Cell Tiss. Res. 170, 275-287 (1976)
Cell and Tissue Research ,~q by Springer-Verlag 1976
Digestive Enzyme Secretion in Stomoxys calcitrans (Diptera: Muscidae) * M.J. Lehane Department of Pure and Applied Zoology, The University of Leeds, England
Summary. Enzyme assays and morphological and histological studies show that the opaque zone midgut cells of the haematophagous fly Stomoxys calcitrans are responsible for the production of proteolytic digestive enzymes and that these are secreted into the gut lumen via membrane bound vesicles (MBV). The secretory cycle can be summarized as follows; initially the rough endoplasmic reticulum is stacked and the apices of the cells are packed with MBV. This is followed by a period of release characterized first by cytoplasmic extrusions containing high densities of MBV, then by microvesiculation of the microvilli combined with a progressive distribution of rough endoplasmic reticulum and lightening of the cellular cytoplasm. Glycogen appears in the cells at this stage and is gradually lost as the rough endoplasmic reticulum becomes stacked once more and the numbers of MBV build up again. The cycle which occurs regularly and synchronously in the cells of the zone repeats itself many times up to the completion of digestion of the blood meal. The secretory cycle is discussed with reference to activity in other secretory tissues.
Key words: Insect midgut - Enzyme secretion - Ultrastructure.
Introduction Digestive enzyme secretion in insects has been the subject of many light microscopical investigations, which have been reviewed by Wigglesworth (1972). Many of the investigators described a process of vacuolation in the apical halves of the midgut cells and the subsequent release of the vacuoles as globules and cytoplasmic extrusions or the breakdown of the whole cell with the release Sendoflprint requests to." Dr. M.J. Lehane, Department of Entomology, London School of Hygiene
and Tropical Medicine, London WC1E 7HT, U.K. *
The author is indebted to the Science Research Council for financial support
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of its contents. These processes were accepted as the histological basis of the secretory cycle until Day and Powning (1949) reported that the occurrence of cytoplasmic extrusions from midgut cells was not correlated with an increase in the digestive enzyme content of the gut lumen of the insects they studied, and indeed that the greatest concentrations of enzymes occurred when the midgut epithelium was cytologically uniform. Later Khan and Ford (1962) showed that cytological changes involving vacuolation occurred as a response to starvation in the gut cells of Dysdercusfasciatus. With the advent of electron microscopy the detailed interpretation of those processes reported as secretory from the earlier light microscopical studies became possible and in vertebrates Caro and Palade (1964) and Jamieson and Palade (1967a, b)working on the mammalian exocrine pancreas showed that the cell producing protein for export contains a well developed rough endoplasmic reticulum which synthesizes the protein, partitions it from the rest of the cellular cytoplasm, and transports it to a prominent Golgi apparatus which packages it into membrane bound vesicles (MBV) which carry the product to the secretory border of the cell for export. In the ultrastructural studies on the midgut cells of insects carried out so far, a well developed rough endoplasmic reticulum and Golgi apparatus has often been described but the presence of large numbers of MBV, or indeed the vacuolation processes described by light microscopists have not, and consequently the way in which the enzymes presumably synthesized by these cells are transported to the lumen of the insect gut is uncertain (Smith, 1968). The aim of the present study was to follow the ultrastructural changes in the secretory cells of an insect's midgut during the synthesis and release of digestive enzymes. The stablefly Stomoxys calcitrans is a discontinuously blood feeding insect in which the gut is separated into several distinct histological regions including a sharply defined secretory zone (Lotmar, 1949). Consequently the secretory cycle of this group of easily identifiable cells can be studied during the course of a single meal without the need to introduce an artificial starvation period with the attendant danger of producing artefacts (Khan and Ford, 1962). Champlain and Fisk (1956) have reported maximal proteolytic activity 13 h after a citrated blood meal in the midguts of stableflies; consequently changes in midgut ultrastructure have been followed throughout this period.
Materials and Methods Stomoxys calcitrans was cultured in the laboratory by the method outlined by Lehane (1975). As the age of a fly and vitellogenesis in the female both affect the cytology of the midgut, only three to five-day old adult males were used in the investigation. Experimental animals were kept at 25 ~ C. Animals unfed for 40 h to clear the gut were given an observed meal of blood. The flies were killed either unfed or at the following time intervals after feeding: 5 min; 15 min ; 40 rain; l h ; 1.1/2h; 2 h ; 3 h ; 4 h ; 5 h ; 6 h ; 7 h ; 8 h ; 9 h ; l l h ; 1 2 h ; 1 3 h ; 1 4 h ; 1 5 h ; 16h. Midguts from animals previously narcotized with carbon dioxide were dissected out and separated into regions in ice cold fixative. The stablefly stores and concentrates the blood meal in the fore midgut often termed the reservoir, and gradually passes it on via the opaque zone to the posterior midgut. The opaque zone which is 2 mm. long and forms about 8% of the length of the midgut can be located in the unfed fly by the characteristic milkiness of its cells and in the fed fly as it
Digestive Enzyme Secretion in Stomoxys calcitrans
277
surrounds the boundary between the red (undigested) and brown (partially digested) blood meal. Using mitochondrial and microvillar preservation as an index of good fixation it was found that a suitable fixative for the opaque zone cells of unfed animals was glutaraldehyde buffered to pH 7.0 in 0.1 M cacodylate for 3 h followed by a rinse in 0.1 M cacodylate with the osmolarity adjusted to 330 m.Os., then 1 h in t% osmic acid buffered to pH 7.2 with veronal acetate, made up in invertebrate salt solution. This fixative was not satisfactory for the midgut of fed animals and these were fixed in the modification of the Hirsch and Fedorko (1968) fixative as used by De Priester (1971). Tissue was dehydrated in ethanol and embedded in Epon. Silver/pale gold sections were cut, double stained with uranyl acetate and lead citrate and observed in an AEI EM6B. Proteolytic enzyme assays were carried out on portions of midgut dissected out in ice cold 0.1 M tris buffer pH 7.9. Three portions were separated, the opaque zone (M), and the midgut before (anterior, A) and the midgut after it (posterior, H). The wet weight of each portion was determined on a Cahn electrobalance. Homogenates of gut were made up to 5 ml. with buffer; immediately before assay they were heated to 43 ~ C and 25 rag. of 'Azocoll "1 was added to each test solution. Incubation was at 4 3 ~ for 90 rain. Colorimetric readings were carried out on a Beckman D B - G T spectrophotometer.
Results
Ultrastructure of Opaque Zone Cells in the Unfed Fly The structure of an opaque zone cell in the unfed fly is shown in (Fig. 2). The cell contains large quantities of rough endoplasmic reticulum much of which is formed into parallel stacks. The extent and degree of stacking is dependent on the time elapsing since the completion of digestion of the last meal and increases with it, until eventually whorls of cisternae are produced (Fig. 3). Similar responses of the rough endoplasmic reticulum to starvation have been shown by Ratcliffe and King (1970). The apical halves of the cells in the unfed fly are packed with electron dense MBV ca. 0.7 ~tm/in diameter (Fig. 2). Vesicles of a similar diameter but with a flocculent lighter content are found in the vicinity of the Golgi bodies (Fig. 4). The Golgi bodies are numerous but small and are often associated with one end of a rough endoplasmic membrane stack. The mitochondria of the cells are of two morphological types; apically there are large and often oval mitochondria with numerous parallel cristae completely crossing a dense mitochondrial matrix. In addition to these there are smaller sausage-shaped mitochondria distributed throughout the cells, but more abundant basally. In the cells of the unfed fly, many of the larger oval mitochondria show signs of disorganisation (Lehane, 1975).
Ultrastructural Changes Following a Blood Meal Five minutes after a blood meal, the regular arrangement of the apical border of the cells has begun to break down and the number of MBV in the cells is down by ca. 50%. The apices of the cells stream out into pseudopods containing high densities of MBV, and these are pinched off to give cytoplasmic extru1
Calbiochem : a substrate for the assay of most kinds of proteolytic activity
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tI
"~MBV
II
-Go
f
4
2
2'
,,,,-
1
3
Fig. 1. The secretory cycle of the opaque zone cells. 1. The opaque zone cell of the unfed fly is packed apically with membrane bound vesicles (MBV) and much of the rough endoplasmic reticulum (R) is arranged in organized arrays. Go., Golgi bodies; N., nucleus. 2. Five to fifteen minutes after feeding large portions of the apical cytoplasm containing MBV are pinched off into the gut lumen. Membrane bound vesicles many of which are producing tails at this time, are also fusing with the enclosing plasma membranes causing the eccrine release of their contents into the gut lumen (arrows). Microvesiculation of the microvilli occurs and the microvesicles (v) are released into the gut lumen where they break down. The rough endoplasmic reticulum starts to become distributed. 3. Up to fifty minutes after feeding the rough endoplasmic reticulum becomes very distributed. Only relatively small numbers of membrane bound vesicles are still present at this time in the cells. 4. Up to ninety minutes after feeding the rough endoplasmic reticulum becomes reorganized and glycogen (Gl) appears in the cells. The glycogen then disappears as a new batch of membrane bound vesicles are formed. This cycle then repeats itself to the completion of digestion. It is estimated that the cycle is repeated up to twenty times during the digestion of a single blood meal
Digestive Enzyme Secretion in Stomoxys calcitrans
279
sions (Figs. 6 and 1, 2) floating freely in the lumen of the gut. Although MBV do not fuse with one another or with other cellular organelles, they do fuse with the apical plasma membrane at this time (Fig. 8), most especially in the cytoplasmic extrusions. Many of the MBV, especially those at the cell apex, begin to show a streaming of their contents producing normally one but occasionally two tails ca. 700 A in diameter (Fig. 7). Many of these tails can be traced into the bases of microvilli. At this time small membrane bound vesicles ca. 650 A, in diameter with an electron density the same as the MBV first appear in the microvillar cores (Fig. 5). The origin of these microvesicles is uncertain, they have a similar diameter to microvesicles budded by the rough endoplasmic reticulum (Fig. 4) and to vesicles budded by the MBV (Fig. 5). They might also conceivably arise by budding of the MBV tails (which have a similar diameter to the microvesicles). The microvesicles are extruded from the sides and tips of the microvilli contained in small encircling membrane bound plasma envelopes ca. 1300 A in diameter (Fig. 5). These microvesicles are certainly exocytosed as they are to be found in various stages of disintegration in the gut lumen. Cytoplasmic extrusions become progressively fewer as secretion goes on, and the rough endoplasmic reticulum becomes more distributed, there are many more tailed zymogen granules in the cells and more microvesiculation in the microvilli up to a peak after ca. fifteen minutes. After ca. one hour the rough endoplasmic reticulum is widely distributed and only very few loosely stacked parallel arrays remain (Fig. 1, 3). The numbers of MBV are greatly reduced, some cytoplasmic extrusions may still be present and at the points at which they occur the density of MBV is highest. The microvesiculation observed earlier is now occurring at a slower rate, and glycogen begins to appear in the cells (Fig. 9). At one and a half hours after feeding the rough endoplasmic reticulum is reformed into parallel stacks, especially in the bases of the cells. The microvilli are quite regular with very few microvesicles present, glycogen is still present in small quantities in the cells and apically the cells are again becoming packed with MBV. Fig. 2. Transverse section through a relatively short opaque zone cell of an unfed fly. The apex of the cell is packed with M B V and basally much of the rough endoplasmic reticulum is formed into stacks, (R). x 5,230 Fig. 3. After a longer period of starvation than was the case with the fly of Figure 2, whorls of rough endoplasmic reticulum appear. Often M B V encircled in rough endoplasmic reticulum also appear as shown here (arrow). x 14,235 Fig. 4. The endoplasmic reticulum (R) synthesizes and carries the enzymes to the Golgi region to which they are passed in small vesicles (v) budded from regions of the rough endoplasmic reticulum which have lost their ribosomes (arrows). Condensing vesicles (C) are formed which mature into membrane bound vesicles (MBV). x 16,490
Fig. 5. Five minutes after feeding microvesicles first appear in the microvilli (v). Some M B V may bud vesicles (arrow) which have a similar diameter to the microvesicles of the microvilli, the relationships of the various vesicles in the cytoplasm is uncertain. • 57,600
280
Captions see p. 279
M.J. Lehane
Digestive Enzyme Secretion in Stomo.v~'s calcitrans
Captions see p. 279
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The cells of the opaque zone undergo these changes in synchrony and the cycle completes itself, under the conditions of this experiment in ca. 110 minutes. The cycle is continuously repeated until the completion of digestion of the blood meal (ca. 36 h) with the exception of a variation after nine to ten hours when glycogen is replaced in the cycle by small lipoid spheres (which do not show the regular periodicity within the cycle displayed by glycogen and are often seen 'out of phase'), and also at about this time the regularity of the cycles becomes disrupted for about one hour as the large apical mitochondria undergo changes similar to those seen in the unfed fly, the cytoplasm becomes packed with free ribosomes and the cells which have progressively shortened during the repeated secretory cycles regenerate to nearly their full original size.
Proteolytic Enzyme Assay The results of the proteolytic enzyme assay given in Table 1 show that the proteolytic enzyme activity of the fore midgut is never more than 12% of total midgut activity, these figures in conjunction with the maintained integrity of blood cells stored in this region preclude the fore midgut from an effective role in proteolytic digestive enzyme synthesis. By far the majority of proteolytic activity in the midgut of the unfed fly is in the opaque zone cells and the high concentration of enzymes is especially marked when expressed in terms of activity per gut unit. This high activity in the opaque zone of unfed flies coincides with the presence of large numbers of MBV in the cells, the equalization of activities of the posterior midgut and opaque zone up to 30 minutes coincides with a period of release of the contents of these MBV into the gut lumen and the build-up in the proteolytic enzyme activity of the opaque zone to one hour is accompanied by the appearance of a new batch of MBV in the opaque zone cell apices. This evidence in conjunction with the darkening of the blood and disruption of the blood cells as they pass the opaque cell zone indicates strongly that the opaque zone cells synthesize and secrete the proteolytic digestive enzymes found in the insect's midgut lumen. The decline in proteolytic activity fifteen minutes after feeding is very probably explained by the presence of inhibitors of proteolytic activity in the blood meal such as those described by Yang and Davies (1971).
Fig. 6. Five minutes after feeding large portions of cytoplasm containing MBV are streaming from the cell apex to be pinched off into the gut lumen, x 4,000 Fig. 7. Streaming of a membrane bound vesicle to produce a tail (arrow). x 52,500 Fig. 8. Some secretory release occurs from both the cytoplasmic extrusions (shown in Fig. 6) and from the apical border of the whole cell by the fusion of membrane bound vesicles with the plasma membrane (arrow). x 24,850 Fig. 9. The apex of the cell one hour after feeding contains few vesicles; glycogen (G) appears in the cell at this stage. Note the distributed rough endoplasmic reticulum and the absence of smooth endoplasmic reticulum from the cell. x 10,800
Digestive Enzyme Secretion in Stomoxvs cah'itrans
283
60
30
15
0
as % 0.3
as % 4.17
Total Activity 96.66 i 12.00
Activity/0.1 mg gut 1.33 + 0.33
as % 0.53
as % 5.41
Activity/0.1 mg gut 2.00 +_ 0.58
Total Activity 83.33 • 18.55
as % 1,95
as % 11.95
Total Activity 90.00 • 15.27
Activity/0.1 mg gut ' 2.66 + 0.66
as % 1.75
Activity/0.1 mg gut 6.40 + 1.03
Activity/0.1 mg gut 224.33 + 20.69
Total Activity 1036.00 +_ 63.59
Activity/0,1 mg gut 195.00 + 22,10
Total Activity 640.00 4- 43.58
Activity/0.1 mg gut 97,66 + 12.19
Total Activity 506.66 • 31.79
Activity/0.1 mg gut 286.93 • 36.86
Total Activity 603.33 • 102.12
Total Activity 42.00 • 16.98
as % 4.21
Opaque midgut
Anterior midgut
as % 50.00
as % 44.68
as % 52.05
as % 41.56
as % 71,47
as % 67.26
as % 78.51
as % 60.41
Activity/0. l m g gut 223.00 • 9.70
Total Activity 1186.00 _+ 73.10
Activity/0.1 mg gut 177.66 + 17.80
Total Activity 816.66 • 20.27
Activity/0.1 mg gut 36.33 + 4.09
Total Activity 156.66 +_ 32.82
Activity/0.1 mg gut 72.13 + 10.55
Total Activity 353.33 • 49.81
Posterior midgut
as % 49.7
as % 51.15
as % 47.42
as % 53.03
as % 26.59
as ~,,; 20.8
as % [9.74
as % 35.38
3
3
3
15
Replicates
Table 1. Results of the assay for proteolytic enzymes in unfed flies and flies at 15, 30 and 60 minutes after feeding. Activity is expressed in arbitrary units derived from absorbance readings by the spectrophotometer. Activities are expressed as the means of the replicates + or - the standard error. Percentage activities are expressed as percentages of the sum of the means disregarding the standard errors
V
4~
Digestive EnzymeSecretionin Stomoxys calcitrans
285
Discussion The opaque zone cells described here are typical of cells undertaking the synthesis and export of proteins (de Robertis et al,, 1970). The ultrastructural evidence shows that they do synthesise and export materials but it does not show that these are digestive enzymes. However, the correlation of the cyclical synthesis and release of material via the MBV with varying proteolytic activities of the opaque and posterior midgut zones (Table 1) and the morphological evidence of primary digestion in the darkening of the blood meal as it passes the opaque cell zone, makes it extremely likely that the opaque zone cells synthesise and secrete the majority of the proteolytic enzymes of the gut. The ultrastructural results show that the route of synthesis and packaging of proteins in the opaque zone cells follows the classical outline of rough endoplasmic reticulum to Golgi bodies drawn by Caro and Palade (1964) and Jamieson and Palade (1967a, b). A parallel between the energy requirements of the process in the vertebrate and invertebrate cell is indicated by the cyclical appearance of glycogen in the opaque zone cells immediately before, and its disappearance during the production of new batches of MBV. This can be held as evidence for the presence of an energy dependent lock between the rough endoplasmic reticulum and the Golgi bodies such as that shown in the vertebrate exocrine pancreatic cells by Jamieson and Palade (1968). Some of the MBV of fed S. calcitrans produce tails (Fig. 7). The fact that tailing in MBV can be induced by cyclic AMP as shown by Schramm et al. (1972) may be a clue to part of the mechanism triggering secretion following a blood meal taken by S. caleitrans and to the mechanism controlling the sequence of events in the secretory cycle. Whorls of rough endoplasmic reticulum like those reported here have been reported in Aedes aegypti by Bertram and Bird (1961) and in A. aegypti and three species of Anopheles by St/iubli et al. (1966). The repeated cyclical distribution and reconstitution of rough endoplasmic reticulum during the digestion of a single meal as reported here does not occur in the mosquitoes, instead the whorls undergo only one cycle of distribution and reconstitution during the digestion of one blood meal. Bertram and Bird (1961) do not speculate on the function of the whorls but St/iubli et al. (1966) have proposed that they secrete and store digestive enzymes. Several lines of evidence suggest whorls do not either actively secrete or store secreted materials. Siekevitz and Palade (1958) showed that decreasing secretory activity corresponded with an increasingly organized rough endoplasmic reticulum in guinea pig pancreatic cells and Emmelot and Benedetti (1960) and Smuckler et al. (1965) have shown whorling of rough endoplasmic reticulum in vertebrate liver cells after treating them with chemicals which completely inhibit amino acid incorporation. In addition neither Stfiubli et al. (1966) nor Bertram and Bird (1961) nor this investigation have described accumulations of electron dense material or granules in the cisternae of whorled rough endoplasmic reticulum such as those commonly found in the early stages of secretory activity (Smith, 1968) and which would presumably become particularly evident if the cisternae were storing secretory products. The distribution of the whorls is a reflection of increased synthetic
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activity in the reticulum of S. calcitrans (Table 1), an observation which is in agreement with the results of Siekevitz and Palade (1958). The cyclical reorganization of the cisternae in S. calcitrans presumably interrupts the synthetic activity, but it is proposed that reorganization and orientation of the cisternae is necessary for the transfer of the synthesis products to the Golgi bodies in S. calcitrans. The single cycle of whorling seen in the mosquitoes then correlates with the absence of production of large numbers of Golgi generated MBV in these insects. The absence of large numbers of MBV in the midgut cells of mosquitoes (and to the author's knowledge, of all other insects, except S. calcitrans studied to date) shows fundamental differences between the method of storage and secretion of digestive enzymes in S. calcitrans and that in other insects. The ultrastructural evidence suggests that storage of digestive enzymes in S. calcitrans is in MBV, the secretion of the stored material occurs by three methods; budding of regions of apical cytoplasm containing large numbers of MBV into the gut lumen, eccrine fusions of MBV with the apical plasma membrane or microvesiculation of the microvilli. Digestive enzyme storage, if there is any in insects which do not produce MBV, must presumably be in the rough endoplasmic reticulum, but to the author's knowledge no report of intracisternal granules or electron dense material in the rough endoplasmic reticulum of insect midgut cells has been made and, therefore, on the arguments presented above storage in the rough endoplasmic reticulum seems unlikely. The way in which digestive enzymes are transported to and secreted from the secretory border of other insect midgut cells remains uncertain (Smith, 1968), but vesiculation of the microvilli as reported here has been described in the midgut cells of other insects, for example by Baccetti (1962) in Dacus olea and Blaps gibba, Marshall and Cheung (1970) in Fulgora candelaria, De Priester (1971) in Calliphora erythrocephala and Nopanitaya and Misch (1974) in Sarcophaga bullata, the microvesicles described by these authors have a diameter similar to those of vesicles budded from the rough endoplasmic reticulum as do those of S. calcitrans. As the microvesicles are certainly secretory in S. calcitrans and are possibly derived from the rough endoplasmic reticulum it seems possible that transportation of digestive enzymes in vesicles derived from the rough endoplasmic reticulum and their release from microvilli might be a general secretory mechanism in insect midgut cells which if it were so would account for the paucity of Golgi system generated MBV found to date.
References Baccetti, B. : Ricerche sull'ultrastruttura dell'intestino degli insetti. II. La cellula epiteliale del mesentero in un ortotero, un coleottero e un dittero adulti. Redia 46, 157-165 (1962) Bertram, D.S., Bird, R.G.: Studies on mosquito-borne viruses in their vectors 1. The normal fine structure of the midgut epithelium of the adult female Aedes aegypti (L.) and the functional significance of its modification following a blood meal. Trans. R. Soc. trop. Med. Hyg. g5, 404-423 (1961) Caro, L.G., Palade, G.E.: Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20, 473 495 (1964)
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Champlain, R.A., Fisk, F.W.: The digestive enzymes of the stablefly Stomoxys cah'itrans. Ohio J. Sci. 56, 52 62 (t956) Day, M.F., Powning, R.F.: A study of the processes of digestion in certain insects. Aust. J. Sci. Res. (B) 2, 175 215 (1949) Emmelot, P., Benedetti, E.E.: Changes in the fine structure of rat liver cells brought about by dimethylnitrosamine. J. biophys, biochem. Cytol. 7, 393 403 (1960) Hirsch, J.G., Fedorko, M.E.: Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "post fixation" in uranyl acetate. J. Cell Biol. 38, 615 627 (1968) Jamieson, J.D., Palade, G, E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. I, Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34, 577 596 (1967a) Jamieson, J.D., Palade, G.E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. lI. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34, 597 615 (1967b) Jamieson, J.D., Palade, G.E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requiremenls. J. Cell Biol. 39, 589 603 (1968) Khan, M.R., Ford, J.B. : Studies on digestive enzyme production and its relationship to the cytology of the midgut epithelium of Dysdercus.li, sciatus (Hemiptera, Pyrrhocoridae). J. Insect Physiol. 8, 597 608 (1962) Lehane, M.J. : Observations on the structure and function of the gut in Stomoxys calcitrans (Insecta: Diptera) Ph.D. thesis, University of Leeds (1975) Lotmar, R.: Beobachtungen fiber Nahrungsaufnahme und Verdauung bei Stomoxys calcitrans. Mitt.schweiz. ent. Ges. 22, 97 115 (1949) Marshall, A.T., Cheung, W.W.K,: Ultrastructure and cytochemistry of an extensive plexiform surface coat on the midgut cells of a Fulgorid insect. J. Ultrastruct. Res. 33, 161 172 (1970) Nopanitaya, W., Misch, D.W.: Developmental cytology of the midgut in the flesh fly, Sarcophaga bullata (Parker). Tissue and Cell 6, 487 502 (1974) Priester, W. De: Ultrastructure of the midgut epithelial cells in the fly, Calliphora er)'throcephala. J. Ultrastruct. Res. 36, 783 805 (1971) Ratcliffe. N.A., King, P.E.: The effect of starvation on the fine structure of the venom system in Nasonia vitripennia. J. Insect Physiol. 16, 885-903 (1970) Robertis, E.D.P. de, Nowinski, W.W., Saez, F . A : Cell biology, 555 pp. Philadelphia - London Toronto: Saunders W.B. 1970 Schramm, M., Selinger, Z., Salomon, Y., Eytan, E., Batzri, S. : Pseudopodia formation by secretory granules. Nature (Lond.) 240, 203 205 (1972) Siekevitz, P., Palade, G.E. : A cyto-chemical study on the pancreas of the guinea pig. I1. Functional variations in the enzymatic activity of microsomes. J. biophys, biochem. Cytol. 4, 309 333 (1958) Smith, D.S.: Insect cells, their structure and function. 372 pp. Edinburgh: Oliver and Boyd 1968 Smuckler, R.E., Ross, R., Bendett, E.P.: Effects of carbon tetrachloride on guinea pig liver. Exp. molec. Path. 4, 328 339 (1965) StS.ubli, W., Freyvogel, T.A., Suter, J.: Structural modification of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopie $, 189 204 (1966) Wigglesworth. V.B.: The principles of insect physiology, 827pp. London: Chapman and Hall 1972 Yang, Y.J., Davies, D.M.: Trypsin and chymotrypsin during metamorphosis in Aedes aegypti and properties of the chymotrypsin. J. Insect Physiol. 17, 117 132 (1971)
Receiw, d March 25, 1976
Cell and Tissue Research ,~q by Springer-Verlag 1976
Digestive Enzyme Secretion in Stomoxys calcitrans (Diptera: Muscidae) * M.J. Lehane Department of Pure and Applied Zoology, The University of Leeds, England
Summary. Enzyme assays and morphological and histological studies show that the opaque zone midgut cells of the haematophagous fly Stomoxys calcitrans are responsible for the production of proteolytic digestive enzymes and that these are secreted into the gut lumen via membrane bound vesicles (MBV). The secretory cycle can be summarized as follows; initially the rough endoplasmic reticulum is stacked and the apices of the cells are packed with MBV. This is followed by a period of release characterized first by cytoplasmic extrusions containing high densities of MBV, then by microvesiculation of the microvilli combined with a progressive distribution of rough endoplasmic reticulum and lightening of the cellular cytoplasm. Glycogen appears in the cells at this stage and is gradually lost as the rough endoplasmic reticulum becomes stacked once more and the numbers of MBV build up again. The cycle which occurs regularly and synchronously in the cells of the zone repeats itself many times up to the completion of digestion of the blood meal. The secretory cycle is discussed with reference to activity in other secretory tissues.
Key words: Insect midgut - Enzyme secretion - Ultrastructure.
Introduction Digestive enzyme secretion in insects has been the subject of many light microscopical investigations, which have been reviewed by Wigglesworth (1972). Many of the investigators described a process of vacuolation in the apical halves of the midgut cells and the subsequent release of the vacuoles as globules and cytoplasmic extrusions or the breakdown of the whole cell with the release Sendoflprint requests to." Dr. M.J. Lehane, Department of Entomology, London School of Hygiene
and Tropical Medicine, London WC1E 7HT, U.K. *
The author is indebted to the Science Research Council for financial support
276
M.J. Lehane
of its contents. These processes were accepted as the histological basis of the secretory cycle until Day and Powning (1949) reported that the occurrence of cytoplasmic extrusions from midgut cells was not correlated with an increase in the digestive enzyme content of the gut lumen of the insects they studied, and indeed that the greatest concentrations of enzymes occurred when the midgut epithelium was cytologically uniform. Later Khan and Ford (1962) showed that cytological changes involving vacuolation occurred as a response to starvation in the gut cells of Dysdercusfasciatus. With the advent of electron microscopy the detailed interpretation of those processes reported as secretory from the earlier light microscopical studies became possible and in vertebrates Caro and Palade (1964) and Jamieson and Palade (1967a, b)working on the mammalian exocrine pancreas showed that the cell producing protein for export contains a well developed rough endoplasmic reticulum which synthesizes the protein, partitions it from the rest of the cellular cytoplasm, and transports it to a prominent Golgi apparatus which packages it into membrane bound vesicles (MBV) which carry the product to the secretory border of the cell for export. In the ultrastructural studies on the midgut cells of insects carried out so far, a well developed rough endoplasmic reticulum and Golgi apparatus has often been described but the presence of large numbers of MBV, or indeed the vacuolation processes described by light microscopists have not, and consequently the way in which the enzymes presumably synthesized by these cells are transported to the lumen of the insect gut is uncertain (Smith, 1968). The aim of the present study was to follow the ultrastructural changes in the secretory cells of an insect's midgut during the synthesis and release of digestive enzymes. The stablefly Stomoxys calcitrans is a discontinuously blood feeding insect in which the gut is separated into several distinct histological regions including a sharply defined secretory zone (Lotmar, 1949). Consequently the secretory cycle of this group of easily identifiable cells can be studied during the course of a single meal without the need to introduce an artificial starvation period with the attendant danger of producing artefacts (Khan and Ford, 1962). Champlain and Fisk (1956) have reported maximal proteolytic activity 13 h after a citrated blood meal in the midguts of stableflies; consequently changes in midgut ultrastructure have been followed throughout this period.
Materials and Methods Stomoxys calcitrans was cultured in the laboratory by the method outlined by Lehane (1975). As the age of a fly and vitellogenesis in the female both affect the cytology of the midgut, only three to five-day old adult males were used in the investigation. Experimental animals were kept at 25 ~ C. Animals unfed for 40 h to clear the gut were given an observed meal of blood. The flies were killed either unfed or at the following time intervals after feeding: 5 min; 15 min ; 40 rain; l h ; 1.1/2h; 2 h ; 3 h ; 4 h ; 5 h ; 6 h ; 7 h ; 8 h ; 9 h ; l l h ; 1 2 h ; 1 3 h ; 1 4 h ; 1 5 h ; 16h. Midguts from animals previously narcotized with carbon dioxide were dissected out and separated into regions in ice cold fixative. The stablefly stores and concentrates the blood meal in the fore midgut often termed the reservoir, and gradually passes it on via the opaque zone to the posterior midgut. The opaque zone which is 2 mm. long and forms about 8% of the length of the midgut can be located in the unfed fly by the characteristic milkiness of its cells and in the fed fly as it
Digestive Enzyme Secretion in Stomoxys calcitrans
277
surrounds the boundary between the red (undigested) and brown (partially digested) blood meal. Using mitochondrial and microvillar preservation as an index of good fixation it was found that a suitable fixative for the opaque zone cells of unfed animals was glutaraldehyde buffered to pH 7.0 in 0.1 M cacodylate for 3 h followed by a rinse in 0.1 M cacodylate with the osmolarity adjusted to 330 m.Os., then 1 h in t% osmic acid buffered to pH 7.2 with veronal acetate, made up in invertebrate salt solution. This fixative was not satisfactory for the midgut of fed animals and these were fixed in the modification of the Hirsch and Fedorko (1968) fixative as used by De Priester (1971). Tissue was dehydrated in ethanol and embedded in Epon. Silver/pale gold sections were cut, double stained with uranyl acetate and lead citrate and observed in an AEI EM6B. Proteolytic enzyme assays were carried out on portions of midgut dissected out in ice cold 0.1 M tris buffer pH 7.9. Three portions were separated, the opaque zone (M), and the midgut before (anterior, A) and the midgut after it (posterior, H). The wet weight of each portion was determined on a Cahn electrobalance. Homogenates of gut were made up to 5 ml. with buffer; immediately before assay they were heated to 43 ~ C and 25 rag. of 'Azocoll "1 was added to each test solution. Incubation was at 4 3 ~ for 90 rain. Colorimetric readings were carried out on a Beckman D B - G T spectrophotometer.
Results
Ultrastructure of Opaque Zone Cells in the Unfed Fly The structure of an opaque zone cell in the unfed fly is shown in (Fig. 2). The cell contains large quantities of rough endoplasmic reticulum much of which is formed into parallel stacks. The extent and degree of stacking is dependent on the time elapsing since the completion of digestion of the last meal and increases with it, until eventually whorls of cisternae are produced (Fig. 3). Similar responses of the rough endoplasmic reticulum to starvation have been shown by Ratcliffe and King (1970). The apical halves of the cells in the unfed fly are packed with electron dense MBV ca. 0.7 ~tm/in diameter (Fig. 2). Vesicles of a similar diameter but with a flocculent lighter content are found in the vicinity of the Golgi bodies (Fig. 4). The Golgi bodies are numerous but small and are often associated with one end of a rough endoplasmic membrane stack. The mitochondria of the cells are of two morphological types; apically there are large and often oval mitochondria with numerous parallel cristae completely crossing a dense mitochondrial matrix. In addition to these there are smaller sausage-shaped mitochondria distributed throughout the cells, but more abundant basally. In the cells of the unfed fly, many of the larger oval mitochondria show signs of disorganisation (Lehane, 1975).
Ultrastructural Changes Following a Blood Meal Five minutes after a blood meal, the regular arrangement of the apical border of the cells has begun to break down and the number of MBV in the cells is down by ca. 50%. The apices of the cells stream out into pseudopods containing high densities of MBV, and these are pinched off to give cytoplasmic extru1
Calbiochem : a substrate for the assay of most kinds of proteolytic activity
278
M.J. Lehane
tI
"~MBV
II
-Go
f
4
2
2'
,,,,-
1
3
Fig. 1. The secretory cycle of the opaque zone cells. 1. The opaque zone cell of the unfed fly is packed apically with membrane bound vesicles (MBV) and much of the rough endoplasmic reticulum (R) is arranged in organized arrays. Go., Golgi bodies; N., nucleus. 2. Five to fifteen minutes after feeding large portions of the apical cytoplasm containing MBV are pinched off into the gut lumen. Membrane bound vesicles many of which are producing tails at this time, are also fusing with the enclosing plasma membranes causing the eccrine release of their contents into the gut lumen (arrows). Microvesiculation of the microvilli occurs and the microvesicles (v) are released into the gut lumen where they break down. The rough endoplasmic reticulum starts to become distributed. 3. Up to fifty minutes after feeding the rough endoplasmic reticulum becomes very distributed. Only relatively small numbers of membrane bound vesicles are still present at this time in the cells. 4. Up to ninety minutes after feeding the rough endoplasmic reticulum becomes reorganized and glycogen (Gl) appears in the cells. The glycogen then disappears as a new batch of membrane bound vesicles are formed. This cycle then repeats itself to the completion of digestion. It is estimated that the cycle is repeated up to twenty times during the digestion of a single blood meal
Digestive Enzyme Secretion in Stomoxys calcitrans
279
sions (Figs. 6 and 1, 2) floating freely in the lumen of the gut. Although MBV do not fuse with one another or with other cellular organelles, they do fuse with the apical plasma membrane at this time (Fig. 8), most especially in the cytoplasmic extrusions. Many of the MBV, especially those at the cell apex, begin to show a streaming of their contents producing normally one but occasionally two tails ca. 700 A in diameter (Fig. 7). Many of these tails can be traced into the bases of microvilli. At this time small membrane bound vesicles ca. 650 A, in diameter with an electron density the same as the MBV first appear in the microvillar cores (Fig. 5). The origin of these microvesicles is uncertain, they have a similar diameter to microvesicles budded by the rough endoplasmic reticulum (Fig. 4) and to vesicles budded by the MBV (Fig. 5). They might also conceivably arise by budding of the MBV tails (which have a similar diameter to the microvesicles). The microvesicles are extruded from the sides and tips of the microvilli contained in small encircling membrane bound plasma envelopes ca. 1300 A in diameter (Fig. 5). These microvesicles are certainly exocytosed as they are to be found in various stages of disintegration in the gut lumen. Cytoplasmic extrusions become progressively fewer as secretion goes on, and the rough endoplasmic reticulum becomes more distributed, there are many more tailed zymogen granules in the cells and more microvesiculation in the microvilli up to a peak after ca. fifteen minutes. After ca. one hour the rough endoplasmic reticulum is widely distributed and only very few loosely stacked parallel arrays remain (Fig. 1, 3). The numbers of MBV are greatly reduced, some cytoplasmic extrusions may still be present and at the points at which they occur the density of MBV is highest. The microvesiculation observed earlier is now occurring at a slower rate, and glycogen begins to appear in the cells (Fig. 9). At one and a half hours after feeding the rough endoplasmic reticulum is reformed into parallel stacks, especially in the bases of the cells. The microvilli are quite regular with very few microvesicles present, glycogen is still present in small quantities in the cells and apically the cells are again becoming packed with MBV. Fig. 2. Transverse section through a relatively short opaque zone cell of an unfed fly. The apex of the cell is packed with M B V and basally much of the rough endoplasmic reticulum is formed into stacks, (R). x 5,230 Fig. 3. After a longer period of starvation than was the case with the fly of Figure 2, whorls of rough endoplasmic reticulum appear. Often M B V encircled in rough endoplasmic reticulum also appear as shown here (arrow). x 14,235 Fig. 4. The endoplasmic reticulum (R) synthesizes and carries the enzymes to the Golgi region to which they are passed in small vesicles (v) budded from regions of the rough endoplasmic reticulum which have lost their ribosomes (arrows). Condensing vesicles (C) are formed which mature into membrane bound vesicles (MBV). x 16,490
Fig. 5. Five minutes after feeding microvesicles first appear in the microvilli (v). Some M B V may bud vesicles (arrow) which have a similar diameter to the microvesicles of the microvilli, the relationships of the various vesicles in the cytoplasm is uncertain. • 57,600
280
Captions see p. 279
M.J. Lehane
Digestive Enzyme Secretion in Stomo.v~'s calcitrans
Captions see p. 279
281
282
M.J. Lehane
The cells of the opaque zone undergo these changes in synchrony and the cycle completes itself, under the conditions of this experiment in ca. 110 minutes. The cycle is continuously repeated until the completion of digestion of the blood meal (ca. 36 h) with the exception of a variation after nine to ten hours when glycogen is replaced in the cycle by small lipoid spheres (which do not show the regular periodicity within the cycle displayed by glycogen and are often seen 'out of phase'), and also at about this time the regularity of the cycles becomes disrupted for about one hour as the large apical mitochondria undergo changes similar to those seen in the unfed fly, the cytoplasm becomes packed with free ribosomes and the cells which have progressively shortened during the repeated secretory cycles regenerate to nearly their full original size.
Proteolytic Enzyme Assay The results of the proteolytic enzyme assay given in Table 1 show that the proteolytic enzyme activity of the fore midgut is never more than 12% of total midgut activity, these figures in conjunction with the maintained integrity of blood cells stored in this region preclude the fore midgut from an effective role in proteolytic digestive enzyme synthesis. By far the majority of proteolytic activity in the midgut of the unfed fly is in the opaque zone cells and the high concentration of enzymes is especially marked when expressed in terms of activity per gut unit. This high activity in the opaque zone of unfed flies coincides with the presence of large numbers of MBV in the cells, the equalization of activities of the posterior midgut and opaque zone up to 30 minutes coincides with a period of release of the contents of these MBV into the gut lumen and the build-up in the proteolytic enzyme activity of the opaque zone to one hour is accompanied by the appearance of a new batch of MBV in the opaque zone cell apices. This evidence in conjunction with the darkening of the blood and disruption of the blood cells as they pass the opaque cell zone indicates strongly that the opaque zone cells synthesize and secrete the proteolytic digestive enzymes found in the insect's midgut lumen. The decline in proteolytic activity fifteen minutes after feeding is very probably explained by the presence of inhibitors of proteolytic activity in the blood meal such as those described by Yang and Davies (1971).
Fig. 6. Five minutes after feeding large portions of cytoplasm containing MBV are streaming from the cell apex to be pinched off into the gut lumen, x 4,000 Fig. 7. Streaming of a membrane bound vesicle to produce a tail (arrow). x 52,500 Fig. 8. Some secretory release occurs from both the cytoplasmic extrusions (shown in Fig. 6) and from the apical border of the whole cell by the fusion of membrane bound vesicles with the plasma membrane (arrow). x 24,850 Fig. 9. The apex of the cell one hour after feeding contains few vesicles; glycogen (G) appears in the cell at this stage. Note the distributed rough endoplasmic reticulum and the absence of smooth endoplasmic reticulum from the cell. x 10,800
Digestive Enzyme Secretion in Stomoxvs cah'itrans
283
60
30
15
0
as % 0.3
as % 4.17
Total Activity 96.66 i 12.00
Activity/0.1 mg gut 1.33 + 0.33
as % 0.53
as % 5.41
Activity/0.1 mg gut 2.00 +_ 0.58
Total Activity 83.33 • 18.55
as % 1,95
as % 11.95
Total Activity 90.00 • 15.27
Activity/0.1 mg gut ' 2.66 + 0.66
as % 1.75
Activity/0.1 mg gut 6.40 + 1.03
Activity/0.1 mg gut 224.33 + 20.69
Total Activity 1036.00 +_ 63.59
Activity/0,1 mg gut 195.00 + 22,10
Total Activity 640.00 4- 43.58
Activity/0.1 mg gut 97,66 + 12.19
Total Activity 506.66 • 31.79
Activity/0.1 mg gut 286.93 • 36.86
Total Activity 603.33 • 102.12
Total Activity 42.00 • 16.98
as % 4.21
Opaque midgut
Anterior midgut
as % 50.00
as % 44.68
as % 52.05
as % 41.56
as % 71,47
as % 67.26
as % 78.51
as % 60.41
Activity/0. l m g gut 223.00 • 9.70
Total Activity 1186.00 _+ 73.10
Activity/0.1 mg gut 177.66 + 17.80
Total Activity 816.66 • 20.27
Activity/0.1 mg gut 36.33 + 4.09
Total Activity 156.66 +_ 32.82
Activity/0.1 mg gut 72.13 + 10.55
Total Activity 353.33 • 49.81
Posterior midgut
as % 49.7
as % 51.15
as % 47.42
as % 53.03
as % 26.59
as ~,,; 20.8
as % [9.74
as % 35.38
3
3
3
15
Replicates
Table 1. Results of the assay for proteolytic enzymes in unfed flies and flies at 15, 30 and 60 minutes after feeding. Activity is expressed in arbitrary units derived from absorbance readings by the spectrophotometer. Activities are expressed as the means of the replicates + or - the standard error. Percentage activities are expressed as percentages of the sum of the means disregarding the standard errors
V
4~
Digestive EnzymeSecretionin Stomoxys calcitrans
285
Discussion The opaque zone cells described here are typical of cells undertaking the synthesis and export of proteins (de Robertis et al,, 1970). The ultrastructural evidence shows that they do synthesise and export materials but it does not show that these are digestive enzymes. However, the correlation of the cyclical synthesis and release of material via the MBV with varying proteolytic activities of the opaque and posterior midgut zones (Table 1) and the morphological evidence of primary digestion in the darkening of the blood meal as it passes the opaque cell zone, makes it extremely likely that the opaque zone cells synthesise and secrete the majority of the proteolytic enzymes of the gut. The ultrastructural results show that the route of synthesis and packaging of proteins in the opaque zone cells follows the classical outline of rough endoplasmic reticulum to Golgi bodies drawn by Caro and Palade (1964) and Jamieson and Palade (1967a, b). A parallel between the energy requirements of the process in the vertebrate and invertebrate cell is indicated by the cyclical appearance of glycogen in the opaque zone cells immediately before, and its disappearance during the production of new batches of MBV. This can be held as evidence for the presence of an energy dependent lock between the rough endoplasmic reticulum and the Golgi bodies such as that shown in the vertebrate exocrine pancreatic cells by Jamieson and Palade (1968). Some of the MBV of fed S. calcitrans produce tails (Fig. 7). The fact that tailing in MBV can be induced by cyclic AMP as shown by Schramm et al. (1972) may be a clue to part of the mechanism triggering secretion following a blood meal taken by S. caleitrans and to the mechanism controlling the sequence of events in the secretory cycle. Whorls of rough endoplasmic reticulum like those reported here have been reported in Aedes aegypti by Bertram and Bird (1961) and in A. aegypti and three species of Anopheles by St/iubli et al. (1966). The repeated cyclical distribution and reconstitution of rough endoplasmic reticulum during the digestion of a single meal as reported here does not occur in the mosquitoes, instead the whorls undergo only one cycle of distribution and reconstitution during the digestion of one blood meal. Bertram and Bird (1961) do not speculate on the function of the whorls but St/iubli et al. (1966) have proposed that they secrete and store digestive enzymes. Several lines of evidence suggest whorls do not either actively secrete or store secreted materials. Siekevitz and Palade (1958) showed that decreasing secretory activity corresponded with an increasingly organized rough endoplasmic reticulum in guinea pig pancreatic cells and Emmelot and Benedetti (1960) and Smuckler et al. (1965) have shown whorling of rough endoplasmic reticulum in vertebrate liver cells after treating them with chemicals which completely inhibit amino acid incorporation. In addition neither Stfiubli et al. (1966) nor Bertram and Bird (1961) nor this investigation have described accumulations of electron dense material or granules in the cisternae of whorled rough endoplasmic reticulum such as those commonly found in the early stages of secretory activity (Smith, 1968) and which would presumably become particularly evident if the cisternae were storing secretory products. The distribution of the whorls is a reflection of increased synthetic
286
M.J. Lehane
activity in the reticulum of S. calcitrans (Table 1), an observation which is in agreement with the results of Siekevitz and Palade (1958). The cyclical reorganization of the cisternae in S. calcitrans presumably interrupts the synthetic activity, but it is proposed that reorganization and orientation of the cisternae is necessary for the transfer of the synthesis products to the Golgi bodies in S. calcitrans. The single cycle of whorling seen in the mosquitoes then correlates with the absence of production of large numbers of Golgi generated MBV in these insects. The absence of large numbers of MBV in the midgut cells of mosquitoes (and to the author's knowledge, of all other insects, except S. calcitrans studied to date) shows fundamental differences between the method of storage and secretion of digestive enzymes in S. calcitrans and that in other insects. The ultrastructural evidence suggests that storage of digestive enzymes in S. calcitrans is in MBV, the secretion of the stored material occurs by three methods; budding of regions of apical cytoplasm containing large numbers of MBV into the gut lumen, eccrine fusions of MBV with the apical plasma membrane or microvesiculation of the microvilli. Digestive enzyme storage, if there is any in insects which do not produce MBV, must presumably be in the rough endoplasmic reticulum, but to the author's knowledge no report of intracisternal granules or electron dense material in the rough endoplasmic reticulum of insect midgut cells has been made and, therefore, on the arguments presented above storage in the rough endoplasmic reticulum seems unlikely. The way in which digestive enzymes are transported to and secreted from the secretory border of other insect midgut cells remains uncertain (Smith, 1968), but vesiculation of the microvilli as reported here has been described in the midgut cells of other insects, for example by Baccetti (1962) in Dacus olea and Blaps gibba, Marshall and Cheung (1970) in Fulgora candelaria, De Priester (1971) in Calliphora erythrocephala and Nopanitaya and Misch (1974) in Sarcophaga bullata, the microvesicles described by these authors have a diameter similar to those of vesicles budded from the rough endoplasmic reticulum as do those of S. calcitrans. As the microvesicles are certainly secretory in S. calcitrans and are possibly derived from the rough endoplasmic reticulum it seems possible that transportation of digestive enzymes in vesicles derived from the rough endoplasmic reticulum and their release from microvilli might be a general secretory mechanism in insect midgut cells which if it were so would account for the paucity of Golgi system generated MBV found to date.
References Baccetti, B. : Ricerche sull'ultrastruttura dell'intestino degli insetti. II. La cellula epiteliale del mesentero in un ortotero, un coleottero e un dittero adulti. Redia 46, 157-165 (1962) Bertram, D.S., Bird, R.G.: Studies on mosquito-borne viruses in their vectors 1. The normal fine structure of the midgut epithelium of the adult female Aedes aegypti (L.) and the functional significance of its modification following a blood meal. Trans. R. Soc. trop. Med. Hyg. g5, 404-423 (1961) Caro, L.G., Palade, G.E.: Protein synthesis, storage and discharge in the pancreatic exocrine cell. An autoradiographic study. J. Cell Biol. 20, 473 495 (1964)
Digestive Enzyme Secretion in Stomoxys calcitrans
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Champlain, R.A., Fisk, F.W.: The digestive enzymes of the stablefly Stomoxys cah'itrans. Ohio J. Sci. 56, 52 62 (t956) Day, M.F., Powning, R.F.: A study of the processes of digestion in certain insects. Aust. J. Sci. Res. (B) 2, 175 215 (1949) Emmelot, P., Benedetti, E.E.: Changes in the fine structure of rat liver cells brought about by dimethylnitrosamine. J. biophys, biochem. Cytol. 7, 393 403 (1960) Hirsch, J.G., Fedorko, M.E.: Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "post fixation" in uranyl acetate. J. Cell Biol. 38, 615 627 (1968) Jamieson, J.D., Palade, G, E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. I, Role of the peripheral elements of the Golgi complex. J. Cell Biol. 34, 577 596 (1967a) Jamieson, J.D., Palade, G.E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. lI. Transport to condensing vacuoles and zymogen granules. J. Cell Biol. 34, 597 615 (1967b) Jamieson, J.D., Palade, G.E. : Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requiremenls. J. Cell Biol. 39, 589 603 (1968) Khan, M.R., Ford, J.B. : Studies on digestive enzyme production and its relationship to the cytology of the midgut epithelium of Dysdercus.li, sciatus (Hemiptera, Pyrrhocoridae). J. Insect Physiol. 8, 597 608 (1962) Lehane, M.J. : Observations on the structure and function of the gut in Stomoxys calcitrans (Insecta: Diptera) Ph.D. thesis, University of Leeds (1975) Lotmar, R.: Beobachtungen fiber Nahrungsaufnahme und Verdauung bei Stomoxys calcitrans. Mitt.schweiz. ent. Ges. 22, 97 115 (1949) Marshall, A.T., Cheung, W.W.K,: Ultrastructure and cytochemistry of an extensive plexiform surface coat on the midgut cells of a Fulgorid insect. J. Ultrastruct. Res. 33, 161 172 (1970) Nopanitaya, W., Misch, D.W.: Developmental cytology of the midgut in the flesh fly, Sarcophaga bullata (Parker). Tissue and Cell 6, 487 502 (1974) Priester, W. De: Ultrastructure of the midgut epithelial cells in the fly, Calliphora er)'throcephala. J. Ultrastruct. Res. 36, 783 805 (1971) Ratcliffe. N.A., King, P.E.: The effect of starvation on the fine structure of the venom system in Nasonia vitripennia. J. Insect Physiol. 16, 885-903 (1970) Robertis, E.D.P. de, Nowinski, W.W., Saez, F . A : Cell biology, 555 pp. Philadelphia - London Toronto: Saunders W.B. 1970 Schramm, M., Selinger, Z., Salomon, Y., Eytan, E., Batzri, S. : Pseudopodia formation by secretory granules. Nature (Lond.) 240, 203 205 (1972) Siekevitz, P., Palade, G.E. : A cyto-chemical study on the pancreas of the guinea pig. I1. Functional variations in the enzymatic activity of microsomes. J. biophys, biochem. Cytol. 4, 309 333 (1958) Smith, D.S.: Insect cells, their structure and function. 372 pp. Edinburgh: Oliver and Boyd 1968 Smuckler, R.E., Ross, R., Bendett, E.P.: Effects of carbon tetrachloride on guinea pig liver. Exp. molec. Path. 4, 328 339 (1965) StS.ubli, W., Freyvogel, T.A., Suter, J.: Structural modification of the endoplasmic reticulum of midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopie $, 189 204 (1966) Wigglesworth. V.B.: The principles of insect physiology, 827pp. London: Chapman and Hall 1972 Yang, Y.J., Davies, D.M.: Trypsin and chymotrypsin during metamorphosis in Aedes aegypti and properties of the chymotrypsin. J. Insect Physiol. 17, 117 132 (1971)
Receiw, d March 25, 1976
