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Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)
J. Insect Physiol. Vol. 29, No. 3, pp. 275 to 280. 1983 Printed irl Great Britairl.
0022-1910/83/030275-06$03.00/O 0 1983 Pergamon Press Ltd
EVIDENCE FOR A SUGAR RECEPTOR (LECTIN) IN THE PERITROPHIC MEMBRANE OF THE BLOWFLY LARVA, CALLIPHORA ERYTHROCEPHALA Mg. (DIPTERA) WERNER PEERS*, HUBERT KOLB~ and VICTORIA KOLB-BACHOFEN~
*Institut fiir Zoologie der Universitit Diisseldorf, TDiabetes-Forschungsinstitut. Diisseldorf, and flnstitut ftir Biophysik
und Elektronenmikroskopie,
UniversitIt
Diisseldorf
(Receined 13 lul.~ 1982; revised 22 September 1982) Abstract--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 er.vthrocephala, 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, Profeus uulgaris 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.
INTRODUCIION
lN INSECTS,as well as in members of other animal phyla, a special secretory product is formed by the midgut cells and is called the peritrophic membrane; it surrounds the food and food residues in the gut (BALBIANI, 1890). Flies and their larvae are of special interest as they have specialized cells at the entrance to the midgut (in the so-called cardia) which secrete a single or up to three morphologically different peritrophic membranes; these are tube-like and extend through the whole midgut and hindgut (for reviews see AUBERTOT. 1934; PETERS, 1976; RICHARDS and RICHARDS, 1977) thus enveloping the incoming food. Smaller molecules and those resulting from enzymatic degradation of food must penetrate these membranes before they reach the midgut epithelium (ZHUZHIKOV, 1964; for a review see PETERS, 1976). Such membranes form a barrier to bacteria and parasites, such as trypanosomes, malaria oijkineres and microfilariae (STOHLER, 1961). As the gut is exposed to many foreign and harmful molecules as well as parasites, it is possible that the plasma membrane of the midgut ceils and/or the peritrophic membranes bear a number of receptors which would allow differentiation between the various components of the food. Such receptors could be carbohydrate, protein or lipid-binding membrane proteins. The following experiments describe the search for D-mannose and D-galactose-binding lectins on the surface of the peritrophic membrane and on the midgut cells of blowfly larvae. These larvae have a single peritrophic membrane which is normally densely populated with bacteria on the lumen side; however,
it is very easy to rear larvae under sterile conditions; the membranes of these larvae are devoid of bacteria. In general, sugar receptors (lectins) can be localized by the electron microscope with the aid of electrondense colloidal gold particles labelled with proteins which have the respective sugar residues (HORISBERGER,1979 ; HORISBERGERand ROSSET,1977; GEOGHEGANand ACKERMAN,1977). Peritrophic membranes and pieces of midgut epithelium were treated with colloidal gold solutions which had been Iabeiled with either horseradish peroxidase or asialofetuin. Horseradish peroxidase is a glycoprotein with terminal mannose residues, which interacts with D-mannosespecific lectins (STAHL et al., 1978). Asialofetuin is a glycoprotein with terminal D-galactose residues, which has been used to demonstrate D-galactosebinding lectins (KOLB-BACHOFENet al., 1983). Competitive-inhibition experiments with monosaccharides were performed to determine the degree of specificity. Furthermore, colloidal gold particles were labelled with a lectin, Concanavalin A, in order to localize mannose and/or glucose residues. MATERIALS
AND
METHODS
Rearing of sterile larvae and preparation membranes
of peritrophic
Blowfly larvae were reared either on beef heart or under sterile conditions. In the latter case the eggs were sterilized for 15 min in a 3% solution of sodium sulphide, washed three times in a 0.5% solution of Zephirol (Bayer), and then rinsed with sterilized distilled water. Sterilized eggs were transferred to an 275
176
W~KNER
P1:TtKs.HCIRERTKOLH and VICTORIA KOLB-BACHWA
autoclaved medium containing: 2 g agar/lOO ml salt 0.52”,,, NaH,PO+ 0.45”,, solution (0.9”,, NaCl, K2HPOJ, 0.02”,, KCI. 0.02”,, CaCI,, 0.02”,, NaHCO,), 4.3 g casein (Serva. Munich, FRG). 4.5 g yeast extract (Serva). 1 g cholesterol (MCGINNIS rt al.. 1956); 1 ml of a solution containing 5000 units of streptomycin and penicillin (Servomed, Munich, FRG) was added to each plate. Peritrophic membranes were dissected out from either normal or sterile third-instar larvae (feeding phase) and rinsed several times in saline until they were cleaned from food residues. Membranes from normal larvae could not be freed from the dense layer of bacteria which gave the whole membrane a whitish appearance. Peritrophic membranes from larvae which had been reared under sterile conditions were translucent. Prepration gates
of’ yold particles
und protrin-gold
conju-
All glassware and centrifuge tubes which came into contact with colloidal gold solutions were siliconized before use as even small amounts of contaminants induce the Aocculation of gold sols. Monodisperse gold sols with a particle size range between 55150 nm are obtained by reducing tetrachloroauric acid: the size of the gold particles can be determined by the method of reduction (FRENS. 1973). In the following, gold colloids with an average particle diameter of 17 + 3 nm were used. The colloid was prepared by heating 200 ml of distilled water with 4 ml of a I”,, sodium citrate solution. 0.5 ml of a 4”. HAuC1, solution were added with rapid mixing and heated to boiling under reflux for 30 min. The colour of the solution turned to red. After cooling the negatively charged gold particles were labelled with proteins (HORISBERGERand ROSSET. 1977; GE~GHEGAN and ACKERMAN, 1977 : GOODMAN rl d., 1981). It has been shown by these authors that efficient adsorption occurs at or near the isoelectric point of the marker protein. and that protein concentration. pH and ionic strength of the solution have to be considered. Therefore, I mg protein (horseradish peroxidase: Serva, Heidelberg; concanavalin A: Medac, Hamburg; asialofetuin was a gift from Prof. Bauer. Free University of Berlin) was dissolved in 1 ml 5 mM NaCI. and the pH was adjusted to 7.2 with 2 mM K,C03. 100 ml of the gold sol were added. and the solution agitated for 2 min. To determine whether the colloid was saturated with adsorbed protein. 0.1 ml of a IO”,, solution of NaCl was added to 0.5 ml of gold sol: if the colour of the solution turned blue as a result of flocculation saturation levels had not been reached. otherwise the colour remained red. In order to avoid flocculation during prolonged storage polyethylene glycol (carbowax) was used. 5 ml of a I”, solution of polyethylene glycol (Serva. Heidelberg) with a molecular weight of 20,000 dalton were filtered through a Millipore filter (0.45 pm pore size) and added to 100 ml gold solution. Then the solution was centrifuged for it3 15 min at 23,000~. The supernatant was discarded and the sediment washed twice with 0.05 M Tris buffer pH 7.2 plus polyethylene glyco1 (see above). Finally the sediment was taken up with 4 ml 0.05 M Tris + 0.15 M NaCI. To avoid bacterial contamination 0.5 mg/ml sodium azide was
added: for the incubation of midgut epithelium freshly prepared gold solutions without sodium azide were used. In the case of concanavalin A. CaCI, and MnCl, were added (final concentration 1 mM) in order to maintain the tetrameric structure of the molecule. The labelled gold solutions were stored at 4-C. Before use they were diluted 1: 5 with phosphate-buffered saline + I mM MgCl, + CaCl, (in the case of concanavalin A plus 1 mM MnClz).
Clean peritrophic membranes (see above) or pieces of midgut epithelium adjacent to the cardia were incubated for 60 min with horseradish peroxidase-gold. asialofetuin-gold or concanavalin--gold followed by rinsing twice with phosphate-buffered saline for 1% 15 min. They were then sampled in small drops of agar. tixed in 2.5”,, glutaraldehyde buffered with 0.1 M cacodylate pH 7.4. postfixed in I”;, 0~0, with the same buffer. dehydrated in a graded series of ethanol and in propylene oxide, and embedded in Araldite. Sections were cut with a diamond knife, mounted on uncoated grids, stained with uranyl acetate and lead citrate, and observed with a Zeiss EM 9-S 2 electron microscope. Specificity of binding was controlled by competitive inhibition with monosaccharides. Clean peritrophic membranes were incubated with sugar solutions at a final concentration of 0.3 M sugar for 30 min at room temperature. Then they were transferred to a medium with 0.3 M of the respective sugar and horseradish peroxidase-gold or concanavalin-gold for another 60 min. After several rinses with phosphate-buffered saline (see above) fixation and embedding followed as described above.
RESULTS If the peritrophic
membranes of larvae of Co//iwhich have been reared under sterile conditions. are treated with colloidal gold labelled with horseradish peroxidase, one can observe without optical aids a positive reaction by the pink colour of the membranes. Transmission electron microscopy reveals a rather dense decoration of the lumen side with opaque gold granules. Only very few granules can be seen on the epithelium side of the membranes (Fig. I). A parallel analysis of the midgut epithelium yielded no gold binding (not shown in Fig. I). If samples of peritrophic membranes are pre-incubated with D-mannose or c+methyl-D-mannoside only a few scattered gold granules can be found (Fig. 3), showing that inhibition is complete. Almost no inhibition is seen with galactose. demonstrating the carbohydrate specificity of binding of horseradish peroxidase. Therefore, a mannose receptor of high specificity seems to be present on the lumen side of this peritrophic membrane, but not on the epithelium side, nor on the surface of the midgut epithelium. If peritrophic membranes or pieces of midgut epithelium were incubated with colloidal gold labelled with asialofetuin there was practically no adsorption of the gold marker, which suggests that a galactose receptor may be absent. Peritrophic membranes from larvae which had been reared on beef heart showed in phora erythrocephalu.
271
Figs 1 and 2. Sections through the peritrophic membrane of larvae of Calliphora were reared under sterile conditions. (Scale: 0.5 pm.)
erythrocephala
which
Fig. 1. After incubation of such membranes with gold marker labelled with horseradish peroxidase the marker is nearly exclusively distributed at the luminal surface (L). Fig. 2. Competitive inhibition with 0.3 M a-methyl-o-mannoside abolishes nearly completely the adsorption of the gold marker. Therefore, it is suggested that a mannoside specific lectin (sugar receptor) is present at the luminal surface of these peritrophic membranes.
278
Figs 3 and 4. Peritrophic membranes of larvae of Cdiphora erythrocephala reared under normal conditions are covered with bacteria on the lumen side. If such membranes are incubated with gold marker labelled with horseradish peroxidase the marker accumulates almost exclusively on the pili of the bacteria. Where the bacteria have been detached during preparation the pili may remain adhered to the membrane leaving only small patches of free peritrophic membrane surface for the adsorption of gold marker (arrow). (Scale: 0.5 pm.)
279
Lectin in the peritrophic membrane of the blowfly larva Table I. Comparison of results Sterile peritrophic membranes
Asialofetuin Horseradish peroxidase Methyl-Man - horseradish peroxidase Gal -t horseradish peroxidase Concanavalin A Methyl-Man + Glut 4 Concanavalin A
the phase contrast light microscope a continuous layer of bacteria on the lumen side. This is confirmed by electron microscopy, although during cleaning and preparation procedures many bacteria were displaced. The bacteria belong to the species Proteus vulgaris and P. morganii. Samples of such membranes showed a conspicuous pink colour after incubation with horseradish peroxidase and gold. The electron microscope revealed that the lumen side of such peritrophic membranes is studded with bacteria which adhere to the membrane by means of fine, long pili (Fig. 3). The latter were covered with gold marker. Despite the cleaning process the adherence between the peritrophic membrane and the pili of these bacteria is so strong that the pili are still present. They seem to cover most of the area where bacteria are no longer present. Only small patches of uncovered membrane remain where the labelled gold markers can be found (Fig. 4 arrow). Elsewhere it is prevented from binding to the peritrophic membrane by the remnants of pili of the bacteria. Competitive inhibition with a-methyl-r>-mannoside did not result in a complete inhibition on the surface of the bacteria; about 20”<, label still adhered to the pili as compared with horseradish peroxidase-gold without inhibitor. Peritrophic membranes of normal larvae which were incubated with concanavalin A and gold exhibited a similar picture, as in Fig. 3. concerning the distribution of gold particles on the lumen side of the membrane and on the surface of the adhering bacteria. Competitive inhibition with a-methyl-D-mannoside and glucose resulted in a nearly complete inhibition in the case of the peritrophic membrane, but only 60--70”,; inhibition in the case of the bacteria. These observations are summarized in Table 1.
DISCUSSION For the first time a sugar receptor (lectin) has been demonstrated on the surface of peritrophic membranes. As far as we know it is also the first electron microscopic demonstration of a sugar receptor in invertebrates. The lectin has a high specificity for D-mannose and is present only on the lumen side of the peritrophic membranes. It is of interest that the same distribution is seen for D-mannoside residues as determined by concanavalin A binding. The mechanism of adherence of pathogenic as well as apathogenic microorganisms has been investigated
Lumen side
Epithelium side
+ + + -
_ -
Midgut epithelium _
by several laboratories (SWANSONet al., 1975; BEACHY and OFFEK, 1976; Hu et al., 1976; JONES and FRETER, 1976; ARON~ON et al., 1979). OFEK eb al. (1977) were able to show using biochemical methods that the adherence of Escherichia coli to human mucosal cells is mediated by a mannose-specific, lectin-like substance, present on the surface of the bacterium which binds to mannose-like residues on the surface of the mucnsa ceil and vice versa. A mutual system of this type appears to exist also in the case of the peritrophic membrane of the larva of Calliphora erJ,throcephala and its adhering bacteria. The lectins of the membranes seem to be less densely distributed than those of the corresponding bacterium. This mutual adherence may be responsible for the fact that it is impossible to clean the adhering bacteria from the membranes of normal larvae by rinsing with buffer. Even on small patches where bacteria have been removed, their pili, and sometimes even parts of their surface. remain attached to the lumen side of the peritrophic membranes. Such a mutual system seems to be in favour of a symbiotic nature of these bacteria. Furthermore, larvae which have been reared under sterile conditions are smaller than infected larvae; their membranes are thinner and more fragile. It is very interesting that the surface of the midgut cells has no lectins with mannose or galactose affinity. This is in agreement with the hypothesis that peritrophic membranes are a special form of the surface coat of midgut cells which is secreted by these cells, reinforced in most cases by chitin-containing microfibrils. and delaminated frequently from the midgut cells in order to envelop food and food residues. In blowfly larvae, a tube-like peritrophic membrane is formed continuously in the cardia by specialized cells at the entrance of the midgut. The remaining midgut cells have no contact with the original food; only small molecules and enzymatic degradation products. as far as they are able to penetrate the peritrophic membrane, can reach them. Therefore, it seems possible that the midgut cells could have lost their lectins during the course of evolution in favour of the peritrophic membrane. Acknowledgements-The authors are very much indebted to Prof. Dr. BAUER,Free University of Berlin. for the supply of asialofetuin. to Dr. HAGEDORN,Institute of Microbiology and Virology, University of Diisseldorf for the determination of the bacteria and to Mrs. INGE LATRA for expert technical assistance.
280
WERNER PETERS. HUBERT K~LB and VICTORIA KOLB-BACHOFEN REFERENCES
ARON~ON M.. MEDALIA 0.. SCHORI L.. MIRELMAN D.. SHARON N. and OFE~ I. (1979) Prevention of colonization of the urinary tract of mice with Escl~rrich~a coli by blocking of bacterial adherence with methyl a-D-mannopyranoside. J. inf>ct. Dis. 139. 329-332. AUBERTOT M. (1934) Recherches sur les membranes p&itrophiques des insectes et en particulier des Dipttres. These, Fat. Sci. Univ. Strasbourg. Nancy (E) No. 44. BALBIANI E. G. (1890) Etudes anatomiques et histologiques sur le tube digestif des Cryptops. Arch. Zoo/. rup. (l&t, 8, 1-82. BEACHY E. H. and 0~6~ 1. (1976) Epithelial cell binding of group A streptococci by lipoteichoic acid on fimbriae denuded of M protein. J. e.uy. Med. 143, 759.-771. FRENS G. (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nuturr Phvs. Sci. 241, 2@22. GEOGHEGAN W. D. and AC~CERMANG. A. (1977) Adsorption of horseradish peroxidase, ovomucoid and antiimmunoglobulin to colloid gold for the indirect detection of concanavalin A. wheat germ agglutinin and goat antihuman immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and apphcation. 1. Histoci:ern. f~tocherf~. 25, I 187-l 200. GOODMAN S. L.. HODGES G. M.. TREJDOSIEWIT%L. K. and LIVINGSTON D. C. (1981) Colloidal gold markers and probes for routine application in microscopy. J. Microscopy 123, 201-213. HORISBERGER M. (1979) Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy. Biol. Ce&tfairr 36, 253- 258. HOR~SBEKGERM. and RossEr J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochenl. Cvrochrrn. 25, 295. 305. Hu P. C.. COLLIER A. M. and BASEMANJ. B. (1976) Interaction of virulent Mycoplasw pwun~or~iur with hamster tracheal organ cultures. I@cf. liuittun. 14, 2 I7 124.
JONES G. W. and
FRE~ER R. (1976) Adhesive properties of I’ibrio clrolerar: nature of the interaction with isolated rabbit brush border membranes and human erythrocytes. i&f. fmfnun. 14, 24s-245. KOLB-BACHOFEN V.. STHLEPPER-SCHAFER J.. VOC;ELL W. and KOLB H. (1983) Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localization and demonstration of lectin-mediated endocytosis. Cell (in press). MCGINNIS A. J.. NEWBUKGH R. W. and CHELDEL~N V. H. (1956) Nutritional studies on the blowfly. Phorrttio reyiru Mg. J. Nutr. 58. 309-322. OFE~ 1.. MIRELMAN D. and SHARON N. t 1977) Adherence of Esclzrrichia co/i to human mucosal cells mediated by mannose receptors. Nantre. Lo&. 265, 623-625. PETERS W. (1976) lnvestigatlons on the peritrophic membranes of Diptera. In The inwct fnteqmutf (Ed. by HEDBURN H. R.). Elsevier. Amsterdam. RICHARDS A. G. and RITHARI>S P. A. (1971) Origin and composition of the peritrophic membrane of the mosquito. Ardrs trqypti. 1. Irtsect Physiol. 17, 2253-2275. RICHARI~SA. G. and RICHARI>S P. A. (1977) The peritrophic membranes of insects. A. Rev. Ent. 22, 219-240. S.TAHL P. D., RODMAN J. S.. MILLFR M. J. and SCHLESINC~ER P. (1978) Evidence for receptor mediated binding of glycoproteins. glycoconjugates and lysosomal glycosidases by alveolar macrophages. Proc. noto. Acrtd. %i. I’..~..~. 75, 1399-1403. ST~HLER H. (1961) The peritrophic membrane of blood sucking Diptera in relation to their role as vectors of blood parasites. Acru trap. 18, 263-266. SWANSON J., SPARKS E.. YOUNG D. and KING G. (1975) Studies on gonococcus infection. X. Pili and leukocyte association factor as mediators of interactions between gonococci and eukaryotic cells ;,I ritro. Infect. Inmun. II. 1352-1361. ZHULHIKO~ D. P. (1964) Function of the peritrophic membrane in Musca dmwstico L. and Cullipl~ortr rr@lrocepkuh Meig. J. I~~srct Plrvsiol. 10, 273- 278.
0022-1910/83/030275-06$03.00/O 0 1983 Pergamon Press Ltd
EVIDENCE FOR A SUGAR RECEPTOR (LECTIN) IN THE PERITROPHIC MEMBRANE OF THE BLOWFLY LARVA, CALLIPHORA ERYTHROCEPHALA Mg. (DIPTERA) WERNER PEERS*, HUBERT KOLB~ and VICTORIA KOLB-BACHOFEN~
*Institut fiir Zoologie der Universitit Diisseldorf, TDiabetes-Forschungsinstitut. Diisseldorf, and flnstitut ftir Biophysik
und Elektronenmikroskopie,
UniversitIt
Diisseldorf
(Receined 13 lul.~ 1982; revised 22 September 1982) Abstract--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 er.vthrocephala, 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, Profeus uulgaris 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.
INTRODUCIION
lN INSECTS,as well as in members of other animal phyla, a special secretory product is formed by the midgut cells and is called the peritrophic membrane; it surrounds the food and food residues in the gut (BALBIANI, 1890). Flies and their larvae are of special interest as they have specialized cells at the entrance to the midgut (in the so-called cardia) which secrete a single or up to three morphologically different peritrophic membranes; these are tube-like and extend through the whole midgut and hindgut (for reviews see AUBERTOT. 1934; PETERS, 1976; RICHARDS and RICHARDS, 1977) thus enveloping the incoming food. Smaller molecules and those resulting from enzymatic degradation of food must penetrate these membranes before they reach the midgut epithelium (ZHUZHIKOV, 1964; for a review see PETERS, 1976). Such membranes form a barrier to bacteria and parasites, such as trypanosomes, malaria oijkineres and microfilariae (STOHLER, 1961). As the gut is exposed to many foreign and harmful molecules as well as parasites, it is possible that the plasma membrane of the midgut ceils and/or the peritrophic membranes bear a number of receptors which would allow differentiation between the various components of the food. Such receptors could be carbohydrate, protein or lipid-binding membrane proteins. The following experiments describe the search for D-mannose and D-galactose-binding lectins on the surface of the peritrophic membrane and on the midgut cells of blowfly larvae. These larvae have a single peritrophic membrane which is normally densely populated with bacteria on the lumen side; however,
it is very easy to rear larvae under sterile conditions; the membranes of these larvae are devoid of bacteria. In general, sugar receptors (lectins) can be localized by the electron microscope with the aid of electrondense colloidal gold particles labelled with proteins which have the respective sugar residues (HORISBERGER,1979 ; HORISBERGERand ROSSET,1977; GEOGHEGANand ACKERMAN,1977). Peritrophic membranes and pieces of midgut epithelium were treated with colloidal gold solutions which had been Iabeiled with either horseradish peroxidase or asialofetuin. Horseradish peroxidase is a glycoprotein with terminal mannose residues, which interacts with D-mannosespecific lectins (STAHL et al., 1978). Asialofetuin is a glycoprotein with terminal D-galactose residues, which has been used to demonstrate D-galactosebinding lectins (KOLB-BACHOFENet al., 1983). Competitive-inhibition experiments with monosaccharides were performed to determine the degree of specificity. Furthermore, colloidal gold particles were labelled with a lectin, Concanavalin A, in order to localize mannose and/or glucose residues. MATERIALS
AND
METHODS
Rearing of sterile larvae and preparation membranes
of peritrophic
Blowfly larvae were reared either on beef heart or under sterile conditions. In the latter case the eggs were sterilized for 15 min in a 3% solution of sodium sulphide, washed three times in a 0.5% solution of Zephirol (Bayer), and then rinsed with sterilized distilled water. Sterilized eggs were transferred to an 275
176
W~KNER
P1:TtKs.HCIRERTKOLH and VICTORIA KOLB-BACHWA
autoclaved medium containing: 2 g agar/lOO ml salt 0.52”,,, NaH,PO+ 0.45”,, solution (0.9”,, NaCl, K2HPOJ, 0.02”,, KCI. 0.02”,, CaCI,, 0.02”,, NaHCO,), 4.3 g casein (Serva. Munich, FRG). 4.5 g yeast extract (Serva). 1 g cholesterol (MCGINNIS rt al.. 1956); 1 ml of a solution containing 5000 units of streptomycin and penicillin (Servomed, Munich, FRG) was added to each plate. Peritrophic membranes were dissected out from either normal or sterile third-instar larvae (feeding phase) and rinsed several times in saline until they were cleaned from food residues. Membranes from normal larvae could not be freed from the dense layer of bacteria which gave the whole membrane a whitish appearance. Peritrophic membranes from larvae which had been reared under sterile conditions were translucent. Prepration gates
of’ yold particles
und protrin-gold
conju-
All glassware and centrifuge tubes which came into contact with colloidal gold solutions were siliconized before use as even small amounts of contaminants induce the Aocculation of gold sols. Monodisperse gold sols with a particle size range between 55150 nm are obtained by reducing tetrachloroauric acid: the size of the gold particles can be determined by the method of reduction (FRENS. 1973). In the following, gold colloids with an average particle diameter of 17 + 3 nm were used. The colloid was prepared by heating 200 ml of distilled water with 4 ml of a I”,, sodium citrate solution. 0.5 ml of a 4”. HAuC1, solution were added with rapid mixing and heated to boiling under reflux for 30 min. The colour of the solution turned to red. After cooling the negatively charged gold particles were labelled with proteins (HORISBERGERand ROSSET. 1977; GE~GHEGAN and ACKERMAN, 1977 : GOODMAN rl d., 1981). It has been shown by these authors that efficient adsorption occurs at or near the isoelectric point of the marker protein. and that protein concentration. pH and ionic strength of the solution have to be considered. Therefore, I mg protein (horseradish peroxidase: Serva, Heidelberg; concanavalin A: Medac, Hamburg; asialofetuin was a gift from Prof. Bauer. Free University of Berlin) was dissolved in 1 ml 5 mM NaCI. and the pH was adjusted to 7.2 with 2 mM K,C03. 100 ml of the gold sol were added. and the solution agitated for 2 min. To determine whether the colloid was saturated with adsorbed protein. 0.1 ml of a IO”,, solution of NaCl was added to 0.5 ml of gold sol: if the colour of the solution turned blue as a result of flocculation saturation levels had not been reached. otherwise the colour remained red. In order to avoid flocculation during prolonged storage polyethylene glycol (carbowax) was used. 5 ml of a I”, solution of polyethylene glycol (Serva. Heidelberg) with a molecular weight of 20,000 dalton were filtered through a Millipore filter (0.45 pm pore size) and added to 100 ml gold solution. Then the solution was centrifuged for it3 15 min at 23,000~. The supernatant was discarded and the sediment washed twice with 0.05 M Tris buffer pH 7.2 plus polyethylene glyco1 (see above). Finally the sediment was taken up with 4 ml 0.05 M Tris + 0.15 M NaCI. To avoid bacterial contamination 0.5 mg/ml sodium azide was
added: for the incubation of midgut epithelium freshly prepared gold solutions without sodium azide were used. In the case of concanavalin A. CaCI, and MnCl, were added (final concentration 1 mM) in order to maintain the tetrameric structure of the molecule. The labelled gold solutions were stored at 4-C. Before use they were diluted 1: 5 with phosphate-buffered saline + I mM MgCl, + CaCl, (in the case of concanavalin A plus 1 mM MnClz).
Clean peritrophic membranes (see above) or pieces of midgut epithelium adjacent to the cardia were incubated for 60 min with horseradish peroxidase-gold. asialofetuin-gold or concanavalin--gold followed by rinsing twice with phosphate-buffered saline for 1% 15 min. They were then sampled in small drops of agar. tixed in 2.5”,, glutaraldehyde buffered with 0.1 M cacodylate pH 7.4. postfixed in I”;, 0~0, with the same buffer. dehydrated in a graded series of ethanol and in propylene oxide, and embedded in Araldite. Sections were cut with a diamond knife, mounted on uncoated grids, stained with uranyl acetate and lead citrate, and observed with a Zeiss EM 9-S 2 electron microscope. Specificity of binding was controlled by competitive inhibition with monosaccharides. Clean peritrophic membranes were incubated with sugar solutions at a final concentration of 0.3 M sugar for 30 min at room temperature. Then they were transferred to a medium with 0.3 M of the respective sugar and horseradish peroxidase-gold or concanavalin-gold for another 60 min. After several rinses with phosphate-buffered saline (see above) fixation and embedding followed as described above.
RESULTS If the peritrophic
membranes of larvae of Co//iwhich have been reared under sterile conditions. are treated with colloidal gold labelled with horseradish peroxidase, one can observe without optical aids a positive reaction by the pink colour of the membranes. Transmission electron microscopy reveals a rather dense decoration of the lumen side with opaque gold granules. Only very few granules can be seen on the epithelium side of the membranes (Fig. I). A parallel analysis of the midgut epithelium yielded no gold binding (not shown in Fig. I). If samples of peritrophic membranes are pre-incubated with D-mannose or c+methyl-D-mannoside only a few scattered gold granules can be found (Fig. 3), showing that inhibition is complete. Almost no inhibition is seen with galactose. demonstrating the carbohydrate specificity of binding of horseradish peroxidase. Therefore, a mannose receptor of high specificity seems to be present on the lumen side of this peritrophic membrane, but not on the epithelium side, nor on the surface of the midgut epithelium. If peritrophic membranes or pieces of midgut epithelium were incubated with colloidal gold labelled with asialofetuin there was practically no adsorption of the gold marker, which suggests that a galactose receptor may be absent. Peritrophic membranes from larvae which had been reared on beef heart showed in phora erythrocephalu.
271
Figs 1 and 2. Sections through the peritrophic membrane of larvae of Calliphora were reared under sterile conditions. (Scale: 0.5 pm.)
erythrocephala
which
Fig. 1. After incubation of such membranes with gold marker labelled with horseradish peroxidase the marker is nearly exclusively distributed at the luminal surface (L). Fig. 2. Competitive inhibition with 0.3 M a-methyl-o-mannoside abolishes nearly completely the adsorption of the gold marker. Therefore, it is suggested that a mannoside specific lectin (sugar receptor) is present at the luminal surface of these peritrophic membranes.
278
Figs 3 and 4. Peritrophic membranes of larvae of Cdiphora erythrocephala reared under normal conditions are covered with bacteria on the lumen side. If such membranes are incubated with gold marker labelled with horseradish peroxidase the marker accumulates almost exclusively on the pili of the bacteria. Where the bacteria have been detached during preparation the pili may remain adhered to the membrane leaving only small patches of free peritrophic membrane surface for the adsorption of gold marker (arrow). (Scale: 0.5 pm.)
279
Lectin in the peritrophic membrane of the blowfly larva Table I. Comparison of results Sterile peritrophic membranes
Asialofetuin Horseradish peroxidase Methyl-Man - horseradish peroxidase Gal -t horseradish peroxidase Concanavalin A Methyl-Man + Glut 4 Concanavalin A
the phase contrast light microscope a continuous layer of bacteria on the lumen side. This is confirmed by electron microscopy, although during cleaning and preparation procedures many bacteria were displaced. The bacteria belong to the species Proteus vulgaris and P. morganii. Samples of such membranes showed a conspicuous pink colour after incubation with horseradish peroxidase and gold. The electron microscope revealed that the lumen side of such peritrophic membranes is studded with bacteria which adhere to the membrane by means of fine, long pili (Fig. 3). The latter were covered with gold marker. Despite the cleaning process the adherence between the peritrophic membrane and the pili of these bacteria is so strong that the pili are still present. They seem to cover most of the area where bacteria are no longer present. Only small patches of uncovered membrane remain where the labelled gold markers can be found (Fig. 4 arrow). Elsewhere it is prevented from binding to the peritrophic membrane by the remnants of pili of the bacteria. Competitive inhibition with a-methyl-r>-mannoside did not result in a complete inhibition on the surface of the bacteria; about 20”<, label still adhered to the pili as compared with horseradish peroxidase-gold without inhibitor. Peritrophic membranes of normal larvae which were incubated with concanavalin A and gold exhibited a similar picture, as in Fig. 3. concerning the distribution of gold particles on the lumen side of the membrane and on the surface of the adhering bacteria. Competitive inhibition with a-methyl-D-mannoside and glucose resulted in a nearly complete inhibition in the case of the peritrophic membrane, but only 60--70”,; inhibition in the case of the bacteria. These observations are summarized in Table 1.
DISCUSSION For the first time a sugar receptor (lectin) has been demonstrated on the surface of peritrophic membranes. As far as we know it is also the first electron microscopic demonstration of a sugar receptor in invertebrates. The lectin has a high specificity for D-mannose and is present only on the lumen side of the peritrophic membranes. It is of interest that the same distribution is seen for D-mannoside residues as determined by concanavalin A binding. The mechanism of adherence of pathogenic as well as apathogenic microorganisms has been investigated
Lumen side
Epithelium side
+ + + -
_ -
Midgut epithelium _
by several laboratories (SWANSONet al., 1975; BEACHY and OFFEK, 1976; Hu et al., 1976; JONES and FRETER, 1976; ARON~ON et al., 1979). OFEK eb al. (1977) were able to show using biochemical methods that the adherence of Escherichia coli to human mucosal cells is mediated by a mannose-specific, lectin-like substance, present on the surface of the bacterium which binds to mannose-like residues on the surface of the mucnsa ceil and vice versa. A mutual system of this type appears to exist also in the case of the peritrophic membrane of the larva of Calliphora erJ,throcephala and its adhering bacteria. The lectins of the membranes seem to be less densely distributed than those of the corresponding bacterium. This mutual adherence may be responsible for the fact that it is impossible to clean the adhering bacteria from the membranes of normal larvae by rinsing with buffer. Even on small patches where bacteria have been removed, their pili, and sometimes even parts of their surface. remain attached to the lumen side of the peritrophic membranes. Such a mutual system seems to be in favour of a symbiotic nature of these bacteria. Furthermore, larvae which have been reared under sterile conditions are smaller than infected larvae; their membranes are thinner and more fragile. It is very interesting that the surface of the midgut cells has no lectins with mannose or galactose affinity. This is in agreement with the hypothesis that peritrophic membranes are a special form of the surface coat of midgut cells which is secreted by these cells, reinforced in most cases by chitin-containing microfibrils. and delaminated frequently from the midgut cells in order to envelop food and food residues. In blowfly larvae, a tube-like peritrophic membrane is formed continuously in the cardia by specialized cells at the entrance of the midgut. The remaining midgut cells have no contact with the original food; only small molecules and enzymatic degradation products. as far as they are able to penetrate the peritrophic membrane, can reach them. Therefore, it seems possible that the midgut cells could have lost their lectins during the course of evolution in favour of the peritrophic membrane. Acknowledgements-The authors are very much indebted to Prof. Dr. BAUER,Free University of Berlin. for the supply of asialofetuin. to Dr. HAGEDORN,Institute of Microbiology and Virology, University of Diisseldorf for the determination of the bacteria and to Mrs. INGE LATRA for expert technical assistance.
280
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