Mechanism of haemolysis of erythrocytes by haemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

J. Insecr Physiol. Vol. 37, No. 1 I, pp. 851-861, Printed in Great Britain

0022-1910/91 $3.00 + 0.00 Pergamon Press plc

1991

MECHANISM OF HAEMOLYSIS OF ERYTHROCYTES BY HAEMOLYTIC FACTORS FROM STOMOXYS CALCITRANS (L.) (DIPTERA: MUSCIDAE) H. J. KIRCH,’ G. SPATES,’ R. DROLESKEY,’W. J. KLOFT* and J. R. DELOACH’ ‘Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Route 5, Box 810, College Station, TX 77845, U.S.A. and *Institute for Applied Zoology, University of Bonn, An der Immenburg 1, D-5300 Bonn 1, Fed. Rep. Germany (Received 23 May 1991)

Abstract-Haemolysis in vitro of bovine erythrocytes by posterior midgut homogenate from stable flies displayed a sigmoidal pattern. The addition of soybean trypsin inhibitor to mixtures of midgut homogenate and erythrocytes reduced haemolytic and proteolytic activity equally. Scanning electron microscopy revealed that erythrocytes exposed to homogenate immediately transformed into echinocytes which released microvesicles from their membrane spicules. Subsequently, membrane spicules became blunt and cells developed membrane invaginations. Finally, at the onset and during the phase of rapid cell lysis, smooth spherocytes were formed. Some of these spherocytes had a single hole of about 1 pm in their plasma membrane which allowed intracellular material to escape. The morphological changes during the process of haemolysis were influenced by soybean trypsin inhibitor. However, the inhibitor did not impair the initial formation of echinocytes, but membrane invaginations and membrane holes were not noticed. Furthermore, cell ghosts remained in the presence of the soybean trypsin inhibitor even after haemolysis was completed. Haemolytic activity of homogenates was markedly reduced when bovine serum albumin was added to the assay medium. Albumin noticeably suppressed the formation of echinocytes. A mechanism for the haemolysis of bovine erythrocytes by midgut homogenate including detergent-like and enzymatic activities is presented. Key

Word Index:

Erythrocytes;

echinocytes;

spherocytes;

haemolysis;

blood

digestion;

stable fly

INTRODUCTION

Successful reproductive performance of blood-feeding parasites depends on optimal utilization of the imbibed blood meal. Erythrocytes, the major cellular fraction of mammalian blood, are of special significance since they provide a substantial quantity of digestible protein which basically is haemoglobin. Furthermore, the plasma membrane of erythrocytes contains nutritionally important constituents. For the stable fly, Stomoxys calcitrans, it was shown that essential dietary components are associated with the lipid fraction of red blood cell membrane (Kapatsa et al., 1989). Hence, haemolysis of erythrocytes is a prerequisite for the sustained longevity of this bloodsucking fly. However, the haemolysis of erythrocytes in the midgut of blood feeding insects is not fully understood. Investigations on specific haemolysins were confined to effects of whole or partially purified gut homogenates on erythrocytes in vitro (Geering, 1975; Gooding, 1977; Spates, 1981; Azambuja et al., 1983). Haemolytic factors from the posterior midgut of stable flies have first been characterized by Spates and

DeLoach (1980). Their studies revealed that the haemolytic principle of the homogenate was linked to the proteolytic activity. Later, Spates et al. (1982) proposed that stable fly midgut homogenate haemolyse erythrocytes with the aid of fatty acids. This theory was based on the fact that the haemolytic activity of midgut homogenates was directly correlated with the free fatty acid content of the homogenate. In the present study attention was given to the morphology of bovine erythrocytes undergoing haemolysis by posterior midgut homogenate. The shape transformations of erythrocytes as a consequence of the action of the lytic material should provide more data to identify the factor(s) and the mechanism responsible for haemolysis. MATERIALS AND METHODS

Chemicals

Trypsin (10,200 BAEE units/mg protein), soybean trypsin inhibitor and azocasein (E,,,,, in 0.1 N NaOH = 36) were purchased from Sigma Chemical 851

H. J. KIRCH et al

852

Co. (St Louis, MO.). Bovine serum albumin was purchased from U.S. Biochemical Corporation (Cleveland, Ohio). Isoton II was obtained from Coulter Diagnostics (Hialeah, Fla). Insects

Adult stable flies were taken from a laboratory colony. Maintenance of flies has been reported elsewhere (Bridges et al., 1984). Flies emerging over a 4 h period were collected, sexed and placed in plastic cups with screen tops and bottoms (15 females and 6 males per cage). Flies were fed daily ad libitum on heparinized bovine blood. After the 6th blood meal female flies were used for experiments. Preparation of posterior midgut homogenate

Posterior midguts were removed according to Spates (1981). Flies were gassed with carbon dioxide, pinned to paraffin wax dishes, washed with distilled water and covered with ice-cold saline. Midguts were removed and transferred into an ice-cold tissue homogenizer (Dual, 2ml). Afterwards they were homogenized for 1 min at 4°C in isotonic phosphate buffer (pH 7.0) and centrifuged at 1OOOgfor 4min to sediment particulate material. Supernatants were adjusted to 20 midguts/ml by adding isotonic buffer and stored at -65°C before further use. Erythrocytes

Bovine blood was obtained by venipuncture and collected into 10 ml vacutainer blood tubes containing heparin as anticoagulant. Red blood cells were isolated by centrifugation at 1OOOgand then washed three times in three volumes of isotonic phosphate buffer. Washed packed cells were adjusted to 5.0 x 10’ cells/ml by addition of buffer and used for haemolytic assays. Fresh erythrocyte solutions were prepared daily. Determination of mean cell volume of erythrocyte spheres and estimation of surface area of normal bovine erythrocytes

Mean cellular volume of normal erythrocytes and erythrocyte spheres was determined by Coulter

Counter equipped with a computerized channel analyser (Coulter Electronics, Hialeah, Fla). Electrical sizing of erythrocytes during haemolysis does not unequivocally prove a change in mean cell volume since the magnitude of the electrical signal from the counter depends on the actual particle size and the cell shape (Waterman et al., 1975). As our system was calibrated for normal discocytes, values for spheres had to be corrected (divided) by a shape factor of 1.338 (Coulter Electronics, Hialeah, Fla). The surface area of the bovine erythrocytes used in our experiments was estimated as follows: the mean cell diameter of 6.15 pm from two populations of 200 cells each was determined with a light microscope. An approximate estimate of the erythrocyte’s surface area would be the surface of a cylindrical object having a diameter of 6.15 pm and a volume of 49.5 p’ (Table 1) which is 91.7 pm*. Another estimation was made on the basis of geometrical equations given by Beck (197.5). Variables needed for this calculation are the mean cell volume, cell diameter and cell thickness. The latter were arbitrarily varied between 1.6-2.5 pm for the maximal cell height (cell rim) and between 0.7-1.5 pm for the minimal cell height (cell centre). By using these variables the surface area was approximated to range between 81 and 83 pm’. Haemolytic and proteolytic assays

Assays for the haemolytic activity of midgut homogenates were performed after the procedure of Spates and DeLoach (1980). Three midgut equivalents of homogenate were mixed with 2.5 x 10’ red blood cells in buffer to a final volume of 1 ml in disposable plastic cuvettes. Reaction were carried out at 300 mosmol/l, pH 7.0 and at 27°C. The red blood cells were added last. Haemolysis of erythrocytes was recorded by measuring the decrease in absorbance at 700 nm (LKB Ultraspec 4050 photometer). The time required for complete hemolysis was taken as a measure for haemolytic activity. To determine haemolytic activity in the presence of soybean trypsin inhibitor and bovine serum albumin homogenates were prepared from triplicate sets of 45 flies at 18-24 h after feeding. Haemolytic activity was measured as described above except that the

Table 1. Mean cell volume (pm3) of spherocytes produced erythrocytes during in &ro haemolysis by posterior midgut from S. calcitrans

Cell type Discocytes Spherocytes Ghosts

No SBTI ._~ _.____~ Coulter* Correctedt 59.8 + 2.4 -

from bovine homogenate

Plus SBTI Coulter’

49.5 * 1.7 76.3 $- 6.0 44.7 + 1.8 75.0 + 9.4

Correctedt 57.0 & 4.5 56.1 k 7.0

Cell size was determined in the absence and in the presence of soybean trypsin inhibitor (SBTI). *Means&SD of two experiments based on triplicate determinations; values measured by direct electrical sizing. TValues corrected for perfect spheres by a shape factor of 1.338 (Coulter Electronics. Hialeah, Fla).

Mechanism of erythrocytes haemolysis

assay contained different concentrations of either the inhibitor or bovine serum albumin. Proteolytic activity of assay mixtures was determined 4 min after mixing homogenate with erythrocytes. Two aliquots (100 ~1 each) were transferred from the assay medium into polyethylene microfuge tubes. Then 250 pl of a I%-azocasein solution, freshly prepared in isotonic phosphate buffer, were added to each aliquot. Controls contained azocasein and buffer alone. The tubes were vortexed and placed into a shaking water bath for 1 h at 30°C. The incubation was stopped by adding 0.5 ml trichlor acetic acid (0.5 M). After 15 min at ambient temperature samples were centrifuged for 3 min at 7000g to sediment the precipitated proteins. The supernatants were transferred into 5 ml culture tubes and mixed with the equal volume of 0.5 N sodium hydroxide. Thereafter the absorbance was measured at 440 nm (LKB Ultraspec 4050 photometer). Proteolytic activity was measured in trypsin-units. One unit was defined to cause the same change in absorbance as 1 fig trypsin (10,200 BAEE units/mg protein) under the same conditions. Haemolysis in vitro of erythroeytes

Posterior midgut homogenate was prepared from 150 flies at 22 h after feeding. The equivalent of 25 midguts was mixed with 3.1 x 10’ washed bovine erythrocytes and isotonic buffer in a 20ml screw capped media bottle. Total volume of reaction mixture was 12.5 ml. The bottle was kept in a water bath at 27°C and gently inverted several times to prevent erythrocyte sedimentation. To monitor haemolysis, 1 ml of reaction mixture was taken every 3-5 min and the absorbance measured at 440 nm. Thereafter the sample was recombined with the reaction mixture. The mean cell volume of erythrocytes was determined in parallel by taking aliquots of 10~1 of assay medium. Aliquots were diluted with 20 ml of Isoton II electrolyte solution and analysed immediately. To document the morphological changes of erythrocytes during haemolysis samples (750~1) were taken from the reaction mixture at varying time intervals and processed for scanning electron microscopy (see below). The same assay was also carried out by adding 30 fig soybean trypsin inhibitor per midgut. The whole experiment was repeated. Erythrocyte morphology was also examined immediately after exposure to midgut homogenate in the presence of bovine serum albumin. The equivalent of one midgut was mixed with 2.5 x 10’ erythrocytes and 600 pg bovine serum albumin. Total volumes of mixtures were adjusted to 1 ml. The same was done without bovine serum albumin. Immediately after mixing (approx. 5 s later) erythrocytes were fixed with an isotonic glutaraldehyde solution and prepared for scanning electron microscopy.

853

Scanning electron microscopy

For scanning electron microscopy erythrocytes subjected to midgut homogenates and erythrocyte controls were fixed in a 1.48%-glutaraldehyde solution (1 v 8% glutaraldehyde, 2.63 v isotonic phosphate buffer pH 7.0, 1.83 v distilled water, pH 7). To ensure complete fixation, erythrocytes were mixed for at least 1 h at ambient temperature. Fixed erythrocytes were centrifuged and the supernatant discarded. Cells were resuspended in 4 ml of isotonic buffer and mixed for 10 min before they were centrifuged again. The washing procedure was repeated twice with distilled water and once with 50% ethanol. The resulting pellet was finally resuspended in 75% ethanol. A drop of this solution was then placed onto a piece of cover slip which was mounted on an aluminum stub with silver conducting paint. Samples were air dried, coated with gold and examined in a Cambridge Stereoscan 200 scanning electron microscope. Dehydration of erythrocytes in ethanol with subsequent air drying produces a shrinkage artefact in cell size (Bessis and Weed, 1972). In our erythrocyte preparations normal discocytes shortened by approx. 30% in diameter compared to unfixed cells which were suspended in a 1: 1 mixture of isotonic phosphate buffer and fresh bovine blood plasma. Transmission electron microscopy

Preparations were carried out after Tsang et al. (1982). Glutaraldehyde-fixed cells were washed in 0.5 M phosphate buffer (pH 7.4) and then postfixed in 1% osmium tetroxide at 4°C for l-2 h. After positive staining with 0.5% aqueous uranyl acetate, samples were washed and dehydrated in graded ethanol and acetone. Finally, samples were embedded in epoxy resin and sectioned (0.6-0.8 ,um). RESULTS

Relationship between proteolytic and activity of posterior midgut homogenate

haemolytic

In the presence of 0.1-2 pg soybean trypsin inhibitor per midgut equivalent of homogenate, the proteolytic activity of assay mixtures was inhibited in a concentration-dependent manner (Fig. 1). But with increasing amounts of inhibitor the inhibition curve plateaued. Under assay conditions, the inhibitor was not able to reduce proteolysis by more than 75%. In parallel to decreasing proteolytic activity the time required for haemolysis increased. The inhibition of haemolysis showed almost the same profile that was observed for proteolytic activity. Haemolytic activity could not be reduced by more than 70% under assay conditions. Haemolysis in vitro of erythrocytes proceeds after a lag time. Moreover, the time course of haemolysis displays a sigmoidal-like pattern. Figure 2 shows that after a lag period the lysis of erythrocytes gradually

854

H. J.

pg

KIRCH et al.

SBTl/midgut

Fig. 1. Inhibition of proteolytic and haemolytic activity in mixtures of bovine erythrocytes with posterior midgut homogenate from S. calcitruns by soybean trypsin inhibitor (SBTI). Reaction mixtures contained 2.5 x IO’ bovine erythrocytes, three midgut equivalents of homogenate and different concentrations of the inhibitor. Each datapoint represents the mean of three determinations.

increased and finally reached a maximal, almost linear rate. This characteristic profile was observed in soybean trypsin inhibitor-free and inhibitor-containing assays. However, the lag period and the phase of rapid lysis were significantly shorter without the inhibitor. Morphological changes of erythrocytes in vitro haemolysis

undergoing

Normal discocytes [Fig. 3(A)] immediately transformed into echinocytes [Fig. 3(B)] after exposure to posterior midgut homogenate. At the tips of some spikes contours of microvesicles appeared just before they split off. Thereafter, at the end of the lag phase, cell spines appeared shorter and had increased in number as compared to O-h cells but the formation of microvesicles still proceeded [Fig. 3(c)]. Before the cell population entered the stage of rapid lysis, spines became blunt and some cells had large membrane invaginations [arrow, Fig. 3(D)]. Subsequently, cells developed a spherical shape and bore numerous

Cell size measurements of spherocytes during in vitro haemolysis

125 0 100

Plus SBTI

+@a -o-

.I B

.

No SBTI

75 -

‘7’

;

I

c

I”

so-

w

25

0

I <

9.’

0

50

100

150 Time

200

250

300

dimples instead of membrane projections [Fig. 3(E)]. At this stage of haemolysis, also smooth spherocytes were present. When cells entered the phase of rapid lysis, the population consisted mostly of smooth spherocytes [Fig. 3(F)]. Some of them had amorphous, circular-shaped material attached to their membranes (arrows). In order to clarify the nature of this material, spherocytes were examined by transmission electron microscopy. Figure 4 shows a section through spherocytes which had a single area of broken plasma membrane of approx. 1 pm in diameter. The extrusion of intracellular material proceeded through these holes. This intracellular material is suggested to be identical with the circularshaped amorphous matter on the cell surface of some spheres [Fig. 3(F)]. Erythrocytes mixed with midgut homogenate in the presence of soybean trypsin inhibitor were also transformed into echinocytes immediately [Fig. 5(A)]. No notable differences to cells in the inhibitor-free assay occurred. Likewise, the release of microvesicles accompanied echinocytosis [Figs S(A) and (B)]. Before the cell population entered the stage of rapid lysis, membrane projections became more rounded and blunt. Occasionally, smooth spherocytes were present [Fig. 5(C)]. But cells were large membrane invaginations and cells having numerous dimples on their surface as seen in inhibitor-free assays were not detectable. During the phase of rapid lysis the cells were rather heterogeneous in appearance, consisting of spiculated cells, smooth spherocytes and cell ghosts [Fig. 5(D)]. The latter were rarely seen during haemolysis in inhibitor-free assays. But in the presence of soybean trypsin inhibitor, cell ghosts were obligatory and sustained in the assay medium even after lysis was completed. Such remaining cell ghosts were not observable in inhibitor-free assays. Whereas circular-shaped amorphous material on the surface of spherocytes was not noticed in assays containing the inhibitor.

350

[min]

Fig. 2. Time course of in vitrohaemolysis of bovine erythrocytes by posterior midgut homogenate from S. calcitrans. Reaction mixtures contained 3.1 x lOa bovine red blood cells and 25 midgut equivalents of homogenate in a total volume of 12.25 ml. Assays were performed without soybean trypsin inhibitor (SBTI) and in the presence of 30 pg inhibitor per midgut equivalent of homogenate used. Each datapoint represents the mean of two experiments.

Cell size of spherocytes was measured at the end of the haemolytic process. Provided that the spherocytes are not deformable during the sizing procedure, the mean cell volume of spheres in the soybean trypsin inhibitor-free assay was 44.7pm3 (Table 1). This corresponds to a surface area of 60.9 pm*. Compared to a normal bovine erythrocyte which has a surface area of approx. 81-83 or 92pm*, respectively, if discocytes are treated as cylindrical objects, the erythrocytes lost a considerable amount of plasma membrane. Determination of the mean cell volume in the inhibitor-containing assay revealed a corrected sphere size of 57 pm3 which corresponds to a surface area of 71 pm*. In the presence of the inhibitor, cell volume did not change significantly after lysis was completed because ghosts were almost the same size.

Fig. 3. Sequence of changes in cell morphology of bovine erythrocytes undergoing in vitro haemolysis by posterior midgut homogenate from S. calcitrans. (A) Normal washed erythrocytes in isotonic phosphate buffer. (B) Erythrocyte immediately after exposure to midgut homogenate. (C) Erythrocyte during lag phase. (D) and (E) Cell populations at the onset and during the first phase of rapid cell lysis. (F) Cell population during the period of rapid cell lysis. Reaction mixtures contained 3.1 x IO* bovine red blood cells and 25 midgut equivalents of homogenate in a total volume of 12.25ml.

855

Fig. 4. Thin section through erythrocyte spheres produced by the action of posterior midgut homogenate from S. calcitrans. The cell surfaces show locally restricted areas of damaged plasma membrane which allows the extrusion of intracellular material.

856

Fig. 5. Sequence of changes in cell morphology of bovine erythrocytes undergoing in vitro haemolysis by posterior midgut homogenate from S. calcitrans in the presence of soybean trypsin inhibitor. (A) Immediately after exposure to midgut homogenate. (B) Erythrocytes during lag phase. (C) Cell population at the onset of rapid ccl1 lysis. (D) Cell population during the period of rapid cell lysis. Reaction mixtures contained 3. I x 10s bovine red blood cells, 25 midgut equivalents of homogenate and 30 pg of inhibitor per midgut in a total volume of 12.25 ml.

857

Fig. 7. Bovine erythrocytes immediately after exposure to posterior midgut homogenate from S. calcifrans. (A) Control erythrocytes; (B) 2.5 x 10’ erythrocytes mixed with one midgut equivalent of homogenate and (C) same as in (B) but in the presence of 600 ng bovine serum albumin. After the addition of homogenate erythrocytes were fixed immediately with the same volume of a 1.47% glutaraldehyde solution.

858

Mechanism of erythrocytes haemolysis

30

60 pg

90

120

BSA/midgut

Fig. 6. Effect of bovine serum albumin on the in vitro haemolysis of bovine erythrocytes by posterior midgut homogenate from S. calcitruns. Reaction mixtures contained 2.5 x 10’ bovine red blood cells, three midgut equivalents of homogenate and different concentrations of bovine serum albumin (BSA) (lo-100 pg per midgut equivalent). Insert: inhibition of proteolytic activity of reaction mixtures of bovine serum albumin (BSA) (8-32mg per midgut equivalent). Each datapoint represents the mean of three determinations. Influence of bovine serum albumin on erythrocyte haemolysis The addition of bovine serum albumin to haemolytic assays caused a marked reduction in haemolytic activity (Fig. 6) which was related to the concentration of albumin used. Proteolytic activity, measured with azocasein as substrate, was not significantly affected when 5-100 pg bovine serum albumin/midgut were added to assay mixtures (insert of Fig. 6). Addition of albumin to assay mixtures also influenced the effect of the midgut homogenate on erythrocyte morphology (Fig. 7). The instant transformation from normal discocytes [Fig. 7(A)] to echinocytes [Fig. 7(B)] was markedly suppressed in the presence of bovine serum albumin [Fig. 7(C)]. DISCUSSION

The involvement of proteases in the haemolysis of erythrocytes by the blood-feeding stable fly has been inferred by Spates and DeLoach (1980). They demonstrated that the addition of trypsin to midgut homogenates causes an increase in haemolytic activity. Whereas for the tsetse fly, Glossina morsitans, it is assumed that no relationship between proteolytic and haemolytic activity exists (Gooding, 1977). Our results showed that soybean trypsin inhibitor reduced proteolytic and haemolytic activity of stable fly midgut homogenate equally. Also Geering (1975) reported, that this inhibitor reduces the haemolytic activity of midgut blood clots from the mosquito Aedes aegypti. However, proteases are thought not to haemolyse intact mammalian erythrocytes (Steck et al., 1971) and therefore the significance of proteolytic enzymes in haemolysis has to be explained by other means than direct proteolysis of membrane components. A new perspective for a participation of proteases might be deduced from the fact that the

859

midgut homogenate of stable flies contains sphingomyelinase activity (Spates et al., 1990). Because it was shown that preincubation of bovine erythrocytes with pronase enhances the absorption of sphingomyelinase onto the plasma membrane, the hydrolysis of sphingomyelin (the major phospholipid in the outer membrane leaflet) begins in advance compared to untreated controls (Tomita et al., 1987). Hence, proteases might improve the accessibility of membrane sphingomyelin and thereby the action of sphingomyelinase. However, the entire role of proteases in erythrocyte haemolysis remains obscure. For human erythrocytes, e.g. it is known that proteolytic enzymes binding to soybean trypsin inhibitor are located in the erythrocyte plasma membrane (Golovtchenko-Matsomoto et al., 1982). Therefore, it cannot be excluded that even membrane bound enzymes might contribute to haemolysis. Yet we could not clarify if the inhibitor is absorbed unspecifically onto the surface of the bovine erythrocytes and thereby passively prevents membrane perturbation. It is known that the shape of erythrocytes responds to changes of the physico-chemical environment such as exposure to hypotonic or hypertonic solutions and deviations from neutral pH (Weed and Chailley, 1973). Since our experiments were conducted at isotonic conditions and at pH 7.0, morphological changes could be attributed to the impact of the midgut homogenate alone. When bovine erythrocytes were mixed with homogenate, they immediately transformed into echinocytes. Conditions other than hypertonic solutions which transform discocytes into echinocytes are: treatment with phospholipase A, if erythrocytes contain a phosphoglyceride in the outer membrane leaflet (Fujii and Tamura, 1979) loss of intracellular ATP (Nakao et al., 1960) or the exposure to detergents (Deuticke, 1968). Since bovine erythrocytes contain only sphingomyelin in the outer membrane leaflet, phospholipase A should not be responsible for the formation of echinocytes. Moreover, a phospholipase A activity has not been detected in midgut homogenates of stable flies (Spates et al., 1990). ATP-loss involves a time factor of at least a couple of hours (Shohet and Haley, 1973) which would contradict the immediate (5 s) shape change seen with midgut homogenate. Therefore, endogenous detergent-like substances of the homogenate are more likely to initiate echinocytosis. Firstly, detergent-induced echinocytes are formed almost instantaneously (Sheetz, 1983). Secondly, when bovine serum albumin is added to the midgut the expression of echinocytes is homogenate, markedly suppressed. Albumin, being a strong absorbent for amphiphiles (Helenius and Simons, 1975), could bind detergents of the homogenate and prevent them from reacting with the erythrocyte membrane. Spates et al. (1982) showed that the homogenate of stable flies contains free fatty acids and thus potent detergent material. The use of detergents in haemolysis

is certainly

a powerful

tool. In sufficient

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H. J. KIRCH etal

concentrations detergents themselves are haemolysins while in sublytic concentrations they act in destabilizing the architecture of the plasma membrane thus rendering membrane components more vulnerable to enzymatic treatments (Roelofsen et al., 1971). Membrane invaginations which appeared after echinocytosis in soybean trypsin inhibitor-free assays could be explained by degradation of membrane sphingomyelin by sphingomyelinase. This has been shown for sheep erythrocytes (Ikezawa et al., 1980). Therefore, the invaginations of erythrocytes might be a morphological marker for the participation of sphingomyelinase activity in membrane degradation. This might also apply for the formation of numerous dimples across the plasma membranes in the case of inhibitor-free assays. However, we cannot mechanistically explain the formation of the dimples. Spherocytosis, the generation of smooth erythrocyte spheres was attained just before cell lysis. In soybean trypsin inhibitor-free assays cell lysis was achieved by the formation of a single hole in the plasma membrane (Fig. 4). Baker (1967) described a similar phenomenon when erythrocytes were exposed to hypotonic solutions. Due to water influx and cell expansion the cells haemolyse by the formation of a membrane hole of about 1 pm in diameter. But during haemolysis by the inhibitor-free homogenate there was no indication of a serious cell swelling. Moreover, erythrocytes shrank in size and lost a considerable amount of surface area. The membrane loss might be a consequence of microvesiculation processes during echinocytosis. It is reported also that profound sphingomyelinase action causes cell shrinkage (Low and Freer, 1977). Spherocytes from the inhibitor-free assay had a mean cell volume of 44.7/*m3 (Table I). This corresponds to a surface area of 60.9 pm* which is approx. 26 or 34%, respectively-if discocytes are treated as cylindrical objects-less than the surface area of normal discocytes. These values correlate with the data of La Celle et al. (1973). They removed surface area from human erythrocytes in the form of microvesicles and found that a reduction by more than 28% lead to the formation of small spherocytes which were not deformable and very susceptible to haemolysis. Therefore, we conclude that haemolysis in vitro by midgut homogenate is triggered by a significant decrease in cell surface area. This induces heavy mechanical stress inside the membrane and finally, analogous to the hypoosmotic-like haemolysis, leads to membrane rupture and the formation of a hole. Addition of soybean trypsin inhibitor to assay mixtures had no noticeable influence on the formation of echinocytes. This might indicate that the inhibitor would not have an immediate masking effect, at least not for detergent molecules. Membrane invaginations and dimples were not observed when proteolytic enzymes were inhibited by the inhibitor. The same is true for membrane holes. Nonetheless, microvesiculation occurred during echinocytosis as

well, and an indefinite amount of surface area is certainly withdrawn. However, membrane loss might not exceed a critical level to produce a membrane break. We assume that haemolysis in assays containing soybean trypsin inhibitor was mainly dependent on the continued action of detergents which finally solubilized the membrane lipids. Furthermore, this mode of haemolysis and the reduced proteolytic activity might be responsible for the preservation of cell ghosts which remained after haemolysis was completed. In summary we suggest that detergent-like substances, sphingomyelinase activity and proteolytic enzymes are involved in the in vitro haemolysis of bovine erythrocytes by posterior midgut homogenate from stable flies. However, the precise interaction of the participating haemolytic components and the role of the erythrocyte’s own membrane dynamics during haemolysis need further clarification. This is especially true for the function of proteases. As the process of haemolysis appears not to be one single step but a rather complex sequential mechanism, proteases might act at different stages of erythrocyte breakdown. Acknowledgements-The authors wish to thank E. Moore for expert technical assistance with the scanning electron microscopy. This research was partially supported by the German Academic Exchange Service.

REFERENCES

Baker R. F. (1967)Ultrastructure of the red blood cell. Fed. Proc. 26, 1785-1801. Beck J. (1978) Relations between membrane monolayers in some red cell shape transformations. J. theor. Biol. 75, 4877501. Bessis M. and Weed R. I. (1972) Preparation of red blood cells (RBC) for SEM. A survey of various artifacts. Proc. Work -shop Biological Specimen Preparation Techniques /or Scanning Electron Microscop.v. IIT Research Institute Chicago, Ill., April. Bridges A. C., Summerlin S. W. and Spates G. E. (1984) A new and more economical base medium for rearing larvae of the stable fly, hornfly, and housefly. Swesr. Enr. 9, 3888391. De Azambuja P.. Guimares J. A. and Garcia E. S. (1983) Haemolytic factor from the crop of Rhodnius prolixus: evidence and partial characterization. J. Insecr Physiol. 29, 833-837. Deuticke B. (1968) Transformation and restoration of biconcave shape of human erythrocytes induced by amphiphilic agents and changes of ionic environment. B&him. biophys. Actn 163, 494-500. Fujii T. and Tamura A. (1979) Asymmetric manipulation of the membrane lipid bilayer of intact human erythrocytes with phospholipase A, C or D induces a change in cell shape. J. Biochem. 86, 1345-1352. Geering K. (1975) Haemolytic activity in the blood clot of Aedes aegypti. Acta Tropica 22, I45 - I5 I. Golovtchenko-Matsomoto A. M., Matsumoto I. and Osawa T. (1982) Degradation of band-3 glycoprotein in vitro by a protease isolated from human erythrocyte membranes. Eur. J. Biochem. 121, 463-467. Gooding R. H. (1977) Digestive processes of haematophagous arthropods. XIV. Haemolytic activity in the

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