Inhibitory effects of cyclodepsipeptides, destruxins, from the fungus Metarhizium anisopliae, on cellular immunity in insects

J. In.wcr Physiol Printed

in Great

Vol. 35, No. 2, pp. 97-105,

1989 Copyright

Britain. All rights reserved

0

0022-1910/89 $3.00 + 0.00 1989 Pergamon Press plc

INHIBITORY EFFECTS OF CYCLODEPSIPEPTIDES, DESTRUXINS, FROM THE FUNGUS METARHIZIUM ANKWPLIAE, ON CELLULAR IMMUNITY IN INSECTS I. M. HUXHAM*, A. M. LACKIE*~and N. J. MCCoRKlNoALEt *Department of Zoology and TDepartment of Chemistry, The University of Glasgow. Glasgow G12 8QQ. Scotland (Rewired

17 March

1988;

rerised 20 June 1986)

Abstract-The effects of a mixture of destruxins A and B, cyclic depsipeptides produced by the fungal pathogen of insects, Metarhizium anisopliae, were tested on various aspects of the behaviour of haemocytes (the freely circulating cells associated with the immune response) of the cockroach Periplanera americana and the locust Schisfocerca gregaria. In general, destruxins inhibited or reduced haemocytic activation induced by /JI,3-glucans such as laminarin or zymosan supernatant; thus, the number of zymosan supematant-induced haemocytic aggregates (nodules) formed in uiuo was reduced by preinjection with destruxins, and the laminarin-stimulated locomotory and aggregation behaviour of a separated subpopulation (‘band 5’) of locust haemocytes was suppressed by co-incubation with destruxins. The morphology and intracellular arrangement of actin filaments, visuahsed by Ruorescently-labelled phalloidin, differed markedly between these laminarin-stimulated and destruxin-suppressed band 5 cells. Laminarin activates the cell-associated prophenoloxidase system to produce phenoloxidase. which converts L-DOPA to the black pigment, melanin; this effect and the release of protein by laminarinstimulated locust cells, both of which can be quantified, was reduced by the destruxin mixture. Helvolic acid, a major secondary metabolite secreted by the fungus. was without effect. Key Word Index: Destruxins. cyclic depsipeptides, haemocytes, cell locomotion, phalloidin, phenoloxidase, Schisiocerca, Periplaneta

p 1,3-glucans such as laminarin or zymosan supernatant, as shown by a decrease in the production of the cell-associated enzyme, phenoloxidase (Huxham and Lackie, 1986). Phenoloxidase is the enzyme responsible for production of melanin, often found at the core of haemocytic aggregates, and several interesting biological effects (Smith and Soderhiill, 1986; Takle and Lackie, 1986) are coincident upon stimulation of the Ca*+ -dependent enzyme sequence that is involved in activation of the pro-enzyme. prophenoloxidase. Although activation of haemocytes undoubtedly stimulates systems unrelated to the prophenoloxidase sequence, the latter system serves as a useful indicator of the stimulation of several aspects of haemocyte behaviour. Any molecule that suppresses activation of the prophenoloxidase sequence is thus likely to modulate some manifestations of cellular immunity. The results of preliminary work suggested that, of the several important secondary metabolites secreted by Metarhizium, the suppressive effect of hyphae-conditioned medium was most likely due to the action of destruxins. The experiments described here are thus designed to investigate the effects of purified destruxins on certain immune phenomena associated with haemocytes in vioo, and on several aspects of haemocyte behaviour in vitro. In vivo, the effect of destruxins on j? 1,3-glucan-induced haemocytic aggregation (nodule formation) is assessed; in vitro, their effects on /? 1,3-glucan-induced prophenoloxidase-activation in living haemocytes and haemocyte lysates, on protein release and on haemocyte locomotory behaviour, are examined.

lNTRODUCTlON

Cyclodepsipeptides, a family of molecules that comprise cyclically-linked amino acids, are produced as secondary metabolites by various fungi, and include destruxins, roseotoxin B, and the mammalian immunosuppressant, cyclosporin A (Taylor, 1970; Klaus and Chisholm, 1986). The destruxins, which contain 5 amino acid residues, are produced by a fungal pathogen of insects, Metarhizium anisopliae, and cause paralysis and death in insects (Kodaira, 1962; Paprowski et al., 1985; Vey et al., 1986; Samuel5 et al., 1986). It is likely, however, that their effects on cockroaches and locusts are more subtle, since these insects may not die until 3 or 4 days after infection, at which time the mycelium has begun to ramify through the body cavity. Prior to invasion of the haemocoele, fungal spores must germinate on the surface of the hard external cuticle, and then penetrate the cuticle itself. Even before the growing hyphae reach the haemocoele, the cells that mediate the immune response, the haemocytes, become activated (Gunnarsson et al., 1989) and aggregate beneath the site of hyphal penetration (Gunnarsson, 1989). Despite this, the haemocytes are apparently unable to contain fungal growth once penetration has occurred and fail to adhere to or encapsulate the hyphae. Recent work has shown that hyphae-conditioned medium suppresses the in vitro activation of haemocytes by :To whom correspondence should be addressed 91

98

I. M.

HUXHAM et

MATERIALS AND METHODS

Insects Adult male locusts (Schistocerca gregaria) were obtained from Larujon (Wales) and were maintained under a 12-h photoperiod at 32 _+ 1‘C (day), 28 f 1“C (night), on a diet of dried bran and water. Adult male cockroaches (Periplaneta americana) were obtained from BioServ (U.K.) and were maintained at 28 t_ 1-C, under a 12-h photoperiod, on a diet of rat cake and water. Culture of‘ Metarhizium anisopliae Isolate ME1 was kindly provided by Dr A. K. Charnley of Bath University, England. During the course of our studies it was passaged twice through adult male locusts by intrahaemocoelic inoculation of mycelial fragments (20-50 pg/locust). Spores were collected aseptically and maintained on Sabouraud’s dextrose agar slants by conventional methods. A seed culture was made from spores collected from two 5 ml slant cultures and germinated in a 500 ml flask containing 150 ml broth (Czapek-Dox, Difco) supple1.4 mg/ml mented with 0.5% bactopeptone, FeS0,.7Hz0. and 0.8 mg/ml ZnS0,,7H,O. After culture for 3 days at 30-C on a rotary shaker (about 160 rpm in darkness), twenty 500 ml flasks each containing 150 ml broth, were each inoculated with 2 ml of the seed medium. Cultures were maintained at 263O’C for a further 14 days with continuous rotary shaking. Isolation of’ the destrusin mixture After freezing and thawing, the clear supernatant above the mycelial fraction was aspirated and extracted continuously overnight with methylene chloride. Extracts were concentrated in vacua to produce l-l .6 g of material and were then dissolved in approx 250 ml boiling diethyl ether. Acidic components were removed by washing the extract with saturated sodium bicarbonate. The remaining extract, containing neutral and basic components, was washed with water, brine and then concentrated in vacua. Ether extracts (about 0.6g) were chromatographed on two Kieselgel HFzS4 preparative thin-layer plates (1 m x 20 cm x 1 mm) using chloroform-methanol (19: 1. v/v). Destruxin-rich fractions within R, values 0.54.6 were removed from the supporting silica using ethyl acetate and pooled (15c-250 mg). The extract was re-chromatographed on a Kieselgel 60zj4 plate (20 cm x 20 cm x 1 mm) 3 times using chloroform-light petrol (9: 1, v/v). Destruxins A and B remained close to the origin (R, &O. 125) whilst the other major contaminants (ergosterol and fatty acids) eluted with R, values greater than 0.25. Destruxinrich bands were removed with ethyl acetate (5-10 mg) and then subjected to oil-pump evacuation to remove residual solvent and water to yield a yellow-white solid. Fractional crystallisation was carried out using benzene-hexane at O’C. Nuclear magnetic resonance spectroscopy was performed in deuterated chloroform on a Bruker WP 200SY spectrometer using tetramethyl silane as internal standard, and showed that the yellow-white solid was a mixture of destruxins A and B. The samples were dissolved in dimethylsulphoxide prior to use.

al.

Effect of destruxins on zymosan-induced haemocytic aggregation (nodule formation) in vivo Zymosan (Sigma, U.K.) [ 10 mg/ml] was suspended in HEPES-buffered cockroach saline (Huxham and Lackie, 1986) and whirlimixed for 3 min. Following centrifugation at 1500 g, the mixture was whirlimixed and spun again. The supernatant was used for injection. Nodules were induced in cockroaches by injection of 20 ~1 zymosan supernatant; the haemolymph of each insect was flushed out 3 h later with 2ml of citrate-EDTA anticoagulant buffer, pH 4.6. into separate 5cm Petri dishes, and the number of nodules per insect counted (Gunnarsson and Lackie, 1985). The effect of destruxins on nodule formation was investigated by injecting the insects 2 h before the zymosan injection with either a mixture of destruxins A and B (2Opg/g b.wt) in 10% dimethylsulphoxide or, as controls, 20~1 of either 10% dimethylsulphoxide or HEPES buffered saline alone. Data were analysed using an analysis of variance. Cockroaches were used in preference to locusts for this part of the investigation, since the number of nodules formed after stimulation of cockroaches with a zymosan supernatant was larger than from locusts (Gunnarsson and Lackie, 1985). Effects of destruxins on activation of prophenoloxidase within locust and cockroach haemocyte monolayers Monolayers of locust and cockroach haemocytes were prepared according to the methods of Huxham and Lackie (1986). Briefly, cells were collected in citrate-EDTA anticoagulant buffer at 4-C. washed, and then resuspended in either locust medium (pH 6.7, 305 mosM) or cockroach medium (pH 7.2. 340mosM; see Huxham and Lackie’s medium in Crompton and Lassiere, 1987). Cells were plated out in 3 cm tissue culture dishes to give a density of 5 x IO5cells/dish (locusts) or IO6cells/dish (cockroaches). Heat-inactivated foetal calf serum (10%) was added to the medium (= SLM). Cell-associated prophenoloxidase was activated by addition of the /3 1.3-glucan, laminarin (Sigma) [l mg/ml], and the presence of active phenoloxidase visualised by addition of the substrate, L-DOPA (0.5 mg/ml final concentration); the number of cells or cell aggregates which subsequently turned black, following conversion of L-DOPA to melanin, was counted and calculated per unit area, as described previously (Huxham and Lackie, 1986). The effect of the destruxin mixture on this laminarin-stimulated prophenoloxidase activation was assessed by preincubation of haemocyte monolayers in 1 ml medium containing a mixture of destruxins A and B for 1 h at room temperature, then co-incubation with destruxins and the stimulant for a further 1 h before the addition of L-DOPA. The effects of one destruxin mixture at three concentrations (2, 5 and 10 pg/ml) and of helvolic acid (30 pg/ml. BDH) were determined. Effects of destruxins A and B on activation of prophenoloxidase within a locust haemocyte lysate The effect of one mixture of destruxins A and B on laminarin- or zymosan supernatant-stimulated activation of the prophenoloxidase sequence in the supernatant fraction from a locust haemocyte lysate was

Immunosuppression by destruxms examined. Locust haemocyte lysate was prepared as described previously (Huxham and Lackie, 19W, the supernatant providing a source of the enzymes responsible for the prophenoloxidase activation sequence (Dularay and Lackie, 1985). Aliquots (10 ~1) of the haemocyte lysate supernatant were added to 100 ~1 HEPES buffered saline and 50~1 zymosan supematant or 50 ~1 laminarin (1 mg/ml) and incubated for 30min at 30°C with or without 0.2% dimethylsulphoxide or 20 pg destruxins. Activity was determined by measuring the absorbance at 490 nm following the addition of 0.5 mg L-DOPA in HEPES buffered saline and was expressed as U = A 490 nm 0.001 /min/mg protein. Quanttjication of protein released into the culture medium by haemocyte monolayers

Monolayers of locust and cockroach haemocytes (1 million/dish in 3 cm dishes) were prepared as above, but without foetal calf serum. Four cultures were compared following incubation at 30°C; 0.2% dimethylsulphoxide for 2 h, 0.2% dimethylsulphoxide for 1 h with 1 mg/ml laminarin for the second hour, 20 pgg/ml destruxins (in 0.2% dimethylsulphoxide) for 2 h, or 20 pgg/ml destruxins for 2 h with 1 mg/ml laminarin for the second hour, in 1 ml culture medium. Two further cultures without dimethylsulphoxide and with or without laminarin, were included as additional controls, so that any direct effect from dimethylsulphoxide could be determined. Culture media were collected and spun at 450g for 10 min and then used directly for spectrophotometric analysis of phenoloxidase activity (see below) or added to 4.5 vol of cold methanol (AR grade, BDH). The protein precipitate, formed at - 15°C overnight, was pelleted at 1500 g. Pellets from individual dishes were dissolved in distilled water (100 ~1) for an analysis of total protein using a modification of the methods used by Lowry et al., 1951. Phenoloxidase activity within 1 ml samples of culture medium was assayed by adding 100 ~1 HEPES buffered saline containing 0.5 mg L-DOPA as a substrate, and measuring the absorbance at 490 nm for 30 min (locust) or 5 h (cockroach), either directly (laminarin-containing samples) or after the addition of 1 mg laminarin (control cultures). Activity was expressed as U = A 490 nm *O.OOl/min/ml, as the average of three readings. Effects of destruxins A and B on the locomotory behaviour of‘ separated subpopulations of locust haemocytes

Locust haemocytes were separated on a 10 ml discontinuous Percoll gradient, with 10% steps from 25-65% as described previously (Huxham and Lackie, 1988). Haemocyte fractions from two of the interfaces were used in the analysis of locomotory behaviour. Those from the interface between 55 and 65% Percoll (= band 5 cells) comprised a homogeneous fraction of densely-granular plasmatocyte-like cells that were strongly associated with the enzyme phenoloxidase. Of the cells from the 45/55% interface (= band 4 cells). 95% were plasmatocyte-like haemocytes with moderate granularity, while the remain-

99

der within this band showed characteristics of the band 5 fraction. Cells in SLM were aliquoted into 3 cm dishes at a density of approx 5000cells/mm2 and allowed to adhere and spread for 15-30 min at 30°C. The monolayers were then incubated for I h with SLM containing combinations of dimethylsulphoxide, laminarin and destruxins as described in the previous section for unseparated haemocyte monolayers. Following this period, the behaviour of the cells was filmed by time-lapse cinemicrography for a further 2 h at 28-3O”C, using an Olympus inverted microscope, a Bolex tine camera (1 frame/40 s) and Kodachrome 40 film. Outlines of the cell periphery and nucleus were traced directly onto paper from a projected image at the beginning of filming and again after 30, 60 and 120 min. Approximately 12 cells were visible per field at the density and magnification used. Cell distribution was frequently heterogeneous so each held was chosen to include both separated and clustered, fully spread cells. Each experiment was repeated 3 times using cells from 8 separate Percoll gradients. Effects of destruxins A and B on the distribution of F-actin in the densely granular haemocyte fraction

Haemocytes from the interface between 55 and 65% Percoll (band 5 cells) were prepared as above. After removal of the Percoll, cells were plated out into Nunc 3 cm-dia. culture dishes in SLM and allowed to spread for 2 h at 28°C. The medium was replaced with fresh medium containing either (1) 0.1% dimethylsulphoxide, or (2) 50 pg/ml destruxins A and B in 0.1% dimethylsulphoxide for 90 min. or (3) 0.1% dimethylsulphoxide for 60 min then 1 mgjml laminarin with 0.1% dimethylsulphoxide for 30 min, or (4) 50 pg/ml destruxins A and B then the addition of 1 mg/ml laminarin for a further 30 min. Cells were fixed with 2% paraformaldehyde in HEPES buffered saline at 4°C for 1 h, washed in the saline, permeabilised with 0.5% Triton X-100, washed again and then incubated with FITC-Phalloidin (5 U/ml, Sigma) for 30 min at 22°C. After mounting in 50% glycerol-50% 0.15 M Tris-HCl buffer pH 8, cells were observed on a Leitz Ortholux microscope using epifluorescence. RESULTS The effect of destruxins on nodule formation in viro

Figure 1 shows that pre-injection of cockroaches with the destruxin mixture significantly reduced the number of haemocytic aggregates formed in response to an injection of zymosan supernatant. Dimethylsulphoxide had no effect. Data points for individual insects were scattered, but the mean number of nodules from insects pre-injected with a mixture of destruxins A and B was significantly less than from both controls (P = 0.01). Suppression of prophenoloxidase struxins

activation by de -

Incubation with destruxins A and B reduced the number of phenoloxidase-positive cells and cell aggregates in monolayers prepared from both locust and cockroach haemocytes (Fig. 2; Table 1); a reduction in the staining intensity was also observed in

I. M. HUXHAMet al.

100 1000

900

cockroach haemocyte monolayers following culture with lOpg/ml destruxins (Fig. 2d). The suppressive effect was concentration-dependent and was not attributable to helvolic acid, a common contaminant of destruxin preparations, since purified helvolic acid was without visible effect on locust cell monolayers up to a concentration of 30 pg/ml. At 2 or 10 pg/ml, four other preparations of destruxins A and B had similar effects on locust cell-associated phenoloxidase (data not shown). Interestingly, in contrast to their inhibitory effect on prophenoloxidase activation in living cells, destruxins did not inhibit prophenoloxidase activation within a locust haemocyte lysate supernatant (Table 2).

. . . . . : .

0; : . :

t

.

m : .

Protein release by haemocytes in monolayer culture

. .

. B .

200

100

l

0.

When incubated without foetal calf serum to stabilise the cells, monolayers of both locust or cockroach haemocytes released protein into the culture medium (Table 3). The amount of protein released by cockroach cells was greater than that released by locust haemocytes cultured at the same density (141,000 cells/cm’). Incubation with laminarin enhanced the amount of protein released by locust haemocytes but this release was reduced by co-culture of monolayers with destruxins. The relative decrease (30%) in the amount of protein released by locust cells following culture with destruxins and laminarin was approximately proportional to the decrease (37%) in phenoloxidase activity detected within the culture medium. In contrast, no differences were found between the amounts of protein released into the culture medium of cockroach haemocytes incubated in the presence of laminarin, or destruxins alone or destruxins plus laminarin. The amount was. in all cases, high

0; i.

.

0 OMSO

Saline

Osrtruxins

Pre-injection

Fig. 1. Effect of destruxins A and B on zymosan-induced nodule formation in the cockroach Periplaneta americana. Saline; pre-injection of insects with 20 p I HEPES buffered saline. DMSO; pre-injection of insects with 20~1 of 10% dimethylsulphoxide in HEPES buffered saline. Destruxins; pre-injection of insects with 20pg/mg of mixture of destruxins A and B dissolved in 10% dimethyl-sulphoxide in HEPES buffered saline. Closed circles represent values from individual insects and the open circles the mean value for each group. ANOVAR: Destruxins vs Saline; F = 27.4 (P = O.Ol), Destruxins vs dimethylsulph-oxide; F = 24 (P = 0.01).

Table

I. Effect

of a mixture

of destruxins positive

~~~

A and

haemocytes

B on the wsualisation

mean/mm*

.~.

of phenoloxldase-

in monolayer

( + SE)

%

Control

n

27

Locust Control

without

Control

plus laminarin’

2~g:ml

(destruxins

0.25 (0.03)

2

10.02 (0.27)

100

33

4.55 (0.20)

45

45

3.74 (0.16)

37

26

1.35(0.10)

I3

35

10.58 (0.75)

100

38

laminarin’ A and

9)

5 Pg/ml IO/lg;ml 30 pg/ml

(helvohc

acid)t

Cockroach Control

wthout

Control

plus laminarin’

2pg:ml

(destruxins

5

lammarin* A and

B)

pgiml

0.25 (0.04)

I

77

16.25 (0.27)

100

93

7.06 (0.25)

43

40

6.37(0.13)

39

30

9

60

I.51 (0.10)

IOpg/ml n: Number *All

cultures

tThe

effects medium

Table

of fields

counted

contained of

from

helvolic

acid

obtained

by re-crystallisation

2. Effects of a mixture

commercxally

of destruxins

fraction

Stimulant 2s

6.3 U

Laminarin

0.5 u

or

purified

using cold

A and B on actwation

haemocyte

lysate

Stimulant

Control (no stunulation)

from methanol

our

value

the average lysate.

culture identical.

of prophenoloxidase

Stimulant

+

Stimulant destruxins

DMSO

alone 492.7 U

459.0 u

448.5 U

286.0 U

294.4 U

31l.OU

of three

readings

within

supernatant

supernatant. represents

U = A 490 nm O.OOl/min/mg

own

were

dimethylsulpboxide.

Zs: zymosan Each

experiments.

of the TLC

a locust

DMSO:

three

0,2%/dimethylsulphoxide.

taken

from

three

lysates

+

Fig. 2. Inhibition of laminarin-induced prophenoloxidase activation in monolayers of cockroach haemocytes by destruxins A and B. Phenoloxidase-positive cells were few in number in cultures without laminarin (a) but common in cultures stimulated with laminarin (b). Co-culture with a mixture of destruxins A and B decreased the number of phenoloxidase-positive cells (see Table 2) both at 2 pg/ml (c) and 10 pg/ml (d). Staining intensity was generally weak and sometimes granular. Bar = 100 pm.

101

Fig. 4. Distribution of F-actin in the densely granular, plasmatocyte-like haemocytes from band 5. The morphology and F-actin localization in haemocytes cultured in medium alone (a) or with destruxins (b) was similar. Stimulation with laminarin (c) resulted in a rapid change in morphology and in the distribution of F-actin. Few F-actin filaments were visible, and most fluorescence was either diffuse or concentrated into punctate areas. Pre-incubation of cells with destruxins prior to stimulation (d), resulted in the formation of filopodia in which F-actin was concentrated (arrows). Bar = 20nm.

102

lmmunosuppression Table 3. Quantitation of the amount of protein and phenoloxidase activity in the culture medium of stimulated and unstimulated locust haemocyte monolayers ,ng protein released/IO6 haemocytes mean (+ SE) Culture medium only Medium (+0.2% DMSO) Plus I mg/ml laminarin Plus lam. + 0.2% DMSO Plus destruxins (Dx)t Plus Dx and lam.

48.6 (2.2) 38.6 (5.3) 71.8 (2.0) 70.2 (2.2) 42.5 (3.3) 49.7 (4.7)

Phenoloxidase activity of medium (U) (average of 3 readings) 1.5* 10.0 I .62* 6.25

n = 15 dishes from three experiments, monolayers in I ml culture medium without foetal calf serum. ‘Laminarin and L-DOPA were added after collection of the medium, in order to assay for phenoloxidase (see text). t0.2% DMSO present as solvent for destruxins (Dx).

(97-125 pg) compared to locust haemocyte monolayers. A small amount of phenoloxidase activity was detected in the culture medium of cockroach cells stimulated with laminarin (0.5 U after 5 h), but phenoloxidase activity was not detected in any other culture medium. Monolayers of cockroach haemocytes cultured in the absence of serum are more unstable than locust haemocytes. 0 ml”

by destruxins

103

Locomotory behaviour of separated locust haemocytes

Unstimulated haemocytes cultured in SLM (plus 0.2% dimethylsulphoxide) or in SLM plus the destruxin mixture remained firmly attached to the substratum, but the leading edges of their plasma membranes and the granules within their cytoplasm were in a state of continuous movement. Conversely, marked differences were observed in the behaviour of laminarin-stimulated band-5 cells within 30 min of the start of filming (Fig. 3). Stimulated haemocytes rounded up from the tissue culture dish, moved upon the substratum and then formed tight cell aggregates. However, 6@90 min after addition of laminarin, the haemocytes once more spread upon the culture dish and regained their pre-stimulation appearance. Degranulation was not observed. In contrast, although co-culture with destruxins and laminarin did not affect ruffling of the plasma membrane or movement of the cytoplasmic granules, locomotion and aggregation was suppressed. Laminarin was without effect on the behaviour of plasmatocytes from bands 2, 3 and 4 (data not shown). Cells remained attached to the substratum and moved slowly during all culture conditions. Thus, laminarin-induced aggregation is only associ-

30 min

60 min

SLM

+ LAM 60 ml”

DX + LAM

Fig. 3. Effect of a mixture of destruxins A and B on laminarin-induced aggregation of the Percollfractionated granular, plasmatocyte-like haemocytes (band-5 cells) from the locust Schistocerca gregaria. In the absence of laminarin (SLM), haemocytes coalesced but did not aggregate during a 3 h filming period. Aggregation of haemocytes took place with 30 min from the addition of laminarin (+ lam), but this response was inhibited by co-culture with destruxins (Dx + lam). In all cases, haemocytes spread and resumed slow locomotion upon the tissue culture plastic surface after further culture.

I. M. HUXHAM et al.

104

ated with the sub-population phenoloxidase-rich plasmatocytes

of very granular, found in band 5.

Changes in the distribution of F-actin within separated haemocytes after treatment with destruxins and laminarin In cells incubated with dimethylsulphoxide or the destruxin mixture, F-a&n was arranged as a complex network of filaments at many focal planes (Fig. 4a and b), emphasising the fibroblast-like morphology of these spread cells in culture. Since laminarin caused rounding-up and a transient reduction in adhesion to the tissue culture plastic many cells were lost during the preparative procedures. The partially adherent cells that remained, however, showed a mixture of diffuse, punctate and filamentous staining patterns at different focal planes (Fig. 4c). In contrast, haemocytes pre-incubated with destruxins A and B and then co-incubated with destruxins and laminarin remained widely spread with most (more than 75%) showing brightly fluorescent filopodia (Fig. 4d).

DISCUSSION

In all the essays described here, the destruxin suppressed the stimulatory effect of mixture fl 1,3-glucans on living haemocytes, whether it was activation of cell-associated prophenoloxidase, protein release (locust cells), haemocyte locomotion and aggregation. or in vivo nodule formation. On unstimulated haemocytes, or where laminarin had no detectable stimulatory effect-as with the locomotory behaviour of locust cells from band 4, or protein release by cockroach haemocytesdestruxins had no observable effect. In addition. laminarin-induced activation of prophenoloxidase within the supernatant from haemocyte lysate was not suppressed by destruxins, a result which might tentatively be interpreted to suggest that destruxins act at the level of the intact haemocyte plasma membrane. Fungal pathogens of insects are potentially highly immunogenic, since the /I 1,3-glucan components of their cell walls activate many different aspects of the immune response. In vitro, addition of laminarin to haemocyte cultures enhances phagocytosis of bacteria (Leonard et al., 1985), endocytosis of latex beads by separated “band-S locust haemocytes (Huxham and Lackie, 1988). haemocyte locomotory activity (Takle. 1985), and activation of the prophenoloxidase system within intact cells (Huxham and Lackie, 1986, 1988; Leonard et al.. 1985) or haemocyte lysate supernatant (Siiderhill, 1981; Huxham and Lackie, 1986, 1988). In vivo, injected pl,3-glucans enhance the phagocytic ability of the haemocyte population (Gunnarsson, 1988; A. Lackie, unpubl. observ.). Nodule formation, which is primarily a mechanism for sequestering particulate materials that enter the haemocoele, is also induced by injected soluble molecules that include /?1,3-glucans and bacterial lipopolysaccharides (Smith et al., 1984; Gunnarsson and Lackie, 1985) and certain glycoproteins (Lackie and Vasta. 1988). The suppression of glucan-induced nodule formation by fungal secondary metabolites,

the destruxins, thus makes sense in terms of the fungal strategy of immune evasion. Using cultured lines of insect cells (nonhaemocytic), Vey and colleagues (1986) have shown that destruxin E is even more potent than destruxin A at suppressing protein synthesis. Whether this derivative also exerts a greater immunosuppressive effect clearly requires investigation. Although the aggregation of locust haemocytes from band 5 in vitro cannot be directly compared with nodule formation in viva. since nodules probably comprise at least 2 classes of haemocyte (Ratcliffe and Gagen, 1977). the effect of laminarin on cell locomotion in vitro raises some interesting questions about the interrelationships between locomotion and adhesion and their relevance to insect immune mechanisms. With siliconised glass as the substratum, to which haemocytes from band 5 adhered but did not spread, laminarin did not enhance the rate of cell locomotion (Huxham and Lackie, 1988), but on tissue-culture plastic, on which the cells spread well and thus moved only slowly, laminarin stimulated the rounding-up of cells, an increase in speed, and enhanced random contact leading to aggregation. Unlike nodule formation, these aggregations were not permanent. possibly because some other stimulus was missing. It was noted earlier that laminarin activated several different aspects of the behaviour of band-5 cells, including production of phenoloxidase (Huxham and Lackie, 1988); destruxins, in addition to suppressing phenoloxidase production in living cells, also suppressed the enhanced locomotory and aggregation behaviour shown by these cells. Moreover, the characteristic appearance of diffuse and punctate regions of F-actin, seen with laminarinstimulated cells, was absent within unstimulated, spread cells and destruxin-treated cells; instead, a meshwork of thin actin filaments similar to that seen in spread Ephestia haemocytes (Davies and Preston, 1987) was observed. Although the mechanisms of the interactions between destruxins, laminarin and phenoloxidase production are not yet clear, it is interesting to note that neither laminarin nor destruxins altered the behaviour of locust band-4 cells, the majority of which have been shown previously to have little phenoloxidase activity (Huxham and Lackie, 1988). Destruxin A affects the membrane polarisation of Lepidopteran muscle (Samuels et al.. 1986) but seems not to have ionophore activity (Abalis, 1981; Samuels et al., 1986). The current results suggest that destruxins may modify events that are likely to occur at the plasma membrane of a specific sub-population of haemocytes. That these cyclodepsipeptides suppress certain manifestations of the cellular immune response of insects is particularly interesting in view of the suppressive effect of cyclosporin A on mammalian cellular immunity.

AcknoM’ledgemPnfs-Thanks arc due to the AFRC (Grant AG171160) for their financial support, to Mrs Pearl Tait for highly competent technical help with the culture and extraction of Mefarhizium, to Liz Currie for technical assistance, to John Laurie for maintaining the locusts and to Peter Rickus for photographic expertise.

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