Phagocytic behavior of the predatory slime mold, Dictyostelium caveatum

E x p e r i m e n t a l Cell R e s e a r c h 159 (1985) 323-334

Phagocytic Behavior of the Predatory Slime Mold, Dictyostelium caveatum Cell Nibbling DAVID R. WADDELL* and GONTER VOGEL Bergische Universitiit GHS, Biochemie, D-5600 Wuppertal 1, FRG

The predatory slime mold, D. caveatum, feeds upon other amoebae by phagocytosis. The D. caveatum amoebae begin feeding upon cells the same size or larger by nibbling pieces of cells. While feeding upon other amoebae as opposed to bacteria, they increase in size. This behavior resembles that of phagocytes in higher organisms. A novel method was used to follow the time course of phagocytosis. A lytic toxin, phallolysin, and mutants resistant to the toxin were utilized in an assay to separate the phagocytes from the prey cells. Since a broad spectrum of cells are sensitive to the toxin, the method has general applicability. © 1985AcademicPress, Inc.

Sensory processes are required by cells to accomplish a variety of tasks. They are involved in chemotaxis, phagocytosis, adhesion, growth control, and morphogenesis. These processes can be conveniently studied using cellular slime mold amoebae [1-3]. Recognition processes involved in phagocytosis have been studied using mutants defective in phagocytosing certain types of particles [4-6]. It has also been possible to select for mutants defective in phagocytosis which exhibit changes in motility [7]. The discovery of a new species of cellular slime mold, D. caveatum, which has the unique capacity to feed and grow upon other amoebae, has provided the opportunity to study self/non-self recognition in this system [8]. By following the time course of phagocytosis, we have discovered two unusual aspects of phagocytosis in D. caoeatum: the first is that initially the D. caveatum amoebae phagocytose only pieces of the prey cells. This type of phagocytosis, which we have named 'nibbling' is apparently necessary for the D. caveatum amoebae to feed upon cells which are the same size or larger than themselves. The second unusual behavior is a dramatic increase in the cell volume of the amoebae as they continue to feed upon other amoebae. This behavior resembles activation of macrophages [9] which can also be induced by phagocytosis of specific particles [10]. To assay phagocytosis it was necessary to develop a method to separate nonphagocytosed prey amoebae from the D. caveatum amoebae. Since D. caoeatum * To whom offprint requests should be sent. Address: Bergische Universit~it GHS, POB 100127, Fachbereich 9, D-5600 Wuppertal 1, FRG. Exp Cell Res 159 (1985)

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Waddell and Vogel

amoebae

and prey amoebae

a r e v e r y s i m i l a r , it w a s n o t p o s s i b l e t o u s e d i f f e r -

e n c e s in s i z e o r p h y s i c a l p r o p e r t i e s f o r t h i s p u r p o s e . T h e r e f o r e ,

we have devel-

oped an assay based upon the use of a lytic toxin, phallolysin, from Amanita mushrooms.

We have selected mutants which are resistant to lysis by this toxin.

In this assay, the non-phagocytosed

cells are rapidly eliminated by lysis with the

toxin and centrifugation. The assay provides a method to compare

self and non-

s e l f r e c o g n i t i o n , s i n c e e i t h e r p h a l l o l y s i n - s e n s i t i v e D. caveatum s t r a i n s o r s e n s i t i v e s t r a i n s o f o t h e r s p e c i e s c a n b e u s e d a s t h e p r e y c e l l s . It s h o u l d b e p o s s i b l e t o adapt this type of assay to other systems.

MATERIAL

AND

METHODS

Growth and M a i n t e n a n c e o f Strains Stock cultures of Dictyostelium caveatum were maintained on lactose peptone agar containing per liter: 2 g Bactopeptone (Oxoid), 2 g lactose, 0.272 g KHzPO4, 0.284 g NazHPO4, 15 g Bactoagar (Difco). New stock cultures were initiated every 2 months from spores stored on silica gel or in liquid nitrogen. For experimental use, cells were grown in liquid suspension cultures in association with a rough strain of Salmonella minnesota (R595) obtained from G. Gerisch (Max Planck Institute for Biochemistry, Martinsried). The bacteria were grown in medium containing per liter: 10 g bactotryptone (Difco), 5 g yeast extract, 10 g dextrose and 5 g NaC1. The bacteria were harvested and washed three times with 17.5 mM phosphate buffer (pH 6.0) and stored at 5°C. To initiate a culture, a suspension of bacteria (A42o= 10-15) was inoculated with spores or amoebae. At 27°C the generation time was 4-5 h. Stock cultures of D. discoideum AX2 ~ATCC 24397) were maintained in the same way as D. caveatum. For experimental use they were grown on axenic medium (14.3g Oxoid peptone, 7.15g Oxoid yeast extract, 18 g maltose, 1.28 g Na2HPO4x 12H20, 0.48 g KHzPO4, per liter HzO, pH 6.7). The cells grew with a doubling time of 8-12 h at 21°C. Cells were harvested at cell densities between 3x 106 and 5x 106 per ml.

Mutagenesis Cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (Sigma). 2-3x108 amoebae were harvested and resuspended at 5 x 106 amoebae per ml in 17.5 mM phosphate buffer. Immediately before use, nitrosoguanidine was dissolved in dimethylsulfoxide and added to the cell suspension to yield a final concentration of 1 mg/ml. After 7 rain the cells were pelleted at 0°C (200 g, 2 rain), washed four times and resuspended with bacteria. Viability after mutagenesis was 1-10 %.

Preparation o f Phallolysin Phallolysin was prepared from Amanita vernar mushrooms following the general procedure of Faulstich et al. [11]. Five hundred grams of mushrooms were homogenized in a Waring blender with 0.9 % NaCI to yield a final volume of 1.21. After stirring for 5 h the homogenate was centrifuged for 30 min at 20000 g. Ammonium sulfate was added to the supernatant to a final concentration of 130 g/1 (23 %). After stirring for 20 min at room temperature, the precipitate was pelleted at 20000 g at 4°C. To the supernatant an additional 100 g/l of ammonium sulfate was added and stirred for 20 min at 4°C. After centrifugation the pellet was redissolved in 50 ml H20 and dialysed extensively against 1.75 mM phosphate buffer (pH 6.0). The solution was cleared by centrifuging for 1 h at 100000 g. The phallolysin was lyophilized and stored at -20°C. The final yield was approx. 50 mg of protein. Further purification was not necessary for use in the assay and selection of mutants. Comparison with electrophoretically-pure phallolysin, which was a kind gift of F. Faulstich, indicated that the partiallypurified phallolysin was about 50 % pure. For experimental use, the lyophilized powder was resuspended at 1 mg/ml (0.5 mg/ml protein) in H20 and sterilized by passing through a membrane fdter (Millex-GV, Millipore, 0.22 lxm pore size).

Exp CellRes 159 (1985)

Phagocytosis in predatory slime mold

325

Selection of Phallolysin-resistant Mutants Each batch of phallolysin was tested at different concentrations to determine the minimum concentration sufficient to lyse 95 % of wild-type amoebae within 20 min. Lysis of wild-type amoebae is very sensitive to the concentration of phallolysin (fig. 1). For the selection of mutants a concentration of between 1 and 2 ttg/ml of the crude phallolysin was used. After recovery from mutagenesis (4-5 days), the cells were washed free of bacteria and resuspended at 7.5×105 per ml in 1.75 mM phosphate buffer (one-tenth the molarity of the buffer they were grown in). The cells were shaken in Erlenmeyer flasks at 100 rpm. After 30 min phaUolysin was added and the cells were shaken with the toxin for 1 h. The surviving cells were pelleted, washed twice with 17.5 mM phosphate buffer, resuspended with bacteria and allowed to grow overnight. The selection was repeated until the cultures were resistant to lysis by microscopic examination and cell counts (approx. 7-10 rounds). The survivors of the final selection were cloned and characterized. Two independently derived mutants were utilized in the experiments reported here: BW2 and BW3. One of these, BW2, is defective in morphogenesis. It does not proceed past the aggregate stage. BW3 exhibits normal morphogenesis. A high proportion of phallolysin mutants selected in D. discoideum are defective in morphogenesis and spore formation (personal observation, G. Vogel).

Quantitation of Phagocytosis by D. caveatum A phallolysin-resistant and a phallolysin-sensitive strain were harvested, washed free of either bacteria or nutrient medium and resuspended at 5x 106-107per ml in phosphate buffer (17.5 mM, pH 6.0). Before mixing the concentration of phallolysin necessary to lyse 95 % of the sensitive ceils within 20 rain was determined. The strains were mixed, and phagocytosis was assayed by diluting 0.2 ml aliquots of the cell suspension with 1.8 ml distilled water. The suspension was dissociated to single cells by vortexing and counted with a Coulter Counter (Coulter Electronics, Harpenden, UK). After 20 rain the number of cells resistant to lysis were counted.

Fluorescamine labelling of D. discoideum D. discoideum (AX2) was harvested from nutrient medium and resuspended at 1× 106 cells/ml in nutrient medium diluted 1 : 5 with 17.5 mM phosphate buffer (pH 6.0). The cell suspension was shaken at 100 rpm for 20 min on a rotating shaker at 0°C. Immediately before use a 0.5 % solution of fluorescamine (Sigma) was prepared in dimethylsulfoxide. While still shaking, 2.5 td of this solution was added per ml of cell suspension. The cells were shaken an additional minute at 0°C and pelleted (1 min, 200 g, 0°C). The cell suspension was washed twice with 17.5 mM phosphate buffer and resuspended in the same buffer.

Electron Miscroscopy D. discoideum and D. caveaturn cells were washed free of nutrient medium or bacteria and suspended in phosphate buffer at 107 cells/ml. After 30 min, the cells were mixed in a 2 : 1 ratio (D. discoideum : D. caveatum). The cell suspension was fixed in 4 ml of 1% glutaraldehyde in 25 mM cacodylate buffer (pH 7.1). Fifteen seconds later, 2 ml of 2 % OsO4 was added and incubated at room temperature for 20 min. The cells were dehydrated at 0°C in ethanol and propylene oxide and embedded in Spurr's resin.

RESULTS

A Lytic Assay for Phagocytosis Employing Phallolysin An

assay

for phagocytosis

selectively lyse approximate

either prey

was

developed

which

cells or phagocytes.

employs

Phallolysin

a lytic drug to is a p r o t e i n

of

m o l e c u l a r w e i g h t 34 000. I t is t h o u g h t t o l y s e c e l l s b y b i n d i n g t o a

r e c e p t o r a n d i n t r o d u c i n g p o r e s in t h e c e l l m e m b r a n e i s o l a t e d f r o m Amanita m u s h r o o m s

[11]. P h a l l o l y s i n w a s i n i t i a l l y

d u e t o its h e m o l y t i c a c t i v i t y . H o w e v e r , it is

Exp CellRes 159 (1985)

326 Waddell and Vogel

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Fig. 1. Concentration dependence of lysis by phallolysin. 0 , Wild-type D. caveatum (BW1), and ©, •, two phallolysin-resistant mutants of D. caveatum (0, BW2; A, BW3) were washed free of

bacteria and suspended in 1.75 mM phosphate buffer at 7.5)< 105 cells/ml. Various concentrations of phallolysin were added to 2 ml portions of each cell suspension. After 30 min the number of cells resistant to lysis were counted. Fig. 2. Phagocytosis of D. discoideum by D. caveatum. (A) Phallolysin-resistant D. discoideum (0, PH1) was mixed with wild-type D. caveatum (A, BW1). (B) Phallolysin-resistant D. caveatum ( A , BW2) was mixed with lysis-sensitive D. discoideum (O, AX2).

also known to lyse a variety of mammalian cells [12] when added in nanomolar concentrations (the same concentrations required to lyse slime mold amoebae). For use in a phagocytic assay, we selected mutants resistant to lysis by phallolysin following mutagenesis with nitrosoguanidine (see Methods). Two independently-derived mutants, BW2 and BW3, were isolated, characterized further and used in the studies described here. A phallolysin-resistant mutant of D. discoideum (HV140) was also utilized. A detailed characterization of this mutant will be reported elsewhere. The kinetics of phagocytosis were followed by mixing a phallolysin-resistant strain and a sensitive strain in shaking suspensions (fig. 2). Under these conditions the cells form loose clumps within 5 min. To assay the number of cells of each type at different times, aliquots were taken, diluted tenfold with distilled water and dissociated to single cells by vortexing. Dissociation to single cells was verified using an inverted microscope. The cell suspensions were then counted and phallolysin was added. After lysis, the number of resistant cells were counted. For up to 3 h after mixing, the prey cell number remained constant. Thereafter, the prey cell number decreased rapidly. Approximately one-third of a prey cell was phagocytosed by a D. caveatum cell in 1 h. Exp Cell Res 159 (1985)

Phagocytosis in predatory slime mold 6-

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Fig. 3. Self vs non-self recognition. D. caveatum strains BW1 and BW3 (phallolysin-resistant) grown

in association with bacteria were washed free of bacteria and resuspended in 17.5 mM phosphate buffer. D. discoideum (AX2) was grown axenically, washed free of nutrient medium and resuspended in phosphate buffer. After 30 rain BW3 was mixed in equal amounts with BWl and with AX2 and phagocytosis was assayed using phallolysin (Methods). BW3 mixed with BWl, O; BW3 mixed with AX2, &; AX2, A; BWl, ©. Fig. 4. Cell volume increase during early phagocytosis. During the experiment represented in fig. 3 the cell volume of the phallolysin-resistant cell type was determined at selected time points. Cell volume distributions were determined on a Coulter Counter by taking successive counts at different thresholds. The threshold scale was calibrated with latex beads of known size (Coulter Electronics, Harpenden, Herts, England). The total cell number represented in the distributions was between 9500-10000. The mean volume was determined from the distributions. D. caveatum (BW3) mixed with &, D. discoideum (AX2); D. caveatum (BW3) mixed with 0 , D. caoeatum (BWl).

Similar results were obtained, regardless of which cell type was lysis-resistant (fig. 2A, B). This indicated that the phallolysin-resistance mutation itself did not dramatically affect phagocytic recognition. The assay allows one to study pure populations of either cell type by casting the appropriate cell in the phallolysinresistant role. With the phallolysin-resistant strain of D. caoeatum it is possible to compare self and non-self recognition using this assay. To make this comparison (fig. 3), a phallolysin-resistant strain (BW3) was mixed in equal numbers with either D. discoideum (AX2) or the wild-type strain of D. caveatum (BWI). The cell numbers in the BWl/BW3 mix remained relatively constant for 7 h. It was not possible to follow this culture further because by 8 h the cells had formed tight cell clumps characteristic of developing cells and could not be easily dissociated. In the AX2/BW3 mix the results were very different. The prey cell numbers remained constant for about 2 h. Thereafter, they declined rapidly so that by 6 h they were eliminated. The BW3 cell numbers increased as they fed upon the AX2 amoebae and also after they were exhausted so that their numbers were approximately doubled by the end of the experiment. Therefore, for each AX2 cell eaten, a new D. caveatum amoeba was produced. This conversion rate is remarkable, even though the AX2 cells were initially larger than the BW3 cells. An earlier Exp Cell Res 159 (1985)

328 Waddell and Vogel

Fig. 5. Phagocytosis of fluorescamine-labelled D. discoideum cells. D. caveatum (BW3) were washed free of bacteria and mixed in a 1 : 1 ratio with fluorescamine-labelled D. discoideum ceils. After 2 h the cells were observed and photographed using an Olympus BH2 fluorescent microscope.

study, in which D. caoeatum amoebae were fed P. pallidum amoebae of comparable size, yielded a conversion rate of one new cell for 2-3 prey cells eaten [8].

Cell 'Nibbling" During the first 2-3 h after mixing with D. caveatum amoebae the prey cell number remained constant. Nevertheless, significant uptake of prey cells takes Exp Cell Res 159 (1985)

Phagocytosis in predatory slime mold

329

Fig. 6. Thin section of a D. caoeatum cell feeding upon a cluster of D. discoideum cells in suspension culture, x960. Fig. 7. Different section through same cells as in fig. 6. x 1500.

place during this period. This can be seen most directly by measuring the cell volume. Normally, vegetative cells washed free of bacteria and resuspended in buffer decrease in size (fig. 4, circles). In contrast the mean cell volume of D. caveatum cells mixed with AX2 cells (fig. 4, triangles) had increased by 13 % within the first 2 h and the maximum mean volume of 0.55 x 10 - 6 ~tl was achieved by 3 h. Thereafter, the mean volume began to decrease slowly until the prey amoebae were completely exhausted (7 h). At this time the volume decreased rapidly due to cell division and starvation. Since a significant volume increase has occurred by 2 h this implies that phagocytosis is already underway during this period. The volume increase is not due to an osmotic effect, since one would have expected a comparable effect in the control amoebae without AX2 cells which were otherwise treated identically. To determine how the D. caoeatum amoebae were feeding during this period, they were fed fluorescently-labelled AX2 amoebae (fig. 5). By 2 h it was evident that the D. caveatum amoebae were filled with fluorescent vesicles. The D. caoeatum amoebae appear to bind the prey and then small fluorescent pieces begin to show up in the cytoplasm of the D. caveatum cells. This type of phagocytosis appears to be necessary for them to feed upon amoebae which are the same size or larger than themselves. Electron microscopy of cells that were fixed after 2 h of feeding confirmed the results with fluorescently-labelled cells (figs 6, 7, 8). In figs 6 and 7 two different sections through a typical cluster of cells are shown. The amoeba in the lower-left hand area can be recognized as a D. caoeatum amoeba by the presence of Exp Cell Res 159 (1985)

330 Waddell and Vogel

Exp Cell Res 159 (1985)

Phagocytosis in predatory slime mold

331

Fig. 9. Cell volume distributions of D. caveatum

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,!o Volume

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cells grown on bacteria and on amoebae. The cell volume distributions were determined on D. caveatum (BW3) cells which were growing exponentially on either (A) bacteria or (B) D. discoideum for 12 h. The AX2 amoebae were eliminated by lysis with phallolysin in 1.75 mM phosphate buffer. After lysis was complete, the cells were washed twice with 17.5 mM phosphate buffer and resuspended in the same buffer prior to determining the volume. Cell volumes were determined as described in fig. 3. The bacterially-grown cells were treated identically after washing free of bacteria.

numerous secondary lysosomes in its cytoplasm. These are probably equivalent to the fluorescent vesicles in the D. caveatum cell in fig. 5. This cell appears to be about to phagocytose another piece of the attached cell. The prey cell has formed a filopod in this area which has been surrounded by the D. caveatum cell. In fig. 8A, a D. caveatum amoeba appears to be about to phagocytose a pseudopod of a prey cell. In fig. 8 B the pseudopods of a larger cell appear to be about to close upon a bite-size piece of another amoeba. Smaller pseudopods emanating from larger areas containing polymerized actin seem to be closing off the neck of cytoplasm remaining between the cell body and the piece of the prey cell. Cell Volume Increase While Feeding upon Amoebae

Since the cells were mixed in equal numbers in the experiment presented in figs 3 and 4, the prey cells were rapidly exhausted and the maximum volume increase was not observed. In fig. 9 the cell volume distribution of cells grown on bacteria is compared with that of cells growing for 12 h on amoebae. The mean cell volume of bacterially-grown cells was 0.462× 10-6 t~1. On the other hand, cells growing for 12 h upon amoebae exhibit a mean cell volume of 0.858x 10 - 6 ~1. The combination of the decrease in the size of prey amoebae due to starvation and nibbling and the increase in the size of the D. caveatum cells probably increases the efficiency of phagocytosis by allowing them to take larger pieces or whole cells, as appears to be the case in feeding upon developing aggregates [8]. Fig. 8. Thin sections showing (A) phagocytosis of a pseudopod; (B) closing of pseudopods Ul~On a

piece of a cell. (A) x 960; (B) x l 300. Exp Cell Res 159 (1985)

332 Waddell and Vogel DISCUSSION Cellular slime molds have proved to be convenient organisms in which to study cell behavior. D. caveatum is particularly attractive as a model system for phagocytic recognition, since it feeds upon cells which are very similar to itself. Here, by the use of a new assay, we have characterized an unusual mode of phagocytosis by cell nibbling. The assay utilizes a lyric toxin and mutants resistant to it. Since a wide spectrum of cells appear to be sensitive to this toxin [12], this method has general applicability. The method may also be valuable in studies other than phagocytosis in which one wishes to measure a parameter of one cell type in a situation in which they are mixed with many other cell types. The current view of the mechanism of phagocytosis, which was derived mostly from studies of phagocytosis in macrophages [13], is that pseudopods receive local signals from the particle being ingested to continue to move across its surface. As long as the appropriate signals are generated, the pseudopod continues to advance. In cases where the signal is generated by a specific ligand it was possible to cap these ligands at one end of the cell and show that the pseudopods only advanced over the capped region of the cell and hence phagocytosis did not occur [14, 15]. A consequence of this model is that the particle determines the size of the phagosome [13]. If the particle is too large to ingest, then the cells flatten out on the surface of the particle. During cell nibbling by D. caveatum the pseudopods do not continue to move over the surface of the prey cell and instead close upon a bite-size piece. Here, local signals may be required to induce the advancement of the pseudopods but at some stage more global signals, perhaps involving the cytoskeleton, cause the pseudopods to fuse and ingest only a piece of the cell. Cell nibbling appears to be a necessary adaptation for D. caveatum amoebae to feed upon cells of their size or larger. It seems likely that the D. caveatum amoebae may employ a more normal type of phagocytosis when the prey cells become small enough to be engulfed. The rigidity of the prey cell may determine the mode of phagocytosis. This property increases as cells become smaller due to a greater surface area to volume ratio. In many cases the D. caveaturn cells appear to phagocytose pseudopods or filopods formed by the prey cells. Perhaps cell nibbling is initiated by the intrusion of filopods of the prey cell into the phagocyte. This suggests that the D. caveatum amoebae may stimulate filopod or pseudopod formation in the prey cells. The increase in cell size when feeding upon other amoebae may also be a special adaptation to feed upon larger cells. This behavior of D. caveatum amoebae resembles the behavior of other phagocytic cells. Macrophages can be induced to undergo a variety of changes when they are 'activated' [16, 17]. One of the primary changes is an increase in cell size [9]. Furthermore, one of the ways in which macrophages can be activated is by phagocytosis of specific particles Exp Cell Res 159 (1985)

Phagocytosis in predatory slime mold

333

[10]. Other phagocytic cells also exhibit comparable phenomena: e.g., a dietrelated increase in the virulence of axenically-grown Entamoeba histolytica can be induced by exposure to bacteria [18], or by passage in hamsters [19]. A diet-related increase in cell size also occurs when axenic mutants of D. discoideum are grown axenically. However, these stains grow at a much slower rate in medium (8-12 h doubling time) than they grow on bacteria (4--5 h doubling time). The difference is probably due to a metabolic change, since axenically grown cells also differ dramatically from bacterially-grown cells in carbohydrate content [20]. In addition, a high percentage of the axenically grown D. discoideum cells are multinucleate [21], which may explain much of the size difference. D. caveatum grows at a similar rate on bacteria or amoebae (4-5 h doubling time). The increase in size also bears a resemblance to the initial phase in the sexual life cycle of cellular slime molds. Here cells of opposite mating types fuse to form a large cell called a giant cell [22]. The giant cell then attracts and phagocytoses other amoebae of the same species to form a very large cell which finally encysts. Perhaps some of the same genes that are involved in the sexual cycle are also employed by D. caveatum amoebae in order to phagocytose other amoebae. However, since so far it is not possible to produce macrocysts in D. caveatum, we can at present only infer this by studying closely related species. The ability to isolate and characterize mutants has proved valuable in understanding phagocytic recognition in D. discoideum [4-6]. We are currently developing D. caveatum as a genetic system in which to study self/non-self recognition. Recently, we have obtained mutants defective in phagocytosing latex and mutants in which self/non-self recognition appears to have broken down. By combining cell, biochemical and genetic methods we hope to reveal the mechanism of this recognition process. We are indebted to the Zoological Laboratory of the University of Leiden for the use of their electron microscope facility and to Pauline Schaap for assistance with the sectioning. This work was supported by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (Vo 348/1-1). The authors thank M. Satre and K. Duffy for critical comments and help with the manuscript.

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Griffin, F M, Jr, Advances in host defense mechanisms 1 (1982) 31. Griffin, G M, Jr, Griffin, J A & Silverstein, S, J exp reed 144 (1976) 788. Griffin, F M, Jr, Griffin, J A, Leider, J E & Silverstein, S, J exp med 142 (1975) 1263. Karnovsky, M L & Lazdins, J K, J immunol 121 (1978) 805. Cohn, Z, J immunol 121 (1978) 813. Wittner, M & Rosenbaum, R M, Amj tropical reed & hygiene 19 (1970) 755. Lushbaugh, W B, Kairalla, A B, Loadholt, C B & Pittman, F E, Amj trop & hygiene 27 (1978) 248. 20. Ashworth, J M & Watts, D J, Biochem j 119 (1970) 175. 21. MacDonald, S A & Durston, A J, J cell sci 66 (1984) 195. 22. Saga, Y & Yanigsawa, K, J cell sci 55 (1982) 341. Received January 15, 1985

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