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Intracellular distribution of ribosomes in midgut cells of the malaria mosquito, Anopheles stephensi (Liston) (Insecta: Diptera) in response to feeding
Int. at. InsectMorphol.& Embryol. 7(3): 267-272.
©PergamonPressLtd. 1978.Printed in Great Britain.
0020-7322/78/0601-0267502.00'0
INTRACELLULAR DISTRIBUTION OF RIBOSOMES IN MIDGUT CELLS OF THE MALARIA MOSQUITO, ANOPHELES STEPHENSI (LISTON) (INSECTA "DIPTERA) IN RESPONSE TO FEEDING HERMANN HECKER
Schweiz. Tropeninstitut, Socinstrasse 57, CH-4051, Basel, Switzerland (Accepted 9 January 1978)
Abstract--In the midgut cells (stomach) of female Anopheles stephensi the numerical density of total free and membrane-bound ribosomes (= number per cytoplasmic volume) is not greatly changed during digestion of the first blood meal. Together with an increase in cytoplasmic volume, the absolute number of ribosomes per epithelial cell is however greatly increased. During digestion, the ratio of membrane-bound-to-free ribosomes is shifted in favour of the former, indicating enhanced synthesis of proteins destined for exocytosis (e.g. digestive enzymes). About 400 ribosomes are bound to 1 /zm2 of rough endoplasmic reticulum (rer). In the first 12 hr after blood meal, this number drops temporarily, pointing to a lag between ribosome synthesis and attachment to the rer and proliferation of the membrane system. Index descriptors (in addition to those in the title): Blood digestion, protein synthesis, cellular dynamics, electron microscopy, morphometry. INTRODUCTION IT HAS been previously demonstrated that during digestion of the first blood meal by A. stephensi the volume of the cells of the stomach epithelium (~-- posterior part of midgut)
increases and most of the organelles and membrane systems of these cells proliferate (Hecker, 1977). Thereby the absolute surface area of the rough endoplasmic reticulum (rer), for instance, becomes enlarged 4-6 times. These morphological findings suggested a considerably raised functional capacity and activity of the midgut epithelium during digestion, which can be correlated to the formation of digestive enzymes (Gooding, 1975) and of the peritrophic membrane (Freyvogel and St/iubli, 1965; Gander, 1968). At the same time, products of digestion are absorbed (Rudin et al., in preparation). Other questions remained open. One of them concerned the ribosomes and their behaviour during blood digestion when the synthesis of structural and secreted proteins is enhanced. Does their numerical density remain constant? Does their absolute number per cell remain constant, or does synthesis of new ribosomes occur? Is there a change in the ratio of membranebound-to-free ribosomes, etc.? In order to answer these questions, we investigated morphometrically the dynamics of the ribosome population within the midgut cells of A. stephensi at various physiological stages before a blood meal, during and after digestion. MATERIAL AND METHODS 1. Mosquitoes and physiological stages A. stephensi derived from a strain kept at the London School of Hygiene and Tropical Medicine and had
been reared in our laboratory since 1971, according to Geigy and Herbig (1955). Midguts of females of the 267
268
HERMANNHECKER
following stages were used: 1. ready for first blood meal = 1 day after emergence (ldae); 2. digestion of blood meal = 1 dae + 0.5 days after blood meal on mice (0.Sdabm); 3. digestion of blood meal = ldae + 1 day after blood meal (ldabm); 4. digestion completed ~ ldae ÷ 3.5 days after blood meal (3.5dabm). 2. Preparation methods and morphometry
The anterior (A-part, A) and posterior parts (P-part, P, stomach) of the midguts were prepared for electron microscopy according to Hecker et al. (1974). Numerical densities of ribosomes (Nvri = number of ribosomes/t~m3 cytoplasm) were evaluated by stereological principles (Weibel, 1973), using the technique of Simar (1973). The following formula was used for the calculations: Nri 1 Nvr~ = A~r " 1) + T - - 2h ; where Nri/Ar = number of ribosome profiles counted on the area of thin section investigated (~mZ); /) ~ diameter of ribosomes ~ 0.02/~m; T = section thickness, determined according to Small (1968) 0.035 ~m in hungry mosquitoes, ~ 0.038 ~m in blood fed mosquitoes; h -- height of smallest recognisable cap sections of ribosome profiles in thin sections -- 1/4/) (Simar, 1973). Numerical densities of m e m b r a n e - b o u n d (Nvrim) and free ribosomes (Nvrif) were evaluated. This also enabled us to calculate the ratio o f membrane-bound-to-free ribosomes (rim/rif). By adding Nvrim to Nvrif the density o f total ribosomes ( N v , ) could be obtained. Absolute cytoplasmic volumes of the epithelial cells (Vcy = t~m3/epithelial cell) and surface densities o f rer (Svre, = /~m 2 membrane/tzm 3 cytoplasm) have been previously investigated (Hecker, 1977). Vcy and N v values were used to calculate approximate numbers o f ribosomes per epithelial cell (Nri, Nrim, Nrif). Nvrim and Svrer were used to calculate the ribosome n u m b e r per unit area o f rer membrane: Nvrim
Svrer
Nrim tLm2rer
Parameter results were compared statistically (T-test, significance limit 2 P < 0 . 0 5 ) . F o r a complete description of the methods the reader is referred to Simar (1973) and Hecker et al. (1974). R E S U L T S (TABLE1) 1. Numerical densities o f ribosomes and ratio o f membrane-bound-to-free ribosomes
In the cytoplasm o f the midgut epithelial cells o f female A. stephensi both membraneb o u n d and free ribosomes are present (Fig. 1). Before the first blood meal the numerical density o f m e m b r a n e - b o u n d ribosomes does not differ significantly between the anterior and posterior parts o f the midgut. During digestion of the first blood-meal the numerical density o f m e m b r a n e - b o u n d ribosomes in the posterior part is significantly increased. After complete digestion the same value as before the blood meal is found. The numerical density o f free ribosomes differs significantly between both midgut parts but shows no significant alterations within the posterior part during digestion. Therefore the numerical density o f total free and m e m b r a n e - b o u n d ribosomes is also not significantly changed during digestion. In every stage examined more free than m e m b r a n e - b o u n d ribosomes are present in the epithelial cells. The ratio o f membrane-bound-to-free ribosomes is similar in both midgut parts before the blood meal. During digestion in the posterior part it is increased but is reduced again when digestion is completed.
269
Intracellular distribution of ribosomes TABLE 1. Stage
Part Nwi
Nvrim Nvm
rim/rif
Vcy*
Nri X 10 6
I dae
A
4000 1300 2700 0.48 450 1.8 200 4- 100 ± 200 ~ 0.05 ± 80 .1_ 0.3
P
5000 1500 3500 0.42 980 300 ± 200 ~ 200 ± 0.05 ± 260
S
ns
0.5dabm P ldabm
P
s
S
ns
ns
s
4.7 ± 1.1
ns
s
5700 1900 3800 0.50 1950 11.I :~_ 300 ± 100 ± 200 ± 0.02 -- 370 ± 2.2 ns ns ns s ns 5800 2200 3600 0.61 3590 20.8 ± 500 ± 200 ± 300 ± 0.04 ± 870 ± 5.3 ns
3.5dabm P
ns
S
ns
S
lqS
5400 1500 3900 0.40 2360 12.7 ± 400 ± 100 ± 300 -L 0.01 __L 310 ± 1.7
Nrim
Nrif
/ 10 6
, 10 6
0.6 ~ 0.1
1.2 ± 0.2
S
1.3 :k 0.3 s
3.6 ± 0.7 ns 7.5 ± 1.7 S
3.6 J_: 0.5
Sv,e,* Nv,im/Sv,e, 3.06 J- 0.23
420 L 40
3.36 -- 0.28
430 :~. 20
6.54 ± 0.28 5.11 ~ 0.22
290 ± 20 s 430 ~ 30
3.90 ~ 0.14
390 ~ 20
S
3.4 .~ 0.9
ns
s
7.5 :- 1.5 ns 13.3 _i: 3.6
s
ns
9.2 ± 1.2
ns
A. stephensi, female, midgut epithelium. Means ± SE of ribosomal parameters. Vertically adjacent values are compared, s = significant, ns = not significant, * = values from Hecker (1977). ldae = mosquitoes ready for first blood meal, 0.5dabm and ldabm = during digestion, 3.5dabm = digestion completed; A = anterior, P = posterior part of midgut; Nv,~ = numerical density of total ribosomes, Nv,m, = of membrane-bound, Nv,~f = of free ribosomes; rim/rif = ratio of membrane-bound-to-free ribosomes; Vcy = volume of cytoplasm per epithelial cell; Nri = number of total ribosomes per cell, Nrim := of membrane-bound, Nrif = of free ribosomes; Sv,e, = surface density of rer; Nv,,m/Sw~r = number of ribosomes per unit area of rer. 2. Absolute number of ribosomes per epithelial cell Before the first b l o o d meal the absolute cell volume is markedly higher in the posterior t h a n in the anterior part of the midgut. By calculating the absolute n u m b e r s o f m e m b r a n e b o u n d a n d free ribosomes, it is obvious that the cells of the posterior part c o n t a i n m o r e ribosomes o f b o t h kinds. D u r i n g digestion in the posterior part a n increase in the absolute ribosome n u m b e r s is observed, followed by a reduction after digestion. Thereby, t h e absolute cell v o l u m e a n d the absolute n u m b e r s of ribosomes per epithelial cell are n o t reduced to the same values measured before the first blood meal. The changes are not always significant which is due to the high standard errors of the absolute parameters.
3. Number of ribosomes per unit area of rer Before the first blood meal nearly the same n u m b e r of ribosomes are b o u n d to I /zm-' o f r e r - m e m b r a n e s in both midgut parts (anterior: 420, posterior: 430). I n the posterior part 0.5 days after the b l o o d meal significantly fewer ribosomes per unit area are found. 1 day after the blood meal, when the epithelial cells are still in full activity, the same ratio as before the blood meal is reached. This also remains practically the same in the stage investigated after digestion. DISCUSSION I n the posterior part of the m i d g u t (stomach) o f A. stephensi, d u r i n g digestion o f the first b l o o d meal, it has been d e m o n s t r a t e d that the absolute n u m b e r s of m e m b r a n e - b o u n d a n d free ribosomes increase drastically per epithelial cell. D u r i n g this phase of high cellular ~IA~ 7 ] 3 - - r
270
HERMANN HECKER
FXG. 1. Anopheles stephensi, female, midgut, P-part, ldae. Basis of epithelial cell. Membrane-
bound ribosomes (A), free ribosomes (A), nucleus (nu), basal labyrinth (lab), basal lamina (bl). ?: 33.000
activity (Bertram and Bird, 1961 ; Freyvogel and St/iubli, 1965; St/iubli et al., 1966; Gander, 1968; Hecker, 1977) synthesis of ribosomes must therefore take place. When digestion is completed, the number of ribosomes is not reduced to the value measured before the blood meal. This runs parallel to the cytoplasmic cell volume which is only partly reduced and which may be an expression of an ongoing differentiation process stimulated by the digestion of the first blood meal. The numerical density of total membrane-bound and free ribosomes is, despite some significant differences (NrrJm), not greatly changed during digestion. The increase in ribosome number per cell is therefore mainly a consequence of the increase in cell volume or vice versa, and it seems that a more or less constant number of ribosomes per unit volume cytoplasm must be present for the functions of the midgut cells.
lntracellular distribution of ribosomes
271
On our micrographs used for morphometry it was sometimes difficult to distinguish between free and membrane-bound ribosomes. This was especially the case when the rer membranes were hit tangentially (cf. Mclntosh and O'Toole, 1976, review article). As we were comparing several physiological stages by the same technique, we assume that the counting errors are similar throughout the investigation. In every physiological stage examined more free than membrane-bound ribosomes are counted. During digestion the ratio of membrane-bound-to-free ribosomes is shifted in favour of the former. This may be correlated with the synthesis of proteins which are secreted by the cells as for instance digestive enzymes (Gooding, 1975; Briegel and Lea, 1975; Yeates, personal communication). Before the first blood meal, about 400 ribosomes are bound to 1/~m 2 of rer membrane in both midgut parts. In the posterior part 12 hr after blood intake this ratio is lowered to about 7 5 ~ indicating that at the beginning of digestion the synthesis of ribosomes and their attachment to the rer takes place after membrane synthesis and proliferation, in spite of the fact that the synthesis of digestive enzymes, in particular proteases, is already increased at that moment (Gooding, 1975; Briegel and Lea, 1975; Yeates, personal communication). Gooding (1973) and Fuchs and Fong (1976) have demonstrated experimentally the "de novo synthesis" of proteases in .4edes aegypti. It may be assumed that in A n o p h e l e s midgut proteases are similarly newly synthesized during digestion (Hecker, 1977). Twenty-four hours after blood meal the pre-blood meal ratio of ribosome number per unit area of rer is established again. Therefore in this phase of digestion, synthesis and attachment of ribosomes is faster than membrane proliferation. When digestion is completed, membrane-bound ribosomes and rer membranes are reduced simultaneously and their ratio is not significantly altered, while the cell volume, the rer and other cellular components are reduced. This could point to an optimal functional unit of the number of ribosomes per unit area of membrane within the midgut cells of .4. stephensi stomach. Acknowledgements--1 thank Profs. T. A. Freyvogel (Swiss Tropical Institute, Basel) and E. S. Gander (Dept. Biol. Cel., Univ. de Brasilia), Drs. P. H. Burri (Anat. Inst., Univ. Bern), W. Rudin and R. Yeates (Swiss Trop. Inst.) for critical discussion of the manuscript. The skillful technical assistance of Miss C. Fauser and the typing of the manuscript by Miss U. Steffen are gratefully acknowledged.
REFERENCES BERTRAM,D. S. and R. G. BIRD. 1961. Studies on mosquito-borne viruses in their vectors. I. The normal fine structure of the midgut epithelium of the adult female Aedes aegypti L. and the functional significance of its modification followinga blood meal. Trans. R. Soc. Trop. Med. Hyg. 55: 404-23. BRIEGEL,H. and A. O. LEA. 1975. Relationship between protein and proteolytic activity in the midgut of" mosquitoes. J. Insect Physiol. 21: 1597-1604. FREYVOGEL,T. A. and W. ST~,UBLI.1965. The formation of the peritrophic membrane in Culicidae. Acta Trop. 22:118-47. FUCHS, M. S. and W. F. FONG. 1976. Inhibition of blood digestion by a-amanitin and actinomycin D and its effect on ovarian development in Aedes aegypti L. J. Insect Physiol. 22: 465-72. GANDER, E. 1968. Zur Histologie und Histochemie des Mitteldarmes von Aedes aegypti und Anopheles stephensi in Zusammenhang mit der Blutverdauung. Acta Trop. 25:133-75. GEIGY, R. und A. HERBIG.1955. Erreger und Uebertr~iger tropischer Krankheiten. Acta Trop. Suppl. 6. Recht und Gesellschaft, Basel. GOODING, R. H. 1973. The digestive processes of haematophagous insects. IV. Secretion of trypsin by Aedes aegypti L. Can. Entomol. 105: 599-603. GOODING,R. H. 1975. Digestive enzymes and their control in haematophagous arthropods. Acta Trop. 32: 96-111. HIECKIER,H. 1977. Structure and function of midgut epithelial cells in Culicidae mosquitoes. Cell Tissue Res. 184: 321-41. HECKLER,H., R. BRUN, C. RIEINHARDTand P. H. BURRI. 1974. Morphometric analysis of the midgut of female Aedes aegypti under various physiological conditions. Cell Tissue Res. 152: 31-49~
272
HERMANNHECKER
MCINTOSH, P. R. and K. O'TOOLE. 1976. The interaction of ribosomes and membranes in animal cells. Biochim. Biophys. Acta 457: 171-212. SIMAR, L. 1973. L'ultrastructure des ganglions lymphatiques au cours des r6actions immunitaires. Th/:se d'agr6gation de l'enseignement sup~rieur, Univ. de Liege. SMALL, J. V. 1968. Measurement of section thickness. Proc. 4th Europ. Conf. EM: 609-10. STAUBLI,W., T. A. FREYVOGELand J. SurER. 1966. Structural modifications of the endoplasmic reticulum ofmidgut epithelial cells of mosquitoes in relation to blood intake. J. Microsc. (Paris) 5: 189-204. WEIBEL, E. R. 1973. Stereological techniques for electron microscopic morphometry, pp. 237-296. In M. A. Hayat (ed.) Principles and Techniques of Electron Microscopy. Van Nostrand-Reinhold, New York.
©PergamonPressLtd. 1978.Printed in Great Britain.
0020-7322/78/0601-0267502.00'0
INTRACELLULAR DISTRIBUTION OF RIBOSOMES IN MIDGUT CELLS OF THE MALARIA MOSQUITO, ANOPHELES STEPHENSI (LISTON) (INSECTA "DIPTERA) IN RESPONSE TO FEEDING HERMANN HECKER
Schweiz. Tropeninstitut, Socinstrasse 57, CH-4051, Basel, Switzerland (Accepted 9 January 1978)
Abstract--In the midgut cells (stomach) of female Anopheles stephensi the numerical density of total free and membrane-bound ribosomes (= number per cytoplasmic volume) is not greatly changed during digestion of the first blood meal. Together with an increase in cytoplasmic volume, the absolute number of ribosomes per epithelial cell is however greatly increased. During digestion, the ratio of membrane-bound-to-free ribosomes is shifted in favour of the former, indicating enhanced synthesis of proteins destined for exocytosis (e.g. digestive enzymes). About 400 ribosomes are bound to 1 /zm2 of rough endoplasmic reticulum (rer). In the first 12 hr after blood meal, this number drops temporarily, pointing to a lag between ribosome synthesis and attachment to the rer and proliferation of the membrane system. Index descriptors (in addition to those in the title): Blood digestion, protein synthesis, cellular dynamics, electron microscopy, morphometry. INTRODUCTION IT HAS been previously demonstrated that during digestion of the first blood meal by A. stephensi the volume of the cells of the stomach epithelium (~-- posterior part of midgut)
increases and most of the organelles and membrane systems of these cells proliferate (Hecker, 1977). Thereby the absolute surface area of the rough endoplasmic reticulum (rer), for instance, becomes enlarged 4-6 times. These morphological findings suggested a considerably raised functional capacity and activity of the midgut epithelium during digestion, which can be correlated to the formation of digestive enzymes (Gooding, 1975) and of the peritrophic membrane (Freyvogel and St/iubli, 1965; Gander, 1968). At the same time, products of digestion are absorbed (Rudin et al., in preparation). Other questions remained open. One of them concerned the ribosomes and their behaviour during blood digestion when the synthesis of structural and secreted proteins is enhanced. Does their numerical density remain constant? Does their absolute number per cell remain constant, or does synthesis of new ribosomes occur? Is there a change in the ratio of membranebound-to-free ribosomes, etc.? In order to answer these questions, we investigated morphometrically the dynamics of the ribosome population within the midgut cells of A. stephensi at various physiological stages before a blood meal, during and after digestion. MATERIAL AND METHODS 1. Mosquitoes and physiological stages A. stephensi derived from a strain kept at the London School of Hygiene and Tropical Medicine and had
been reared in our laboratory since 1971, according to Geigy and Herbig (1955). Midguts of females of the 267
268
HERMANNHECKER
following stages were used: 1. ready for first blood meal = 1 day after emergence (ldae); 2. digestion of blood meal = 1 dae + 0.5 days after blood meal on mice (0.Sdabm); 3. digestion of blood meal = ldae + 1 day after blood meal (ldabm); 4. digestion completed ~ ldae ÷ 3.5 days after blood meal (3.5dabm). 2. Preparation methods and morphometry
The anterior (A-part, A) and posterior parts (P-part, P, stomach) of the midguts were prepared for electron microscopy according to Hecker et al. (1974). Numerical densities of ribosomes (Nvri = number of ribosomes/t~m3 cytoplasm) were evaluated by stereological principles (Weibel, 1973), using the technique of Simar (1973). The following formula was used for the calculations: Nri 1 Nvr~ = A~r " 1) + T - - 2h ; where Nri/Ar = number of ribosome profiles counted on the area of thin section investigated (~mZ); /) ~ diameter of ribosomes ~ 0.02/~m; T = section thickness, determined according to Small (1968) 0.035 ~m in hungry mosquitoes, ~ 0.038 ~m in blood fed mosquitoes; h -- height of smallest recognisable cap sections of ribosome profiles in thin sections -- 1/4/) (Simar, 1973). Numerical densities of m e m b r a n e - b o u n d (Nvrim) and free ribosomes (Nvrif) were evaluated. This also enabled us to calculate the ratio o f membrane-bound-to-free ribosomes (rim/rif). By adding Nvrim to Nvrif the density o f total ribosomes ( N v , ) could be obtained. Absolute cytoplasmic volumes of the epithelial cells (Vcy = t~m3/epithelial cell) and surface densities o f rer (Svre, = /~m 2 membrane/tzm 3 cytoplasm) have been previously investigated (Hecker, 1977). Vcy and N v values were used to calculate approximate numbers o f ribosomes per epithelial cell (Nri, Nrim, Nrif). Nvrim and Svrer were used to calculate the ribosome n u m b e r per unit area o f rer membrane: Nvrim
Svrer
Nrim tLm2rer
Parameter results were compared statistically (T-test, significance limit 2 P < 0 . 0 5 ) . F o r a complete description of the methods the reader is referred to Simar (1973) and Hecker et al. (1974). R E S U L T S (TABLE1) 1. Numerical densities o f ribosomes and ratio o f membrane-bound-to-free ribosomes
In the cytoplasm o f the midgut epithelial cells o f female A. stephensi both membraneb o u n d and free ribosomes are present (Fig. 1). Before the first blood meal the numerical density o f m e m b r a n e - b o u n d ribosomes does not differ significantly between the anterior and posterior parts o f the midgut. During digestion of the first blood-meal the numerical density o f m e m b r a n e - b o u n d ribosomes in the posterior part is significantly increased. After complete digestion the same value as before the blood meal is found. The numerical density o f free ribosomes differs significantly between both midgut parts but shows no significant alterations within the posterior part during digestion. Therefore the numerical density o f total free and m e m b r a n e - b o u n d ribosomes is also not significantly changed during digestion. In every stage examined more free than m e m b r a n e - b o u n d ribosomes are present in the epithelial cells. The ratio o f membrane-bound-to-free ribosomes is similar in both midgut parts before the blood meal. During digestion in the posterior part it is increased but is reduced again when digestion is completed.
269
Intracellular distribution of ribosomes TABLE 1. Stage
Part Nwi
Nvrim Nvm
rim/rif
Vcy*
Nri X 10 6
I dae
A
4000 1300 2700 0.48 450 1.8 200 4- 100 ± 200 ~ 0.05 ± 80 .1_ 0.3
P
5000 1500 3500 0.42 980 300 ± 200 ~ 200 ± 0.05 ± 260
S
ns
0.5dabm P ldabm
P
s
S
ns
ns
s
4.7 ± 1.1
ns
s
5700 1900 3800 0.50 1950 11.I :~_ 300 ± 100 ± 200 ± 0.02 -- 370 ± 2.2 ns ns ns s ns 5800 2200 3600 0.61 3590 20.8 ± 500 ± 200 ± 300 ± 0.04 ± 870 ± 5.3 ns
3.5dabm P
ns
S
ns
S
lqS
5400 1500 3900 0.40 2360 12.7 ± 400 ± 100 ± 300 -L 0.01 __L 310 ± 1.7
Nrim
Nrif
/ 10 6
, 10 6
0.6 ~ 0.1
1.2 ± 0.2
S
1.3 :k 0.3 s
3.6 ± 0.7 ns 7.5 ± 1.7 S
3.6 J_: 0.5
Sv,e,* Nv,im/Sv,e, 3.06 J- 0.23
420 L 40
3.36 -- 0.28
430 :~. 20
6.54 ± 0.28 5.11 ~ 0.22
290 ± 20 s 430 ~ 30
3.90 ~ 0.14
390 ~ 20
S
3.4 .~ 0.9
ns
s
7.5 :- 1.5 ns 13.3 _i: 3.6
s
ns
9.2 ± 1.2
ns
A. stephensi, female, midgut epithelium. Means ± SE of ribosomal parameters. Vertically adjacent values are compared, s = significant, ns = not significant, * = values from Hecker (1977). ldae = mosquitoes ready for first blood meal, 0.5dabm and ldabm = during digestion, 3.5dabm = digestion completed; A = anterior, P = posterior part of midgut; Nv,~ = numerical density of total ribosomes, Nv,m, = of membrane-bound, Nv,~f = of free ribosomes; rim/rif = ratio of membrane-bound-to-free ribosomes; Vcy = volume of cytoplasm per epithelial cell; Nri = number of total ribosomes per cell, Nrim := of membrane-bound, Nrif = of free ribosomes; Sv,e, = surface density of rer; Nv,,m/Sw~r = number of ribosomes per unit area of rer. 2. Absolute number of ribosomes per epithelial cell Before the first b l o o d meal the absolute cell volume is markedly higher in the posterior t h a n in the anterior part of the midgut. By calculating the absolute n u m b e r s o f m e m b r a n e b o u n d a n d free ribosomes, it is obvious that the cells of the posterior part c o n t a i n m o r e ribosomes o f b o t h kinds. D u r i n g digestion in the posterior part a n increase in the absolute ribosome n u m b e r s is observed, followed by a reduction after digestion. Thereby, t h e absolute cell v o l u m e a n d the absolute n u m b e r s of ribosomes per epithelial cell are n o t reduced to the same values measured before the first blood meal. The changes are not always significant which is due to the high standard errors of the absolute parameters.
3. Number of ribosomes per unit area of rer Before the first blood meal nearly the same n u m b e r of ribosomes are b o u n d to I /zm-' o f r e r - m e m b r a n e s in both midgut parts (anterior: 420, posterior: 430). I n the posterior part 0.5 days after the b l o o d meal significantly fewer ribosomes per unit area are found. 1 day after the blood meal, when the epithelial cells are still in full activity, the same ratio as before the blood meal is reached. This also remains practically the same in the stage investigated after digestion. DISCUSSION I n the posterior part of the m i d g u t (stomach) o f A. stephensi, d u r i n g digestion o f the first b l o o d meal, it has been d e m o n s t r a t e d that the absolute n u m b e r s of m e m b r a n e - b o u n d a n d free ribosomes increase drastically per epithelial cell. D u r i n g this phase of high cellular ~IA~ 7 ] 3 - - r
270
HERMANN HECKER
FXG. 1. Anopheles stephensi, female, midgut, P-part, ldae. Basis of epithelial cell. Membrane-
bound ribosomes (A), free ribosomes (A), nucleus (nu), basal labyrinth (lab), basal lamina (bl). ?: 33.000
activity (Bertram and Bird, 1961 ; Freyvogel and St/iubli, 1965; St/iubli et al., 1966; Gander, 1968; Hecker, 1977) synthesis of ribosomes must therefore take place. When digestion is completed, the number of ribosomes is not reduced to the value measured before the blood meal. This runs parallel to the cytoplasmic cell volume which is only partly reduced and which may be an expression of an ongoing differentiation process stimulated by the digestion of the first blood meal. The numerical density of total membrane-bound and free ribosomes is, despite some significant differences (NrrJm), not greatly changed during digestion. The increase in ribosome number per cell is therefore mainly a consequence of the increase in cell volume or vice versa, and it seems that a more or less constant number of ribosomes per unit volume cytoplasm must be present for the functions of the midgut cells.
lntracellular distribution of ribosomes
271
On our micrographs used for morphometry it was sometimes difficult to distinguish between free and membrane-bound ribosomes. This was especially the case when the rer membranes were hit tangentially (cf. Mclntosh and O'Toole, 1976, review article). As we were comparing several physiological stages by the same technique, we assume that the counting errors are similar throughout the investigation. In every physiological stage examined more free than membrane-bound ribosomes are counted. During digestion the ratio of membrane-bound-to-free ribosomes is shifted in favour of the former. This may be correlated with the synthesis of proteins which are secreted by the cells as for instance digestive enzymes (Gooding, 1975; Briegel and Lea, 1975; Yeates, personal communication). Before the first blood meal, about 400 ribosomes are bound to 1/~m 2 of rer membrane in both midgut parts. In the posterior part 12 hr after blood intake this ratio is lowered to about 7 5 ~ indicating that at the beginning of digestion the synthesis of ribosomes and their attachment to the rer takes place after membrane synthesis and proliferation, in spite of the fact that the synthesis of digestive enzymes, in particular proteases, is already increased at that moment (Gooding, 1975; Briegel and Lea, 1975; Yeates, personal communication). Gooding (1973) and Fuchs and Fong (1976) have demonstrated experimentally the "de novo synthesis" of proteases in .4edes aegypti. It may be assumed that in A n o p h e l e s midgut proteases are similarly newly synthesized during digestion (Hecker, 1977). Twenty-four hours after blood meal the pre-blood meal ratio of ribosome number per unit area of rer is established again. Therefore in this phase of digestion, synthesis and attachment of ribosomes is faster than membrane proliferation. When digestion is completed, membrane-bound ribosomes and rer membranes are reduced simultaneously and their ratio is not significantly altered, while the cell volume, the rer and other cellular components are reduced. This could point to an optimal functional unit of the number of ribosomes per unit area of membrane within the midgut cells of .4. stephensi stomach. Acknowledgements--1 thank Profs. T. A. Freyvogel (Swiss Tropical Institute, Basel) and E. S. Gander (Dept. Biol. Cel., Univ. de Brasilia), Drs. P. H. Burri (Anat. Inst., Univ. Bern), W. Rudin and R. Yeates (Swiss Trop. Inst.) for critical discussion of the manuscript. The skillful technical assistance of Miss C. Fauser and the typing of the manuscript by Miss U. Steffen are gratefully acknowledged.
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