Effects of polyoxin D on in vitro synthesis of peritrophic membranes in Calliphora erythrocephala

The synthesis of peritrophic membranes in vitro in adult Calliphora erythrocephala is affected strongly by polyoxin D, an inhibitor of chitin synthesis. Depending on the polyoxin concentrations used, a reduction in membrane dry weight, an inhibition of chitin synthesis, and prominent changes in the fine structure of the membranes were found.     

Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)

For the first time a sugar receptor (lectin) has been localized by electron microscopy in an invertebrate. The peritrophic membrane of the blowfly larva, Calliphora erythrocephala, is shown here to express lectins with high specificity for mannose. The lectin is restricted to the lumen side of the peritrophic membrane. The surface of the midgut epithelium is devoid of mannose-specific lectins. It is suggested that the midgut epithelium has lost these lectins during the course of evolution in favour of the peritrophic membrane which is secreted by specialized cells only at the beginning of the midgut.Peritrophic membranes and the midgut epithelium lack lectins specific for galactose. The lumen side of the peritrophic membrane of the larvae has mannose and/or glucose residues, and it is densely packed with two species of bacteria, Proteus vulgaris and P. morganii. These also have mannose-specific lectins as well as mannose residues on their pili. The existence of mannose-specific receptors and mannose residues on both, peritrophic membranes and bacteria, leads to the assumption of mutual adherence between the two surfaces.     

Anisotropic cross-bands in peritrophic membranes of Diptera

In agreement with previous findings in adult Calliphora erythrocephala a periodic incorporation of glucose-¹⁴C and methionine-S35 has been observed into the peritrophic membranes of C. erythrocephala and Lucilia sericata larvae as well as in peritrophic membranes of adult L. sericata and Musca domestica. The width of the periodic units (=labelled band + unlabelled interval) is of the order of 50 to 80 μm.After staining with the dichroitic dye Congo red, peritrophic membranes exhibit a periodic pattern of anisotropic bands in polarized light, resembling the pattern in autoradiographs. If stained membranes are rotated between crossed polars, every 90° the extinction is shifted from a ‘band’ into an ‘interval’ and vice versa, indicating that in these peritrophic membranes two anisotropic sections are arranged perpendicular to each other in series. There are no differences between band patterns of males and females.A relation between width of the bands and the diameter of the peritrophic membranes seems to be possible, but since correlation factors are only of the order of 0·83 it is not significant. Besides the macrounits (band+interval) with a width of about 50 to 80 μm, smaller units with a width of 10 to 14 μm have been detected only in the peritrophic membranes of adult C. erythrocephala and L. sericata, as well as in the inner membranes of adult M. domestica. The anisotropic structure of these microbands is arranged about 45° with respect to that in macrobands.Peritrophic membranes of a blackfly larva, Odagmia ornata, after staining with Congo red show in polarized light a complicated zig-zag pattern of periodic units with a width of 15 to 20 μm, or patches with a regular orthogonal network.     

SDS gel electrophoresis of proteins and glycoproteins from peritrophic membranes of some Diptera

Peritrophic membranes (PM) of larval and adult Calliphora erythrocephala and Sarcophaga barbata contain proteins and glycoproteins which were extracted by 2.5% SDS and 8 M urea from the matrix. The acid mucopolysaccharide moiety of PM which was demonstrated by the carbazole method remained in the insoluble resudues. After SDS electrophoresis the gels were counterstained with PAS and Coomassie blue; the carbohydrate and protein content of the bands were recorded by dual scanning. Besides molecular weight (MW) determination from the migration rate, the MW of some glycoprotein bands of PM were evaluated also from their retardation coefficients. The methods revealed different results indicating anomalous SDS binding and mobility of these glycoproteins in SDS electrophoresis.The glycoprotein patterns of larvae and of adults of Calliphora as well as of Sarcophaga differed markedly. PM of adults of both species contained only one carbohydrate fraction which migrated in the gel according to an apparent MW of about 200.000 daltons. PM of the larvae, however, showed a variety of bands in the range between 30.000 and 80.000 daltons which had binding capacities for the protein as well as for the carbohydrate stain. On the other hand, the patterns of pure protein bands were similar in the larval and in the adult stage. Obviously, the glycoprotein pattern of PM is altered during development according to special requirements. Also the similarities between both species in the larval and in the adult stage point to a special physiological function of the glycoprotein moiety.     

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.     

Hormone mediated induction of acid phosphatase activity in the fat body of Calliphora erythrocephala prior to metamorphosis

At the end of the larval feeding stage of Calliphora erythrocephala, ecdysteroids are most likely to be responsible for the rapid increase in acid phosphatase activity in the fat body. This is demonstrated by the precocious induction of the enzyme by 20-hydroxyecdysone in ligatured feeding-stage larvae weighing 55–70 mg. The hormone does not influence normal protein accumulation: this is inhibited by the ligature and is not restored by injection of the hormone.     

Effects of Weismann ring gland on RNA synthesis in imaginal wing disks of Calliphora erythrocephala

Imaginal wing disks of Calliphora erythrocephala have been studied in normal animals and in permanent larvae surgically deprived of the ring gland. By the end of larval life, uridine incorporation in the nuclear RNA and the ribosomal RNA increases for a brief period. This phenomenon does not occur in permanent larvae. A quantitative study suggests that renewal of a part of the ribosomal RNA might precede wing evagination.     

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.     

Über das Formunterscheidungsvermögen der Schmeißfliege Calliphora erythrocephala

Zusammenfassung Mit Alternativwahlversuchen wird nachgewiesen, daß folgende Formmerkmale die Zielorientierung (= Telotaxis) von laufenden, männlichen Schmeißfliegen (Calliphora erythrocephala) stimulieren: 1. Dunkle Kontrastflächen (Abb. 1); 2. Vertikale Kontrastgrenzen (Abb. 2-6); 3. Figurengliederung (Abb. 3f und 4).

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On the pattern discrimination of the blow fly Calliphora erythrocephala

Summary Experiments on binary choices of walking male blow flies (Calliphora erythrocephala) demonstrate the stimulatory value of the following pattern characteristics on the targed orientation (= telotaxis): 1. Dark contrasting area (Fig. 1). 2. Vertical edge of contrast (Fig. 2–6). 3. Disruption of shape (Fig. 3f and 4).

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Mit Unterstützung der Deutschen Forschungsgemeinschaft (SFB 45).     

Insecticidal secondary metabolic products from the entomogenous fungus Fusarium larvarum

The known fungal metabolic products monocerin and (+)-mellein and the new natural products 7-O-demethylmonocerin, fusarentin 6-methyl ether, and fusarentin 6,7-dimethyl ether have been obtained from two strains of Fusarium larvarum isolated from widely different localities and hosts. The major metabolites, monocerin and the fusarentin ethers, show insecticidal activity in a bioassay against Calliphora erythrocephala.     

Rezeptorrauschen und Informationskapazität der Sehzellen vonCalliphora erythrocephala undPeriplaneta americana

Zusammenfassung Die mittlere, effektive Rauschamplitude des Belichtungspotentials wird im Soma der Sehzellen vonCalliphora erythrocephala undPeriplaneta americana in Abhängigkeit von der Lichtintensität gemessen. Sie nimmt mit ansteigender Intensität ab. Für eine Intensitätsspanne von 11000 beträgt sie fürCalliphora erythrocephala 0,440,26 mV und fürPeriplaneta americana 0,670,16 mV. Die Amplitudenverteilung des Rauschens ist gaußförmig. Der Einfluß auf die Informationskapazität (bit/sec) wird für verschiedene Kontrastverteilungen diskutiert; die Informationskapazität ist bei geringen mittleren Lichtintensitäten im Soma der Sehzellen vonCalliphora erythrocephala gering, d.h. bei geringen Beleuchtungsstärken werden Kontraste aus der Umwelt nur schlecht übertragen. In den Axonen der Sehzellen 1–6 wird jedoch durch die präsynaptische Summation der Belichtungspotentiale die Übertragung von Kontrasten gerade bei geringen Beleuchtungsstärken erheblich verbessert (Abb. 5). Die Informationskapazitäten im Soma der Sehzellen vonCalliphora erythrocephala undPeriplaneta americana werden verglichen; die Leistung der Sehzellen bei der Übertragung von Kontrasten vor allem bei geringen Beleuchtungsstärken ist beiPeriplaneta americana erheblich schlechter als beiCalliphora erythrocephala.

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Inherent noise and information capacity in the visual cells ofCalliphora erythrocephala andPeriplaneta americana

Summary When the receptor potential from the soma of the visual cells ofCalliphora erythrocephala andPeriplaneta americana is measured we distinguish the r.m.s. voltage of the noise in dependence on the stimulus light intensity. The r.m.s. voltage of the noise decreases with increasing light intensity. The values for an intensity range of 11000 are 0.440.26 mV forCalliphora erythrocephala and 0.670.16 mV forPeriplaneta americana. The amplitude of the inherent receptor noise has a Gaussian Distribution. The influence on the information capacity of the visual cell is discussed for different contrast distributions as stimulus pattern. The information capacity is decreased at low mean light intensities inCalliphora erythrocephala visual-cell somata, i.e. environmental contrasts are transmitted only weakly at low illuminations. In the axons of visual cells 1–6, however, the transmission of contrasts especially at low illumination intensities is considerably improved by the presynaptie summation of receptor potentials (Fig. 5). The information capacities in the visual cell somata ofCalliphora erythrocephala andPeriplaneta americana are compared; the efficiency of visual cells during the transmission of contrasts, especially at low illumination intensities, is far worse inPeriplaneta than inCalliphora.

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Die Experimente wurden mit Sachmitteln durchgeführt, die Herrn Prof. Dr. Dr. h. c. H. Autrum von der Deutschen Forschungsgemeinschaft zur Verfügung gestellt wurden.     

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

Summary The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microseopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5–10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthognal texture of bundles of microfibrils that are thought to contain chitin. The pesence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

The effects of the piroplasm Babesia bigemina on the survival and reproduction of the blue tick,Boophilus decoloratus

Seven strains of the insect pathogenic fungus Verticillium lecanii have been examined in the Calliphora erythrocephala bioassay for the production, in surface culture on Czapek Dox medium, of insecticidal secondary metabolites. One strain which had lost its pathogenicity on storage yielded no active compounds. The remainder yielded dipicolinic acid (pyridine-2,6-dicarboxylic acid) which was responsible for the insecticidal activity of acidic extracts. Neutral extracts from two strains contained novel insecticidal C₂₅ compounds.     

Hormonal control of resilin deposition in the blowfly, Calliphora erythrocephala

The deposition of the resilin tendon in the blowfly Calliphora erythrocephala was investigated in normal and in various experimental conditions. The results showed that the weight of the protein resilin that is deposited is controlled by diet as well as by the hormone secreted by the medial neurosecretory cells.Endocrinologically abnormal Calliphora adults deposited a tendon with normal ultrastructure but showed signs of premature ageing while Calliphora fed on a sugar diet deposited a tendon with abnormal ultrastructure.     

The ribosomal DNA of Calliphora erythrocephala; an analysis of hybrid plasmids containing ribosomal DNA

We have analysed the ribosomal DNA of Calliphora erythrocephala, a Dipteran fly of the same sub-order as Drosophila melanogaster, through a series of rDNA² fragments cloned in a plasmid vector. We have mapped the sites for eight restriction enzymes within these plasmids, and positioned the regions coding for the 18 S and 28 S rRNAs within the maps of selected plasmids using the S₁ endonuclease mapping procedure of Berk & Sharp (1977). This analysis establishes that some rDNA cistrons of C. erythrocephala contain an “intron” (Gilbert, 1978) which interrupts the 28 S coding region at the same position as that of D. melanogaster rDNA. Two introns of 2.85 kilobases in length and part of a longer, sequence-related variant were isolated in these cloned fragments. Restriction enzyme site analysis and preliminary hybridization data indicate that the 2.85 kb intron of C. erythrocephala is largely unrelated in sequence to the two classes of D. melanogaster rDNA introns.     

The constancy and similarity of the amounts of free amino acids in inbred strains of Drosophila and outbred Calliphora

The composition of the free amino acids are compared in adults of two inbred strains of Drosophila subobscura and their hybrids, three inbred lines of Drosophila melanogaster, and in flies from a heterogeneous population of Calliphora erythrocephala. It appears that not only do the amounts of amino acid vary very little from fly to fly, but also very little between inbred lines. Furthermore, the relative amounts of the amino acids in Drosophila are similar to the relative amounts in the blowfly, Calliphora. This characteristic of invariant amounts of the free amino acids in these Diptera occurs in spite of the probably large numbers of genes affecting them.     

Developmental cytology of the midgut in the flesh-fly, Sarcophaga bullata (Parker)

The cytological comparisons of the midgut in Sarcophaga bullata (Parker) between the second instar, the third instar larvae and the adult are made. The adult midgut differs from that of the larvae in the following ways: (1) the peritrophic membrane is thicker than in the larvae and has become multi-layered; (2) epithelial cells are smaller; (3) branched microvilli are present throughout the entire midgut instead of being present only in the posterior region as in the larval midgut; (4) nuclear pores are less frequent; (5) lysosome-type structures occur less frequently; (6) the basal membrane is thicker; (7) the z-bands in the surrounding muscle fibers are more distinct in adults. The possible function and the significance of these structures related to previous observations in Sarcophaga and other Diptera are discussed.     

Continuity of the membrane systems in the cells of imaginal discs

Summary Imaginal discs from mature larvae of the fly Calliphora erythrocephala fixed in permanganate and embedded in Epon 812 were observed by electron microscopy. Irrespective of the type of the disc, the endoplasmic reticulum in the cells was found to be in unbroken contact with the outer nuclear membrane, Golgi areas, the mitochondria and the cell membrane. The imaginai discs are syncytial in that all cells are in cytoplasmic communication through secondary breaks in the cell membranes.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Mrs. Mariann Carleson and Miss Brita Nilsson for technical aid.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

Analysis of sex chromosome homologies in representatives of the family calliphoridae

The distribution of sequences homologous to the Calliphora erythrocephala Mg. sex chromosome was studied in Protophormia terranovae R-D and Lucilia sp. The chromatin structure was found to be similar in regions containing homologous DNA sequences.