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Ultrastructural localisation of cathepsin B in the midgut of Rhodnius prolixus Stål ( Hemiptera : Reduviidae) during blood digestion
Int. J. Insect Morphol. & Embryol., Vol. 17, No. 4/5, pp. 295-3112, 1988 Printed in Great Britain
0020 7322/88 $3.1~) + .00 © 1988 Pergamon Press plc
U L T R A S T R U C T U R A L L O C A L I S A T I O N OF C A T H E P S I N B IN T H E M I D G U T OF RHODNIUS P R O L I X U S ST,~L ( H E M I P T E R A • R E D U V I I D A E ) D U R I N G B L O O D DIGESTION
P. F. BILLINGSLEY* and A. E. R. DOWNE Department of Biology, Queen's University. Kingston, Ontario, Canada K7L 3N6
(Accepted 16 February 1988)
Abstract--The artificial substrate N-benzoyl-Dm-arginine-13-naphthylamine,was used to localise cathepsin B in midgut cells of the haematophagous insect, Rhodnius prolixus Stfd (Hemiptera : Reduviidae), during blood digestion. Cathepsin B was localised primarily in the lysosomes of cells from all 3 midgut regions and in Golgi vesicles of the digestive intestinal regions, but not in association with any other cellular structures. The timing of localisation correlated with previously described cycles of endoproteinase activity and with known ultrastructural modifications to the midgut cells. Secretory vesicles, which originated from the Golgi complexes, were present only in the intestinal regions, and in the anterior intestine, they showed a strong positive correlation (r = 0.939, P = 0.01) with post-feeding cathepsin B activity. Cathepsin B plays a major role in primary extracellular digestion of blood proteins, and is active in the midgut lumen and lysosomes rather than in association with the microvilli. Index descriptors (in addition to those in title): Blood meal digestion; enzyme localisation.
INTRODUCTION CONCURRENT wi'th the p r o d u c t i o n of digestive proteinases ( H o u s e m a n and D o w n e , 1983), the m i d g u t cells of Rhodnius prolixus exhibit several ultrastructural modifications as a result of b l o o d feeding (Bauer, 1981; Billingsley, 1985; Billingsley and D o w n e , 1983, 1985, 1986b). T h e r o u g h endoplasmic reticulum (rer) is present in large whorls before feeding, b e c o m e s dispersed within 2 hr of a blood meal, and subsequently reforms during the digestion pe:riod. T h e lysosomes also display m a r k e d structural changes during the first 2-6 hr after a blood meal and subsequently return to a structure similar to that of lysosomes in the midgut of unfed insects between 6 hr and 5 days after feeding (Billingsley and D o w n e , 1983). These changes are not observed in the anterior midgut (stomach or crop) where no protein digestion takes place (Billingsley and D o w n e , in preparation), but only in the posterior midgut (which is further divided into the anterior and posterior in~testinal regions). A t the apical cell surface, a second apical m e m b r a n e develops o v e r the microvilli 12-24 hr after feeding, and proliferation of this o u t e r *Present address: Department of Pure and Applied Biology, Imperial College, Prince Consort Road, London SW7 2BB, England. 295
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m e m b r a n e results in t h e f o r m a t i o n of c o m p l e x e x t r a c e l l u l a r m e m b r a n e layers ( E C M L ) , which a r e t h o u g h t to function as a p e r i t r o p h i c m e m b r a n e (Billingsley a n d D o w n e , 1985, 1986b). C a t h e p s i n B has n o r m a l l y b e e n d e s c r i b e d as an i n t r a c e l l u l a r p r o t e a s e , r e s t r i c t e d to t h e l y s o s o m e s ( B a r r e t t , 1977) b u t has b e e n localised at the u l t r a s t r u c t u r a l level in s e c r e t i o n g r a n u l e s of rat p a n c r e a t i c cells (Smith a n d van F r a n k , 1975). In the m i d g u t of R. prolixus ( H o u s e m a n a n d D o w n e , 1980, 1982), c a t h e p s i n B is o n e o f 3 acid p r o t e a s e s o f l y s o s o m a l origin t h a t a r e utilised in t h e p r i m a r y d i g e s t i o n o f i n g e s t e d p r o t e i n s . C a t h e p s i n B, c a t h e p s i n D a n d c a r b o x y p e p t i d a s e B a r e m o r e active in the l u m e n t h a n in t h e cells o f t h e m i d g u t a n d activities of all 3 e n z y m e s p e a k at 7 days after f e e d i n g ( H o u s e m a n a n d D o w n e , 1980, 1982, 1983). R. prolixus also utilises a m i n o p e p t i d a s e for t e r m i n a l d i g e s t i o n of the b l o o d m e a l c o m p o n e n t s ( H o u s e m a n a n d D o w n e , 1981). This e n z y m e displays a cycle of activity in r e s p o n s e to b l o o d f e e d i n g ( H o u s e m a n a n d D o w n e , 1983), a n d is localised m a i n l y on microvilli a n d l y s o s o m e s in R. prolixus intestinal cells, b u t is also a s s o c i a t e d with t h e E C M L , rer a n d large i n t r a c e l l u l a r s t o r a g e vesicles (Billingsley a n d D o w n e , 1985). C h a n g e s in t h e l o c a l i s a t i o n o f a m i n o p e p t i d a s e on the microvilli a p p e a r e d to c o r r e l a t e with t h e p e a k o f a m i n o p e p t i d a s e activity, while the c o n t i n u e d l o c a l i s a t i o n o f a m i n o p e p t i d a s e in the l y s o s o m e s p r o b a b l y accounts for the p r o l o n g e d m a i n t a i n a n c e o f e n z y m e activity a b o v e b a s e l i n e levels. T h e p r e s e n t s t u d y is d e s i g n e d to c o m p l e m e n t p r e v i o u s investigations on t h e e n z y m o l o g y a n d u l t r a s t r u c t u r e o f the m i d g u t o f R. prolixus. P a r t i c u l a r l y , the sites o f c a t h e p s i n B activity a r e d e t e r m i n e d a n d the v a r i a t i o n s in l o c a l i s a t i o n a r e u s e d to c o r r e l a t e u l t r a s t r u c t u r a l a n d b i o c h e m i c a l r e s p o n s e s to feeding. T h e sites o f activity o f c a t h e p s i n B (an acid p r o t e a s e ) are c o m p a r e d with those of a m i n o p e p t i d a s e (an a l k a l i n e e x o p e p t i d a s e ) to i m p r o v e u p o n a p r e v i o u s l y d e s c r i b e d m o d e l of b l o o d d i g e s t i o n in R. prolixus (Billingsley a n d D o w n e , 1985).
MATERIALS
AND METHODS
Rhodnius prolixus StS1 were maintained and mated females obtained as previously described (Kwan and Downe, 1977; Houseman and Downe, 1983). Midguts were dissected from mated females before, 2 and 6 hr, and 1, 2, 4-7, 10, 15, 20 and 25 days after a meal of rabbit blood. At every time point, 3-5 midguts were dissected for e~ich of the experimental and control procedures. Blocks of tissue from each midgut region (stomach, anterior intestine and posterior intestine; Wigglesworth, 1943) small enough to allow rapid fixation (Brunings and de Priester, 1971), were fixed for 1 hr in ice cold 2.5% glutaraldehyde in 0.2M sodium cacodylate-HC1 buffer, pH 7.2. After several rinses in the same buffer, cathepsin B was localised using modifications to the methods of Seligman et al. (1970) (Billingsley and Downe, 1985) and Barrett (1972), by incubating the tissue blocks in the following medium: Eight mg/ml N-benzoyl-arginine-13-De-naphthylamine (BANA), 0.5 ml; 0.1M potassium phosphate buffer pH 5.5 containing 5raM ethylenediamine tetraacetate and 3 mM dithiothreitol, 5.0 ml; 2.5 mg/ml freshly diazotised 4-aminophthalhydrazide, 2.5 ml; distilled water, 2.0 ml. (All reagents from Sigma or Fisher Scientific.) The absence of substrate was used as a control, which was found to be suitable in a previous study (Billingsley and Downe, 1985). In contrast to the aminopeptidase localisation, an alternative substrate which reacts with cathepsin B but not the azo-dye, is not commercially available. Prolonged fixation and enzyme inhibitors both reduced the reaction, but were not used as controls because of their lack of specificity. Midguts were incubated for 2 hr at room temperature with one change to freshly prepared medium after 1 hr. The tissue was then rinsed in several changes of buffer over a 30 min period, post-fixed in 2% osmium tetroxide in cacodylate buffer for 1 hr, then dehydrated through an alcohol series. Tissue blocks were embedded in Epon resin, as previously described (Billingsley and Downe, 1985) and unstained thin sections were examined in a Zeiss EM10 electron microscope. Blocks from experimentally- and control-treated tissues were blind coded and codes not broken until after assessment of the results.
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Midgut tissue wzs also prepared for electron microscopyby standard techniques, and stained with uranyl acetate and lead cilrate as previously described (Billingsleyand Downe, 1988). Electron micrographs, which bad been used for a previous morphometric study (Billingsley, 1985), were checked for the presence of secretory vesicles; these represented unbiased samples from all 3 midgut regions, and all areas of the midgut cells in approximat,.~lyequal proportions. The vesicles per electron micrograph were counted and the results subjected to a 2-way linear regression analysis against the post-feedingactivitycycleof cathepsin B (data taken from Fig. 6 of Hou~,.emanand Downe, 1983).
RESULTS
Detection and general distribution of cathepsin B activity No cathepsin B activity is detected in any structures or organelles of the midgut cells before the bloc,d meal, except for trace amounts in the lysosomes (Fig. la, b). After feeding, cathept;in B is localised as a fine, granular, electron-dense precipitate associated mainly with the lysosomes (Fig. lc-f) and occasionally with Golgi vesicles (Fig. lg-i) of the posterior midgut. The lysosomes are usually found grouped together in the midgut cells, and in any group at any time between 2 and 15 days after feeding, the amount of precipitate associated with the lysosomes may vary from trace amounts to a very dense reaction (Fig. ld, e). Precipitate is never detectable on the rough endoplasmic reticulum (rer), on the microvilli or in association with storage vesicles seen in the midgut cells.
Cathepsin B localisation after feeding Only trace anaounts of cathepsin B activity are detectable in the lysosomes but not in any other structures or organelles in any midgut region until between 1 and 2 days after the blood meal. Between 2 and 4 days precipitate begins to appear in association with the lysosomes, and from 5 days to 10-15 days, maximum precipitate is observed overlying the lysosomes in all 3 midgut regions (Fig. ld-f). In the stomach and posterior intestine, only traces of cathepsin B activity are detectable in lysosomes after 15 days, while activity in the lysosomes of the anterior intestine begins to decline between 15 and 20 days, until only trace amounts are detectable 25 days after feeding. Precipitate is often observed overlying small vesicles on the rer side of the Golgi (Fig. l g - h ) , but only in vesicles that are away from the Golgi. Golgi-associated precipitate is only detectable in the intestinal regions from 2 to 14 days after blood feeding. Traces of cathepsin B activity are sometimes observed in the periphery of the posterior midgut lumen (not shown), but no consistent pattern can be determined as the reaction is always very diffuse and faint.
Secretory vesicles in the midgut cells Secretory vesicles in R. prolixus midgut contain electron-dense material (Fig. 2), and originate from the Golgi complexes mostly in the apical third of the epithelial cells (Fig. 2a). Vesicles are found in the apical cell region (Fig. 2b) where they seem to fuse with the apical plasma membrane at the base of the microvilli (Fig. 2c). Possible vesicle contents are observed in the periphery of the midgut lumen and/or in the E C M L (Fig. 2d), but cannot be detected any further into the lumen. Secretory vesicles are never observed in stomach cells but are present in the anterior intestinal cells from 2 hr to 20 days and at all times in posterior intestinal cells (Fig. 3). The correlation coefficients between cathepsin B activity and secretory vesicle numbers are 0.939 (P = 0.01) in the anterior intestine and 0.433 (not significant) in the posterior intestine.
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FIG. 1. Cathepsin B Iocalisation in the lysosomes (a-f) and Golgi (g-i) of Rhodnius prolixus midgut cells, a. Anterior intestine, before feeding, B A N A (× 23,200). A very faint precipitate is detectable in lysosomes, b. Anterior intestine, before feeding, control (× 18,000). Colloidal material present in lysosomes, but no electron-dense precipitate, c. Posterior intestine, 7 days after feeding, B A N A ( x 12,600). Precipitate in variable amounts is detectable overlying lysosomes, d. Anterior intestine, 5 days after feeding, B A N A (× 17,600). Precipitate in some lysosomes may be very heavy whereas others show little or no reaction. Anterior intestine, 5 days after feeding, B A N A (× 13,600). f. Anterior intestine, 6 days after feeding, control (× 19,800). No precipitate on lysosomes of controls, g. Anterior intestine, 7 days after feeding, BANA (× 32,100). Dense-staining vesicles are observed on rer side of Golgi in intestinal cells, h. Anterior intestine, 6 days after feeding, B A N A (× 22,100), Vesicles from the Golgi, regularly exhibit variable amounts of precipitate, i. Anterior intestine, 7 days after feeding, control (× 17,800). No dense-staining vesicles are seen in control tissues.
Cathepsin B in the Midgut of Rhodnius prolixus
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FIG. 2. Secretory vesicles in the intestinal cells of R. prolixus. Tissue fixed by standard techniques and stained with uranyl acetate and lead citrate, a. 12 days after feeding, anterior intestine (× 6,700). Formation of secretory vesicles ( . - ~ ) in Golgi (Go) and their possible route of transport to cell surface, b. 12 days after feeding, posterior intestine (× 10,200). Secretory vesicles (SV) near apical cell surface. Microvilli, MV. c. 2 days after feeding, anterior intestine (x 35,400). Release of secretory vesicle into lumen at base of microvilli (MV). d. 6 days after feeding, anterior intestine (× 19,000). Secretory vesicles (SV) in apical cell region and vesicle-like material (.-~,-) in extracellular membrane layers (ECML).
(28) o • -~
4-
o 3~
21-
>
0
(22)(25)~ 130)~ 0
2h
2d
(18)
N3 6d
12d
20d
Time after blood feeding
FIG. 3. Number of secretory vesicles in the intestinal cells of Rhodnius prolixus. Secretory vesicles were counted on micrographs used for a previous morphometric study and represent at least 5 cells per midgut region, except at 20 days (Billingsley, 1988). Numbers in parentheses are total electron micrographs counted per time point from each midgut region. Cross-hatched bars = anterior intestine, hat zhed bars = posterior intestine. 142 electron micrographs of stomach cells were examined, but no secretory vesicles were observed.
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P.F. BILLINGSLEYand A. E. R. DOWNE
DISCUSSION The results from the present study are consistent with histochemical demonstrations of the cathepsin B in several tissues (Barrett, 1977) and with the ultrastructural localisation of cathepsin B in the B-cells of rat pancreas (Smith and van Frank, 1975). The substrates used for the ultrastructural demonstration of aminopeptidase (Billingsley and Downe, 1985) and cathepsin B (present study) do not show any cross reactivity (Barrett and Poole, 1969). This is reflected in the differences between the ultrastructural localisation of the 2 enzymes in the midgut of R. prolixus; aminopeptidase was localised as both granular and aggregated precipitates (Billingsley and Downe, 1985) which correlated with the detection by column chromatography, of 2 distinct aminopeptidases from R. prol&us midgut tissue homogenates (Houseman and Downe, 1981). Conversely, no such separation of cathepsin B activity has been demonstrated and only 1 form of localisation reaction, a fine granular precipitate, was observed. By washing and centrifuging whole posterior midguts, cathepsin B was shown to be most active in the posterior midgut lumen of R. prolbcus (Houseman and Downe, 1980), although this procedure did not allow for any check on the level of cell disruption. The present study can only partially confirm this luminal activity. A very diffuse luminal localisation was observed (which was difficult to reproduce photographically, and results were not included), but indirect evidence is provided by the localisation of the enzyme in association with the small Golgi vesicles in the posterior midgut but not in the stomach. This is consistent with the proposed extracellular nature of this enzyme in the midgut of R. prolixus, and given the absence of protein digestion in the stomach (Wigglesworth, 1943), suggests that these vesicles may be secretory in nature rather than primary lysosomes. This is confirmed further by the high correlation between cathepsin B activity and the secretory vesicles in the anterior intestine, where blood protein digestion is initiated (Wigglesworth, 1943) and where synthetic and secretory organelles are most concentrated (Billingsley, 1988). Unfortunately, though, cathepsin B localisation was never observed in a secretory vesicle during fusion with the apical plasma membrane. Furthermore, fusion of a vesicle with the plasma membrane would only serve to release the contents into the space between the double membranes, which acts as a closed compartment for digestion of peptides (Billingsley and Downe, 1985; Ferreira et al., in press). It is likely that vesicle number would correlate just as well with other enzymes which show a similar cycle of activity (Houseman and Downe, 1983). Therefore, 2 other possible routes of cathepsin B secretion into the midgut lumen should be considered. In the milkweed bug, Oncopeltus fasciatus, small membranebounded vesicles were observed within the cytoplasm of the microvilli (Baerwald and Delcarpio, 1983). Similar structures have been noted in R. prol&us intestinal microvilli (Billingsley and Downe, 1983, 1985), but no cathepsin B localisation was observed in the microvilli of any midgut cells. Large fluctuations occur in the lysosome populations in both intestinal regions of R. prol&us (Billingsley, 1988), and the structure of the lysosomes at 2 hr after a blood meal is extremely similar to double membrane-bounded vesicles in the midgut of Nepa cinerea (Hemiptera) (Andries and Torpier, 1982), which are apically secreted and are responsible for ECML production. Coupled with the localisation of cathepsin B, the lysosomes are implicated as the most likely source of luminal cathepsin B in the haematophagous Hemiptera. Again, though, the mode of secretion remains to be elucidated, but the present study suggests that, unlike aminopeptidase, secretory cathepsin B is processed in the Golgi complex in preparation
Cathepsin B in Midgut of Rhodnius prolixus
301
for secretion. If the Golgi-associated, cathepsin B-positive vesicles are regarded as primary lysosorrtes, then there must be at least 2 lysosome populations, because activity is observed in the immature lysosomes, meaning that differences in the lysosomal activity within a single cell cannot be due to a maturing process. It is worth noting that the poor vesicular nature of the Golgi in R. prolixus midgut is not a result of the experimental nor preparative procedures used; more "typical", well-organised Golgi can be found in midgut endocrine cells that are adjacent in the epithelium to the digestive cells examined here (Billingsley and Downe, 1986a). Such poorly organised Golgi in the midgut cells of R. prolixus and other haematophagous insects have been noted previously (Hecker, 1977; Bauer, 191gl; Houk and Hardy, 1982; Billingsley and Downe, 1983).
A model for digestion in Rhodnius prolixus Investigations of the midgut cells with respect to ultrastructure (Billingsley and Downe, 1983, 1986a, b), morphometric analysis (Billingsley, 1988), endopolyploidy (Billingsley, in press) and aminopeptidase localisation (Billingsley and Downe, 1985) support the proposed division of the midgut into 3 functional regions (Wigglesworth, 1943) for storage (anterior midgut or stomach), mainly secretion (anterior intestine) and mainly absorption (posterior intestine). The absence of Golgiassociated cathepsin B in the stomach provides further evidence that this region is not involved in protein digestion. Similarly, the presence of secretory vesicles only in the intestine and the strong correlation between the presence of secretory vesicles and cathepsin B in the anterior intestine, further supports the role of the intestine in protein digestion. The ECML have been designated a "peritrophic membrane" role in the Hemiptera (Gutierrez and Burgos, 1978; Lane and Harrison, 1979; Bauer, 1981; Baerwald and Delcarpio, 1983; Billingsley and Downe, 1985, 1986b). In some non-haematophagous insects, well structured peritrophic membranes have been shown to separate digestive events, particularly primary and secondary hydrolytic events (Santos et al., 1983; Terra and Ferreira, 1981, 1983), and it was suggested that the ECML play a similar role in R. prolixus (Billingsley and Downe, 1985). Cathepsin B plus 2 other extracellular proteases, cathepsin D and carboxypeptidase B, are secreted to maximum activity at 6-7 days after feeding (Houseman and Downe, 1983). These enzymes are somehow secreted directly into the lumen, possibly via the double membrane-bounded "lysosomes", where they digest the blood meal away from the luminal wall. The ECML do not completely separate the midgut cells from the lumen until after this time (Billingsley and Downe, 1983, 1986b), and subsequent ECML proliferation may inhibit or retard the passage of further secretoJ:y vesicle material into the midgut lumen, resulting in a decrease in enzyme activity. In contrast, aminopeptidase is retained on the microvilli and in the ECML of the intestinal cells (Billingsley and Downe, 1985; Ferreira et al., in press) being involved in the terminal digestion of the blood meal proteins close to the microvilli. Acknowledgements--These studies were funded by a grant from the Natural Sciences and Engineering Research Council of C a n a d a to Dr A. E. R. Downe. P. F. Billingsley was further supported by a C o m m o n w e a l t h Scholarship from the Association of Universities and Colleges of Canada. We thank Mrs A. M. Hutchinson and Mrs A. Svatek for excellent technical assistance, Dr G. P. Morris for advice, and Dr W. R. Rudin for critical reading of the manuscript.
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0020 7322/88 $3.1~) + .00 © 1988 Pergamon Press plc
U L T R A S T R U C T U R A L L O C A L I S A T I O N OF C A T H E P S I N B IN T H E M I D G U T OF RHODNIUS P R O L I X U S ST,~L ( H E M I P T E R A • R E D U V I I D A E ) D U R I N G B L O O D DIGESTION
P. F. BILLINGSLEY* and A. E. R. DOWNE Department of Biology, Queen's University. Kingston, Ontario, Canada K7L 3N6
(Accepted 16 February 1988)
Abstract--The artificial substrate N-benzoyl-Dm-arginine-13-naphthylamine,was used to localise cathepsin B in midgut cells of the haematophagous insect, Rhodnius prolixus Stfd (Hemiptera : Reduviidae), during blood digestion. Cathepsin B was localised primarily in the lysosomes of cells from all 3 midgut regions and in Golgi vesicles of the digestive intestinal regions, but not in association with any other cellular structures. The timing of localisation correlated with previously described cycles of endoproteinase activity and with known ultrastructural modifications to the midgut cells. Secretory vesicles, which originated from the Golgi complexes, were present only in the intestinal regions, and in the anterior intestine, they showed a strong positive correlation (r = 0.939, P = 0.01) with post-feeding cathepsin B activity. Cathepsin B plays a major role in primary extracellular digestion of blood proteins, and is active in the midgut lumen and lysosomes rather than in association with the microvilli. Index descriptors (in addition to those in title): Blood meal digestion; enzyme localisation.
INTRODUCTION CONCURRENT wi'th the p r o d u c t i o n of digestive proteinases ( H o u s e m a n and D o w n e , 1983), the m i d g u t cells of Rhodnius prolixus exhibit several ultrastructural modifications as a result of b l o o d feeding (Bauer, 1981; Billingsley, 1985; Billingsley and D o w n e , 1983, 1985, 1986b). T h e r o u g h endoplasmic reticulum (rer) is present in large whorls before feeding, b e c o m e s dispersed within 2 hr of a blood meal, and subsequently reforms during the digestion pe:riod. T h e lysosomes also display m a r k e d structural changes during the first 2-6 hr after a blood meal and subsequently return to a structure similar to that of lysosomes in the midgut of unfed insects between 6 hr and 5 days after feeding (Billingsley and D o w n e , 1983). These changes are not observed in the anterior midgut (stomach or crop) where no protein digestion takes place (Billingsley and D o w n e , in preparation), but only in the posterior midgut (which is further divided into the anterior and posterior in~testinal regions). A t the apical cell surface, a second apical m e m b r a n e develops o v e r the microvilli 12-24 hr after feeding, and proliferation of this o u t e r *Present address: Department of Pure and Applied Biology, Imperial College, Prince Consort Road, London SW7 2BB, England. 295
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m e m b r a n e results in t h e f o r m a t i o n of c o m p l e x e x t r a c e l l u l a r m e m b r a n e layers ( E C M L ) , which a r e t h o u g h t to function as a p e r i t r o p h i c m e m b r a n e (Billingsley a n d D o w n e , 1985, 1986b). C a t h e p s i n B has n o r m a l l y b e e n d e s c r i b e d as an i n t r a c e l l u l a r p r o t e a s e , r e s t r i c t e d to t h e l y s o s o m e s ( B a r r e t t , 1977) b u t has b e e n localised at the u l t r a s t r u c t u r a l level in s e c r e t i o n g r a n u l e s of rat p a n c r e a t i c cells (Smith a n d van F r a n k , 1975). In the m i d g u t of R. prolixus ( H o u s e m a n a n d D o w n e , 1980, 1982), c a t h e p s i n B is o n e o f 3 acid p r o t e a s e s o f l y s o s o m a l origin t h a t a r e utilised in t h e p r i m a r y d i g e s t i o n o f i n g e s t e d p r o t e i n s . C a t h e p s i n B, c a t h e p s i n D a n d c a r b o x y p e p t i d a s e B a r e m o r e active in the l u m e n t h a n in t h e cells o f t h e m i d g u t a n d activities of all 3 e n z y m e s p e a k at 7 days after f e e d i n g ( H o u s e m a n a n d D o w n e , 1980, 1982, 1983). R. prolixus also utilises a m i n o p e p t i d a s e for t e r m i n a l d i g e s t i o n of the b l o o d m e a l c o m p o n e n t s ( H o u s e m a n a n d D o w n e , 1981). This e n z y m e displays a cycle of activity in r e s p o n s e to b l o o d f e e d i n g ( H o u s e m a n a n d D o w n e , 1983), a n d is localised m a i n l y on microvilli a n d l y s o s o m e s in R. prolixus intestinal cells, b u t is also a s s o c i a t e d with t h e E C M L , rer a n d large i n t r a c e l l u l a r s t o r a g e vesicles (Billingsley a n d D o w n e , 1985). C h a n g e s in t h e l o c a l i s a t i o n o f a m i n o p e p t i d a s e on the microvilli a p p e a r e d to c o r r e l a t e with t h e p e a k o f a m i n o p e p t i d a s e activity, while the c o n t i n u e d l o c a l i s a t i o n o f a m i n o p e p t i d a s e in the l y s o s o m e s p r o b a b l y accounts for the p r o l o n g e d m a i n t a i n a n c e o f e n z y m e activity a b o v e b a s e l i n e levels. T h e p r e s e n t s t u d y is d e s i g n e d to c o m p l e m e n t p r e v i o u s investigations on t h e e n z y m o l o g y a n d u l t r a s t r u c t u r e o f the m i d g u t o f R. prolixus. P a r t i c u l a r l y , the sites o f c a t h e p s i n B activity a r e d e t e r m i n e d a n d the v a r i a t i o n s in l o c a l i s a t i o n a r e u s e d to c o r r e l a t e u l t r a s t r u c t u r a l a n d b i o c h e m i c a l r e s p o n s e s to feeding. T h e sites o f activity o f c a t h e p s i n B (an acid p r o t e a s e ) are c o m p a r e d with those of a m i n o p e p t i d a s e (an a l k a l i n e e x o p e p t i d a s e ) to i m p r o v e u p o n a p r e v i o u s l y d e s c r i b e d m o d e l of b l o o d d i g e s t i o n in R. prolixus (Billingsley a n d D o w n e , 1985).
MATERIALS
AND METHODS
Rhodnius prolixus StS1 were maintained and mated females obtained as previously described (Kwan and Downe, 1977; Houseman and Downe, 1983). Midguts were dissected from mated females before, 2 and 6 hr, and 1, 2, 4-7, 10, 15, 20 and 25 days after a meal of rabbit blood. At every time point, 3-5 midguts were dissected for e~ich of the experimental and control procedures. Blocks of tissue from each midgut region (stomach, anterior intestine and posterior intestine; Wigglesworth, 1943) small enough to allow rapid fixation (Brunings and de Priester, 1971), were fixed for 1 hr in ice cold 2.5% glutaraldehyde in 0.2M sodium cacodylate-HC1 buffer, pH 7.2. After several rinses in the same buffer, cathepsin B was localised using modifications to the methods of Seligman et al. (1970) (Billingsley and Downe, 1985) and Barrett (1972), by incubating the tissue blocks in the following medium: Eight mg/ml N-benzoyl-arginine-13-De-naphthylamine (BANA), 0.5 ml; 0.1M potassium phosphate buffer pH 5.5 containing 5raM ethylenediamine tetraacetate and 3 mM dithiothreitol, 5.0 ml; 2.5 mg/ml freshly diazotised 4-aminophthalhydrazide, 2.5 ml; distilled water, 2.0 ml. (All reagents from Sigma or Fisher Scientific.) The absence of substrate was used as a control, which was found to be suitable in a previous study (Billingsley and Downe, 1985). In contrast to the aminopeptidase localisation, an alternative substrate which reacts with cathepsin B but not the azo-dye, is not commercially available. Prolonged fixation and enzyme inhibitors both reduced the reaction, but were not used as controls because of their lack of specificity. Midguts were incubated for 2 hr at room temperature with one change to freshly prepared medium after 1 hr. The tissue was then rinsed in several changes of buffer over a 30 min period, post-fixed in 2% osmium tetroxide in cacodylate buffer for 1 hr, then dehydrated through an alcohol series. Tissue blocks were embedded in Epon resin, as previously described (Billingsley and Downe, 1985) and unstained thin sections were examined in a Zeiss EM10 electron microscope. Blocks from experimentally- and control-treated tissues were blind coded and codes not broken until after assessment of the results.
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Midgut tissue wzs also prepared for electron microscopyby standard techniques, and stained with uranyl acetate and lead cilrate as previously described (Billingsleyand Downe, 1988). Electron micrographs, which bad been used for a previous morphometric study (Billingsley, 1985), were checked for the presence of secretory vesicles; these represented unbiased samples from all 3 midgut regions, and all areas of the midgut cells in approximat,.~lyequal proportions. The vesicles per electron micrograph were counted and the results subjected to a 2-way linear regression analysis against the post-feedingactivitycycleof cathepsin B (data taken from Fig. 6 of Hou~,.emanand Downe, 1983).
RESULTS
Detection and general distribution of cathepsin B activity No cathepsin B activity is detected in any structures or organelles of the midgut cells before the bloc,d meal, except for trace amounts in the lysosomes (Fig. la, b). After feeding, cathept;in B is localised as a fine, granular, electron-dense precipitate associated mainly with the lysosomes (Fig. lc-f) and occasionally with Golgi vesicles (Fig. lg-i) of the posterior midgut. The lysosomes are usually found grouped together in the midgut cells, and in any group at any time between 2 and 15 days after feeding, the amount of precipitate associated with the lysosomes may vary from trace amounts to a very dense reaction (Fig. ld, e). Precipitate is never detectable on the rough endoplasmic reticulum (rer), on the microvilli or in association with storage vesicles seen in the midgut cells.
Cathepsin B localisation after feeding Only trace anaounts of cathepsin B activity are detectable in the lysosomes but not in any other structures or organelles in any midgut region until between 1 and 2 days after the blood meal. Between 2 and 4 days precipitate begins to appear in association with the lysosomes, and from 5 days to 10-15 days, maximum precipitate is observed overlying the lysosomes in all 3 midgut regions (Fig. ld-f). In the stomach and posterior intestine, only traces of cathepsin B activity are detectable in lysosomes after 15 days, while activity in the lysosomes of the anterior intestine begins to decline between 15 and 20 days, until only trace amounts are detectable 25 days after feeding. Precipitate is often observed overlying small vesicles on the rer side of the Golgi (Fig. l g - h ) , but only in vesicles that are away from the Golgi. Golgi-associated precipitate is only detectable in the intestinal regions from 2 to 14 days after blood feeding. Traces of cathepsin B activity are sometimes observed in the periphery of the posterior midgut lumen (not shown), but no consistent pattern can be determined as the reaction is always very diffuse and faint.
Secretory vesicles in the midgut cells Secretory vesicles in R. prolixus midgut contain electron-dense material (Fig. 2), and originate from the Golgi complexes mostly in the apical third of the epithelial cells (Fig. 2a). Vesicles are found in the apical cell region (Fig. 2b) where they seem to fuse with the apical plasma membrane at the base of the microvilli (Fig. 2c). Possible vesicle contents are observed in the periphery of the midgut lumen and/or in the E C M L (Fig. 2d), but cannot be detected any further into the lumen. Secretory vesicles are never observed in stomach cells but are present in the anterior intestinal cells from 2 hr to 20 days and at all times in posterior intestinal cells (Fig. 3). The correlation coefficients between cathepsin B activity and secretory vesicle numbers are 0.939 (P = 0.01) in the anterior intestine and 0.433 (not significant) in the posterior intestine.
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FIG. 1. Cathepsin B Iocalisation in the lysosomes (a-f) and Golgi (g-i) of Rhodnius prolixus midgut cells, a. Anterior intestine, before feeding, B A N A (× 23,200). A very faint precipitate is detectable in lysosomes, b. Anterior intestine, before feeding, control (× 18,000). Colloidal material present in lysosomes, but no electron-dense precipitate, c. Posterior intestine, 7 days after feeding, B A N A ( x 12,600). Precipitate in variable amounts is detectable overlying lysosomes, d. Anterior intestine, 5 days after feeding, B A N A (× 17,600). Precipitate in some lysosomes may be very heavy whereas others show little or no reaction. Anterior intestine, 5 days after feeding, B A N A (× 13,600). f. Anterior intestine, 6 days after feeding, control (× 19,800). No precipitate on lysosomes of controls, g. Anterior intestine, 7 days after feeding, BANA (× 32,100). Dense-staining vesicles are observed on rer side of Golgi in intestinal cells, h. Anterior intestine, 6 days after feeding, B A N A (× 22,100), Vesicles from the Golgi, regularly exhibit variable amounts of precipitate, i. Anterior intestine, 7 days after feeding, control (× 17,800). No dense-staining vesicles are seen in control tissues.
Cathepsin B in the Midgut of Rhodnius prolixus
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FIG. 2. Secretory vesicles in the intestinal cells of R. prolixus. Tissue fixed by standard techniques and stained with uranyl acetate and lead citrate, a. 12 days after feeding, anterior intestine (× 6,700). Formation of secretory vesicles ( . - ~ ) in Golgi (Go) and their possible route of transport to cell surface, b. 12 days after feeding, posterior intestine (× 10,200). Secretory vesicles (SV) near apical cell surface. Microvilli, MV. c. 2 days after feeding, anterior intestine (x 35,400). Release of secretory vesicle into lumen at base of microvilli (MV). d. 6 days after feeding, anterior intestine (× 19,000). Secretory vesicles (SV) in apical cell region and vesicle-like material (.-~,-) in extracellular membrane layers (ECML).
(28) o • -~
4-
o 3~
21-
>
0
(22)(25)~ 130)~ 0
2h
2d
(18)
N3 6d
12d
20d
Time after blood feeding
FIG. 3. Number of secretory vesicles in the intestinal cells of Rhodnius prolixus. Secretory vesicles were counted on micrographs used for a previous morphometric study and represent at least 5 cells per midgut region, except at 20 days (Billingsley, 1988). Numbers in parentheses are total electron micrographs counted per time point from each midgut region. Cross-hatched bars = anterior intestine, hat zhed bars = posterior intestine. 142 electron micrographs of stomach cells were examined, but no secretory vesicles were observed.
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P.F. BILLINGSLEYand A. E. R. DOWNE
DISCUSSION The results from the present study are consistent with histochemical demonstrations of the cathepsin B in several tissues (Barrett, 1977) and with the ultrastructural localisation of cathepsin B in the B-cells of rat pancreas (Smith and van Frank, 1975). The substrates used for the ultrastructural demonstration of aminopeptidase (Billingsley and Downe, 1985) and cathepsin B (present study) do not show any cross reactivity (Barrett and Poole, 1969). This is reflected in the differences between the ultrastructural localisation of the 2 enzymes in the midgut of R. prolixus; aminopeptidase was localised as both granular and aggregated precipitates (Billingsley and Downe, 1985) which correlated with the detection by column chromatography, of 2 distinct aminopeptidases from R. prol&us midgut tissue homogenates (Houseman and Downe, 1981). Conversely, no such separation of cathepsin B activity has been demonstrated and only 1 form of localisation reaction, a fine granular precipitate, was observed. By washing and centrifuging whole posterior midguts, cathepsin B was shown to be most active in the posterior midgut lumen of R. prolbcus (Houseman and Downe, 1980), although this procedure did not allow for any check on the level of cell disruption. The present study can only partially confirm this luminal activity. A very diffuse luminal localisation was observed (which was difficult to reproduce photographically, and results were not included), but indirect evidence is provided by the localisation of the enzyme in association with the small Golgi vesicles in the posterior midgut but not in the stomach. This is consistent with the proposed extracellular nature of this enzyme in the midgut of R. prolixus, and given the absence of protein digestion in the stomach (Wigglesworth, 1943), suggests that these vesicles may be secretory in nature rather than primary lysosomes. This is confirmed further by the high correlation between cathepsin B activity and the secretory vesicles in the anterior intestine, where blood protein digestion is initiated (Wigglesworth, 1943) and where synthetic and secretory organelles are most concentrated (Billingsley, 1988). Unfortunately, though, cathepsin B localisation was never observed in a secretory vesicle during fusion with the apical plasma membrane. Furthermore, fusion of a vesicle with the plasma membrane would only serve to release the contents into the space between the double membranes, which acts as a closed compartment for digestion of peptides (Billingsley and Downe, 1985; Ferreira et al., in press). It is likely that vesicle number would correlate just as well with other enzymes which show a similar cycle of activity (Houseman and Downe, 1983). Therefore, 2 other possible routes of cathepsin B secretion into the midgut lumen should be considered. In the milkweed bug, Oncopeltus fasciatus, small membranebounded vesicles were observed within the cytoplasm of the microvilli (Baerwald and Delcarpio, 1983). Similar structures have been noted in R. prol&us intestinal microvilli (Billingsley and Downe, 1983, 1985), but no cathepsin B localisation was observed in the microvilli of any midgut cells. Large fluctuations occur in the lysosome populations in both intestinal regions of R. prol&us (Billingsley, 1988), and the structure of the lysosomes at 2 hr after a blood meal is extremely similar to double membrane-bounded vesicles in the midgut of Nepa cinerea (Hemiptera) (Andries and Torpier, 1982), which are apically secreted and are responsible for ECML production. Coupled with the localisation of cathepsin B, the lysosomes are implicated as the most likely source of luminal cathepsin B in the haematophagous Hemiptera. Again, though, the mode of secretion remains to be elucidated, but the present study suggests that, unlike aminopeptidase, secretory cathepsin B is processed in the Golgi complex in preparation
Cathepsin B in Midgut of Rhodnius prolixus
301
for secretion. If the Golgi-associated, cathepsin B-positive vesicles are regarded as primary lysosorrtes, then there must be at least 2 lysosome populations, because activity is observed in the immature lysosomes, meaning that differences in the lysosomal activity within a single cell cannot be due to a maturing process. It is worth noting that the poor vesicular nature of the Golgi in R. prolixus midgut is not a result of the experimental nor preparative procedures used; more "typical", well-organised Golgi can be found in midgut endocrine cells that are adjacent in the epithelium to the digestive cells examined here (Billingsley and Downe, 1986a). Such poorly organised Golgi in the midgut cells of R. prolixus and other haematophagous insects have been noted previously (Hecker, 1977; Bauer, 191gl; Houk and Hardy, 1982; Billingsley and Downe, 1983).
A model for digestion in Rhodnius prolixus Investigations of the midgut cells with respect to ultrastructure (Billingsley and Downe, 1983, 1986a, b), morphometric analysis (Billingsley, 1988), endopolyploidy (Billingsley, in press) and aminopeptidase localisation (Billingsley and Downe, 1985) support the proposed division of the midgut into 3 functional regions (Wigglesworth, 1943) for storage (anterior midgut or stomach), mainly secretion (anterior intestine) and mainly absorption (posterior intestine). The absence of Golgiassociated cathepsin B in the stomach provides further evidence that this region is not involved in protein digestion. Similarly, the presence of secretory vesicles only in the intestine and the strong correlation between the presence of secretory vesicles and cathepsin B in the anterior intestine, further supports the role of the intestine in protein digestion. The ECML have been designated a "peritrophic membrane" role in the Hemiptera (Gutierrez and Burgos, 1978; Lane and Harrison, 1979; Bauer, 1981; Baerwald and Delcarpio, 1983; Billingsley and Downe, 1985, 1986b). In some non-haematophagous insects, well structured peritrophic membranes have been shown to separate digestive events, particularly primary and secondary hydrolytic events (Santos et al., 1983; Terra and Ferreira, 1981, 1983), and it was suggested that the ECML play a similar role in R. prolixus (Billingsley and Downe, 1985). Cathepsin B plus 2 other extracellular proteases, cathepsin D and carboxypeptidase B, are secreted to maximum activity at 6-7 days after feeding (Houseman and Downe, 1983). These enzymes are somehow secreted directly into the lumen, possibly via the double membrane-bounded "lysosomes", where they digest the blood meal away from the luminal wall. The ECML do not completely separate the midgut cells from the lumen until after this time (Billingsley and Downe, 1983, 1986b), and subsequent ECML proliferation may inhibit or retard the passage of further secretoJ:y vesicle material into the midgut lumen, resulting in a decrease in enzyme activity. In contrast, aminopeptidase is retained on the microvilli and in the ECML of the intestinal cells (Billingsley and Downe, 1985; Ferreira et al., in press) being involved in the terminal digestion of the blood meal proteins close to the microvilli. Acknowledgements--These studies were funded by a grant from the Natural Sciences and Engineering Research Council of C a n a d a to Dr A. E. R. Downe. P. F. Billingsley was further supported by a C o m m o n w e a l t h Scholarship from the Association of Universities and Colleges of Canada. We thank Mrs A. M. Hutchinson and Mrs A. Svatek for excellent technical assistance, Dr G. P. Morris for advice, and Dr W. R. Rudin for critical reading of the manuscript.
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