaltet sind.Die optischen Ganglien werden mit einer Doppelmikroelektrode abgetastet. Da die Spannung zwischen zwei eng benachbarten Elektroden in der Nähe der Spannungsquelle am größten sein muß, kann gezeigt werden, daß die Belichtungsrhythmen wahrscheinlich in der äußeren Körnerschicht des Ganglion opticum II entstehen (Abb. 14 und 15).Als Maß für die Größe der Belichtungsrhythmen wird die größte während einer Belichtung auftretende Amplitude gewählt, die 'Maximalamplitud ; sie hängt stetig und reproduzierbar von der Zahl belichteter Ommatidien, von der Lichtintensität und vom Adaptationszustand des Auges ab (Abb. 5, 6, 7, 8, 10, 11 und 12).Die Amplituden der Belichtungsrhythmen klingen bei längerer Belichtung allmählich ab (Helladaptation), (Abb. 1C, Abb. 5). Die Heiladaptationszeit ist der Maximalamplitude proportional (Abb. 6, 8, 9 und 10). Wird die Belichtung vor dem völligen Abklingen der Rhythmen unterbrochen, so werden sie durch den Aus-Effekt des Retinogramms gehemmt und brechen sofort und vollkommen ab (Abb. 1 D). Die Dunkeladaptation ist selbst nach vorangegangener Belichtung mit sehr hohen Lichtintensitäten nach spätestens einer Minute abgeschlossen (Abb. 6 und 7).Die Frequenz der Belichtungsrhythmen liegt zwischen 100 sec^–1 und 250 sec^–1, sie nimmt mit steigender Temperatur zu (Tabelle 1). Die Frequenz ist unabhängig von der Lichtintensität, vom Adaptationszustand d von der Zahl belichteter Ommatidien.Während der einzelnen Belichtung zeigen die Rhythmen ein verschieden starkes Schwanken der Amplitude, eine Amplitudenmodulation. Die Modulation hängt vom Präparat und vom Präparationszustand ab.Durch den Vergleich der verschiedenen Modulationstypen und durch gleichzeitige Ableitung an mehreren Stellen des Ganglions können die physikalischen Überlagerungsvorgänge untersucht werden. Die Einzelschwingungen physiologischer Einheiten überlagern sich am gemeinsamen Ableitwiderstand zwischen den Elektroden. Durch die Art der Überlagerung wird die Modulationsform bestimmt. Sie hängt im besonderen von der Frequenz und der Phasenlage der Einzelrhythmen und von physiologischen Synchronisationsvorgängen ab (Abb. 1, 2 und 16).Auch wenn ein Bereich der Retina gereizt wird, der nur wenige Sinneszellen umfaßt, treten Belichtungsrhythmen wie bei großen Reizflächen auf (Abb. 12). Deshalb wird die Möglichkeit diskutiert, daß bereits die kleinste physiologische Einheit im Ganglion mit rhythmischer Erregung antwortet, die in ihrer Amplitude, nicht aber in ihrer Frequenz vom Reiz abhängt.Herrn Prof. Dr. H. Autrum danke ich für das stete Interesse, das er den Untersuchungen entgegengebracht hat. Die Untersuchungen wurden zum Teil mit Apparaten durchgeführt, die die Deutsche Forschungsgemeinschaft Herrn Prof. Autrum zur Verfügung stellte.     

Extracellular ribosomes during metamorphosis in the blowfly Calliphora erythrocephala

During metamorphosis of the blowfly Calliphora erythrocephala extracellular ribosomes, in the form of monosomes, appear in the body fluid. The total number of ribosomes, i.e. intracellular and extracellular, remains approximately constant during this period, whereas the proportion of extracellular ribosomes first rises, plateaus, and then declines in a manner suggesting that their appearance is a result of larval tissue breakdown.