larvicidal activity of lectins on lucilia cuprina: mechanism of action

Entomol. exp. appl. 72: 1-10, 1994. 1 (~) 1994 Kluwer Academic Publishers. Printed in Belgium.

Larvicidal activity of lectins on Lucilia cuprina: mechanism of action

C. H. E i s e m a n n , R. A. D o n a l d s o n , R. D. Pea r son , L. C. C a d o g a n , T. V u o c o l o & R. L. T e l l a m CSIRO Division of Tropical Animal Production, CSIRO Private Mail Bag 3, lndooroopilly, 4068, QLD, Australia

Accepted: November 4, 1993

Key words: lectins, peritrophic membrane, Lucilia cuprina, blowfly strike, larvicide, feeding deterrent, wheat germ lectin, lentil lectin, Concanavalin A

Abstract

Larvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum-free medium in the presence of a number of lectins. Lectins with specificities for/3-(1,4)-N-acetylglucosamine (wheat germ lectin) and c~-D-mannopyranosyl and (c~-D-glucopyranosyl residues (lentil lectin and Con A) caused strong concentration-dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 #M and 100% mortality at 25 #M. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated, There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial ceils although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.

Introduction

Lectins are proteins which bind to carbohydrates and are present in a wide variety of animal and plant tissues (Goldstein & Poretz, 1986). Lectins from plants were originally identified by their ability to agglutinate mammalian red blood cells. However, the biological functions of lectins, in general, are poorly defined espe- cially in plants where these proteins can be present in relatively high concentrations (e.g. 2-10% of the total plant protein; Liener, 1979). It has been speculated that the presence of lectins in plant tissues such as seeds and foliage protects those tissues from attack by insects and also accounts for the selective diets of many plant-eating insects (Liener, 1979). Indeed, specific lectins can kill or retard the growth of some plant-eating insects (Janzen et al., 1976; Cavalieri et al., 1991; Osborn et al., 1988; Czapla & Lang, 1990). It is interesting to note the markedly different sensitiv- ities of insect species to the various lectins that have been tested. The mechanism of action of the insectici-

dal activity is not clear. However, it has been shown that Phaseolus vulgaris lectin binds to the apical membranes of midgut cells in larvae of the bruchid Calloso- bruchus maculatus to which it is toxic (Gatehouse et al., 1984).

Larvae of the blowfly Lucilia cuprina feed on tissue and tissue fluids of susceptible sheep ultimately leading to conditions which can cause the death of the sheep and severe economic losses in the sheep and wool industries.

In this manuscript we report strong inhibition of the growth in vitro of larvae of the blowfly L. cuprina by lectins of different specificities. This is the first exam- ple of an insect which feeds on animal rather than plant tissues being adversely affected by ingestion of plant lectins. Evidence is also presented which details possible mechanisms of action of these lectins in retarding growth of the larval stage of this insect. These results, which may also be relevant to many phytophagous insects, suggest a possible model for the insecticidal action of other large molecules that are capable of spe-

cific binding to insect tissues. This model is discussed below in relation to the possibility of immunological control of this economically important pest of sheep.

Materials and methods

Materials. All laboratory reagents were of analytical grade and high quality water (Milli-RO 4 System, Mil- lipore) was used for all solutions. Concanavalin A (Con A), and lectins from Bauhinia purpurea (BPL), Tetragonolobus purpureas (TPL) and Sambucus nigra (SNL) were purchased from Sigma Chemical Co. (St Louis, Mo. U.S.A.). Wheat germ lectin (WGL) and lentil lectin (LL) were prepared by the methods of Vretblad (1976) and Sage & Green (1974), respectively. Table 1 summarises details regarding the lectins used in this study. They were selected on the basis of their range of specificities and availability.

Fluorescein-5-isothiocyanate (FITC; Sigma Chem- ical Co.) was conjugated to wheat germ lectin and ovalbumin (Sigma Chemical Co.) by the method of Johnston et aL (1973). There was a very low labelling ratio for the FITC-wheat germ lectin which necessitat- ed its use at a relatively high concentration (l mg/ml) compared to FITC-Con A and FITC-lentil lectin (10 #g/ml) which had a much higher labelling ratio. Fluo- rescein sodium salt was purchased from Sigma Chem- ical Co. and FITC-ConA and FITC-lentil lectin from ICN Biochemicals (Australia).

Diet components were purchased as follows: yeast RNA (Boehringer Mannheim Australia); bovine serum albumin (CSL, Australia); Wesson's salts (ICN); Van- derzant's vitamin mixture (Roche Chemicals); agar Noble (Difco, U.S.A.); yeast extract (Y-0375) and cholesterol-rich lipids from bovine serum (Sigma Chemical Co.). All larvae of Lucilia cuprina were from a laboratory population originating from fly- struck sheep and maintained on an agar-based larval diet (Singh & Jerram, 1976) for 15-20 generations.

Growth and survival assays. Lectins were incorporat- ed into one of two types of agar-based diet media: (a) 75% normal sheep serum supplemented with soluble yeast extract as described by Eisemann et al. (1990) or (b) synthetic medium, composed of 5 g bovine serum albumin, 300 mg Wesson's salts, 30 mg Vanderzant's vitamin mixture, 530 #1 cholesterol-rich lipids, 150 mg RNA in 70 ml phosphate-buffered saline (PBS) and 25 ml agar Noble (4%) in PBS. The mixture was heated to 45 ~ dispensed and allowed to solidify. All exper-

iments using serum medium were performed with 1 ml aliquots on each of which 10 neonate L. cuprina larvae were grown for 20 h at 34 ~ Five replicates were used for each lectin concentration. Assays with synthetic medium incorporating wheat germ lectin, Con A and lentil lectin were performed as for the serum medium, except that their duration was extended to 24 h in partial compensation for slower larval growth. All lectin concentrations given refer to the final volume of the growth medium.

To allow more efficient testing of a range of lectins using lower total quantities of the lectins, bioassays using a reduced volume of synthetic medium were employed. Briefly, 10 larvae were grown for 24 h at 26 ~ on each of three replicate aliquots for each lectin concentration tested. Each aliquot contained 250 #1 of synthetic medium. After their removal from diet medium, larvae were rinsed to remove adhering diet, blotted dry, counted and weighed.

Measurement of feeding deterrence. Larvae of L. cuprina were grown to second instar (2.0-2.5 mg) on synthetic medium. Groups of 50--60 larvae were counted and transferred to 5 ml aliquots of synthetic medium containing 0.0015% fluorescein (sodium salt) and a range of concentrations of lectin. The larvae were allowed to feed for 1 h at 34 ~ (the approximate time required for complete filling of the gut) before being removed, rinsed, dried and weighed and then ground (1 rnin) in 40 mM Tris-HCl, pH 7.4 (in the ratio of 100 mg of larvae to 3 ml of buffer) using a 25 ml glass tissue grinder. The homogenate was centrifuged at 12000 x g for 3 min and the supernatant removed. Fluorescence values of samples of supernatant were read in a Perkin- Elmer 203 fluorescence spectrophotometer (excitation 495 nm; emission 520 nm). Groups of larvae fed on synthetic medium without fluorescein or lectin were processed similarly for use as blanks. Relative quantities of medium contained in larvae from various lectin treatments were estimated by comparing fluorescence values of the supernatants with the values from supernatants from control larvae (i.e. no lectin) after sub- traction of the blank fluorescence value from each. Standard solutions of fluorescein in supernatant from blowfly larvae were read in the spectrophotometer to confirm linearity of response in the working range.

Fluorescence localization of bound lectins. Pieces of tissue excised from third instar larvae of L. cuprina were washed in PBS and incubated for 1 h at room temperature in either 1 mg/ml FITC-labelled wheat germ

Table 1. Properties of plant lectins used in feeding experiments with larvae of Lucilia cuprina

Lectin I Abbreviation Sugar specificity Mol. Wt (kDa)

Concanavalin A Con A c~-D-mannopyranoside 102 c~-D-glucopyranoside

Lentil lectin LL c~-D-mannopyranoside 49 c~-D-glucopyranoside

Wheat germ lectin WGL /3-(1,4)-N-acetylglucosamine 36 N-acetyl-D-neuraminic acid

Bauhinia purpurea lectin BPL /3-(1,4)-N-acetylgalactosamine 195

D-galactose c~-lactose

Sambucus nigra lectin SNL lactose 140

Tetragonolobus purpureas lectin TPL c~-L-fucose 98

1 For references and further details see Liener et al. (1986).

lectin, 10 #g/ml FITC-labelled Con A or 10 #g/ml FITC-labelled lentil lectin in PBS. The tissue pieces were then washed in 4 changes of PBS for a total of 1.5 h and examined in a fluorescence microscope. Control preparations were either as above with 50 mg/ml N-acetyl glucosamine (for wheat germ lectin) or 50 mg/ml methyl-c~-D-mannopyranoside (for ConA and lentil lectin) added to the incubation solution or 1 mg/ml FITC-labelled ovalbumin in place of FITC- wheat germ lectin. Other larvae were allowed to feed for 3 h at 34 ~ on the synthetic diet medium containing FITC-wheat germ lectin (2 mg/ml), FITC-Con A (20 /~g/ml) or FITC-lentil lectin (20 #g/ml). These larvae were removed at this stage, dissected and the excised tissue washed and examined as described above. Other larvae fed on the synthetic diet and FITC-wheat germ lectin, FITC-Con A or FITC-lentil lectin were transferred to fresh synthetic medium containing no FITC- labelled lectin and allowed to feed for a further 1-1.25 h at 34 ~ before being dissected and the excised tissue treated as above.

Measurement of the permeability of the peritrophic membrane using colloidal gold. Colloidal gold solutions of mean diameter 7.5 nm and 15 nm were pre-

pared by the method of Slot & Geuze (1985). The colloidal gold was conjugated to the B-chain of insulin to avoid aggregation of the gold and to minimize binding of macromolecules to the gold particles and thus maintain a total diameter close to stated values. Larvae of L. cuprina were grown on either (a) serum medium containing 50 #M wheat germ lectin or no lectin for 20 h at 34 ~ or (b) synthetic medium containing either no lectin, 13 #M wheat germ lectin, 13/zM Con A or 50 /~M lentil lectin for 24 h at 34 ~ Larvae were then transferred to a similar medium, containing the same lectin concentrations, but also containing 15-18% (v/v) of a concentrated 7.5 nm colloidal gold preparation and with the synthetic medium an additional 7.5% (v/v) of the 15 nm colloidal gold preparation. The larvae were allowed to feed on these media for 2 h at 28-34 ~ before being removed, dissected open anteriorly and posteriorly (avoiding damage to the gut) and fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature for 1.5 h. After washing in buffer (4 ~ without fixative overnight, the dissected larvae were dehydrated through an alcohol series and embedded in Epon-Araldite (Mollenhauer, 1964). Blocks were sectioned on an LKB Nova ultra- microtome, and the sections taken up on copper grids,

4

3

,...,'-"~ 2.5 t

"~ 2

M 1.5 L

0 I I I I t I I I

Control 0.8 1.5 3 6 13 25 50

Lectin Concentration (p~M)

Fig. 1. Inhibition of the growth of larvae of L. cuprina in the presence of leetins. Larvae were grown on a synthetic diet medium in the presence of wheat germ lectin (~), Con A (e) or lentil lectin ( I ) . The larvae were weighed after 24 h growth in these lectin-containing media. Each data point is the mean of five replicates, each of which initially contained 10 neonate L. cuprina larva.

stained lightly with uranyl acetate and lead citrate and then examined in a Philips EM300 transmission electron microscope.

Results

Adequate growth of larvae was observed on the serum- free synthetic medium, which was formulated to avoid problems of sequestration of lectins by serum glycoproteins. Figure 1 shows the concentration-dependent effects of three lectins on the growth (mean weights) of L. cuprina larvae in this serum-free synthetic medium. The lectin concentrations causing 50% inhibition of larval growth were 2 #M for wheatgerm lectin, 4 /~M for Con A and 7 #M for lentil lectin. Wheat germ lectin is specific for/3-(1,4)-N-acetylglucosamine and N-acetyl-D-neuraminic acid, while Con A and lentil lectin both bind to c~-D-mannopyranosyl and ce- glucopyranosyl residues. Unrelated proteins such as bovine serum albumin, chicken ovomucoid and ovine immunoglobulin G have no effect on larval growth over the same concentration range as used for the lectins.

In general, the shapes of these lectin dose-response curves were similar. Hill plots for the lectin dose- response data (log (mean larval weight) versus log (lectin concentration)) have slopes of 0.60 for Con A, 0.75 for lentil lectin and 0.92 for wheat germ lectin. If each of these lectins was 100% active, then these slopes are consistent with lectin valencies of 1 for wheat germ lectin and 1-2 for both Con A and lentil lectin. All three lectins are kndwn to be multivalent (Liener, 1979). The reason(s) for the lack of correspondence between the known valencies of these lectins and the values determined from the slopes of these log-log plots is not clear. One possibility is that only a single interaction of the lectin with a target molecule is required to exert an effect on the growth rate of Lucilia cuprina larvae. Another possibility is that the valency of these lectins determined using simple sugars is different from that for complex carbohydrates (which may be binding these lectins after ingestion by the larvae).

Larvae which were less than approximately 5% of the weight of larvae grown in the absence of any of these lectins generally did not survive to 24 h in the medium (Fig. 2). This effect occurred with wheat germ lectin and Con A at concentrations of 25 #M and above. Conversely, larval weights in excess of 20% of the control weight resulted in 80-100% survival of the larvae. The data for all three lectin concentration ranges are approximately coincident in this plot, suggesting a common mode of action. Thus, overall these larvae have remarkable resilience to external influences which cause major changes in their growth rates i.e. the presence of exogenously added lectins.

Table 2 summarizes the effects of a number of lectins (each at 3 concentrations: 1.5, 6 and 25 #M) with differing sugar specificities on the mean weight of larvae grown in vitro in the small volume bioas- say. In this experiment the mean weight of control larvae measured in the absence of lectins was reduced to 0.93-t-0.14 mg because of the lowered temperature. Lentil lectin showed a strong concentration-dependent inhibition of larval growth with an approximate half maximal effect at 6-7 # M - the same half-maximal concentration determined from the results shown in Figure 1. Thus, the altered growth conditions (volume and incubation temperature) do not affect the sensitiv- ity of larval growth to lentil lectin.

Sambucus nigra lectin (SNL; lactose specificity) and Tetragonolobus purpureas lectin (TPL; specificity for c~-L-fucose) have little effect (<15%) on mean larval weights at all three concentrations. Bauhiniapurpurea lectin (BPL; specificity for/3-(1,4)-

Table 2. Effect of lectins on the mean weight of larvae (in mg) of Lucilia cuprina grown on a small volume of synthetic medium over a period of 24 h at 26 oC

Lectin Lectin concentration (/~M)

0 1.5 6 25

None (control) 0.934-0.14 l BPL 0.404-0.10 0.494-0.07 0.45-t-0.05 TPL 0.924-0.22 0.844-0.03 0.984-0.23 SNL 0.834-0.16 0.884-0.10 0.794-0.09 LL 0.684-0.10 0.404-0.03 0.114-0.011

I Values presented are means + standard deviations. Each value was derived from three replicates.

100

80

60 1.1

4O

20

OQ

i i i I i 1

O 20 40 60 80 100

% of Control Weight

Fig. 2. Relationship between larval survival and larval weight for L. cuprina larvae grown in the presence of lectins. The conditions for this experiment are described in the cap- tion to Figure 1. Wheat germ lectin (0), Con A (.) and lentil lectin (I) . Concentrations of 25 /~M wheat germ lectin or Con A resulted in 100% mortality of the larvae which were not able to be recovered and weighed. These two data points have been included at the origin in this graph.

N-acetylgalactosamine, D-galactose and c~-lactose) decreased the larval weights by approximately 50% at all three lectin concentrations. The reason(s) for the lack of concentration-dependence in the effect of this lectin is not clear. These results and those shown in Figure 1 indicate that lectins with specificities for /3-(1,4)-N-acetylglucosamine (wheat germ lectin) and

c~-D-mannopyranosyl residues (lentil lectin and Con A) were the most effective in retarding larval growth in the present study.

The specificity of lectins for particular carbohydrate residues suggests that the relatively strong inhibition of larval growth caused by wheat germ lectin and lentil lectin (Con A has a similar monosac- charide specificity to lentil lectin) should be alle- viated by the presence of the appropriate sugars (eg. N-acetylglucosamine and methyl c~-D-mannopyranoside, respectively). Table 3 shows the results for experiments where appropriate sugars were added with wheat germ lectin, lentil lectin and Con A in the growth medium. The sugars, when introduced into the synthetic growth medium in the absence of lectins, affected larval growth, reducing the mean larval weight from 2.90-t-0.21 mg to 1.064-0.18 mg for methyl c~-D-manno-pyranoside (50 mg/ml; 0.26M) and to 0.694-0.07 mg for N-acetylglucosamine (50 mg/ml; 0.23M). The reason(s) for the weight reduction in the presence of the sugars is not known, but may be connected with osmotic or sensory effects on the larvae. Notwithstanding this effect, even at the highest concentrations of wheat germ lectin (25 #M) or lentil lectin (50 #M) in the presence of the appropriate sugars, there was little or no additional inhibitory effect of these lectins on the growth of the larvae. Figures 1 and 2 show that wheat germ lectin at a concentration of 25 #M causes 100% mortality of the larvae and lentil lectin at 50/zM causes a 90% reduction in larval growth. However, c~-D-manno-pyranoside did not protect larvae from growth inhibition caused by Con A. Nevertheless, the presence of the appropriate sugar generally protects the larvae from weight reduc-

tions and mortality caused by wheat germ lectin and lentil lectin introduced into the synthetic growth medium. The larvicidal activity of these lectins is therefore directly dependent upon their specificity for particular sugars. The differences in effect observed with Con A and lentil lectin may reflect differences in their finer specificity for binding to complex oligosaccharides or differences in their structural stability (Con A is much more labile than lentil lectin).

The mechanisms whereby these lectins inhibit larval growth have been investigated. Figure 3 shows that the amount of material ingested by larvae is reduced in the presence of wheat germ lectin and lentil lectin in the synthetic growth medium. This effect does not account fully for the strong growth inhibitory effects of wheat germ lectin and lentil lectin. This can be seen from a comparison of Figures 1 and 3. Wheat germ lectin does not reduce food intake at 1.5 #M (Fig. 3), but the same concentration reduces larval weight by approximately 50% (Fig. 1). Lentil lectin has no significant effect on food intake at 3 #M (Fig. 3), but this concentration reduces larval weight by 30% (Fig. 1). It is noteworthy that the data for both lectins are nearly coincident. With Con A, however, the results (not shown) were variable, probably due to solubility problems associated with this lectin. In addition, Con A is not as stable as wheat germ lectin or lentil lectin (unpublished observations). It is not clear whether the reduced rate of diet ingestion caused by these lectins is a primary effect, perhaps mediated by gustation, or a secondary one, possibly resulting from a reduction in general activity as a consequence of lectin toxicity.

Wheat germ lectin (conjugated to fluorescein-5- isothiocyanate) binds directly to isolated peritrophic membrane (PM) as shown in the fluorescence micrograph shown in Figure 4(a). This interaction could occur directly with the chitin component (poly-/3-(1,4)- N-acetylglucosamine) if this is accessible in the intact membrane, or with specific glycoproteins associated with the membrane. Similar results were obtained with fluorescently labelled Con A and lentil lectin (results not shown). The specificity of the latter two lectins for c~-D-mannopyranosyl residues suggests that the observed interaction of these lectins with the PM occurs via glycoproteins attached to the membrane rather than directly to the complex carbohydrate (poly-/3-(1,4)-N-acetylglucosamine) which makes up the chitin component of the membrane. Western blots of proteins extracted from PM and probed with biotiny- lated lentil lectin (and then streptavidin-peroxidase) demonstrated the presence of a number of glycopro-

120

~-- 100 I l q @

O r..) 80

'~ 60

~ 40

N 20

I

0 i . I . I i i ,~ I

Control 1.5 3 6 13 25 50

Lectin Concentrat ion (~tM)

Fig. 3. Feeding deterrence caused by lectins introduced into the synthetic growth medium. Wheat germ lectin (~), lentil lectin (ll). Open symbols refer to corresponding control experiments performed in the absence of lectin.

teins (results not shown). Control experiments were performed with FITC-labelled lectins in the presence of 50 mg/ml of the appropriate sugar (Fig. 4(b)). The lack of fluorescence in these control samples clearly demonstrates the specificity of the lectins.

All of these lectins (wheat germ lectin, Con A and lentil lectin) also bind to the apical membranes (microvilli) of midgut epithelial cells as shown for wheat germ lectin in Figure 4(c). This is true both of cells incubated with lectin in vitro and of cells in the gut of larvae fed on the fluorescein-labelled lectin. Lar- vae fed on synthetic medium containing FITC-labelled wheat germ lectin or Con A and then fed on medium without lectin to purge lectins contained in the gut before dissection also showed fluorescein-labelled lectin bound to the microvilli of midgut cells. This lectin evidently did not bind after being released from the endoperitrophic space during dissection, as this space (as well as the PM itself) was largely free of fluorescence after purging, and the lectin must therefore have gained access to the midgut cells by pass- ing through the PM after being ingested. Although these lectins bind to the midgut epithelial cells, no evidence for any disruption of these cells was found during examination of sections using electron microscopy.

The effect of ingested wheat germ lectin, Con A and lentil lectin on the permeability of the larval PM to

Table 3. Effects of lectins on the growth of Lucilia cuprina larvae in the presence of sugars in a synthetic medium. Values presented are mean weights (in rag) 4- standard deviation

Addition Lectin concentration (/~M)

0 1.5 3 6 13 25 50

WGL and N-acetyl glucosamine 1'2 0.694-0,07 0.804-0.18

LL and methyl c~-D-mannopyranoside ~ 1.064-0.18

Con A and methyl c~-D-mannopyranoside I 1,064-0.18 0.48i0.04

0.664-0.07 0.534-0.09

1.014-0.18 0.934-0.11 0.934-0.16

0.334-0.05

Each sugar was used at a concentration of 50 mg/ml. 2 The mean weight of larvae grown in the absence of sugar and lectin was 2.904-0,21 mg. Other conditions are described in the Materials and Methods. 3 All the larvae were dead at this concentration of Con A.

gold particles of defined sizes included in the growth medium has been examined (Fig. 5). The electron micrograph in Figure 5(a) shows a section through the midgut of a second instar larva which was fed on the synthetic growth medium containing gold particles of mean diameter 7.5 rim. The arrows point to gold particles which are present in the endoperitrophic space (ENPS), the PM and the ectoperitrophic space (ECPS) surrounding the microvilli of digestive epithelial cells. Thus, gold particles of this size apparently can move freely across the PM, which in this species forms an unbroken partition between the ENPS and ECPS throughout the midgut and often throughout the hindgut as well. When larvae were fed on the synthetic medium containing wheat germ lectin, the gold particles were often restricted to the ENPS - - few or no gold particles were found within the PM or in the ECPS (Fig. 5(b)). In addition, there was usually a layer of unknown composition along the gut lumen side of the PM. The nature of this material is not clear. The thickness of this layer indicates that it does not rep- resent a simple interaction between wheat germ lectin and the PM. One possibility is that this layer repre- sents precipitated wheat germ lectin and other proteins concentrated on the gut lumen side of the PM. Presum- ably, this material restricts the access of gold particles to the PM pores. Thus, ingested wheat germ lectin (and lentil lectin and Con A, results not shown) may restrict the bi-directional movement of nutrients and digestive enzymes across the pores in the PM. In some sections, numerous gold particles were found in the ECPS, However, in most such regions, few or no gold

particles were present in the PM, even where they were also numerous in the ENPS, indicating that the gold particles did not pass through the PM at these places, but penetrated it elsewhere and subsequently spread within the ECPS.

Discussion

The results described herein are the first demonstra- tion of the larvicidal effects of some plant lectins on insect larvae which feed on animal tissue and fluids rather than plant tissues. Of the lectins tested, only those which have specificities for/3-(1,4)-N- acetylglucosamine or c~-D-mannopyranosyl residues have significant effect on the growth and development of L. cuprina larvae. This information, as well as the relatively low concentrations of these lectins required to have an effect on the growth of L. cuprina and the inhibition of the growth retarding activity of these lectins by the presence of the appropriate sugar, suggest a highly specific interaction. However, some of the results with Con A (i.e. the lack of effect of methyl c~-D-mannopyranoside and the variable feeding deterrence) suggest that it may be acting differently from wheat germ lectin and lentil lectin.

The ingestion of specific lectins (eg. wheat germ lectin, lentil lectin and Con A) by L. cuprina larvae causes a reduction in larval growth rate and death of the larvae at higher concentrations of the lectins. These effects are probably caused by three (or more) mechanisms of action: first, a reduced intake of diet; second,

a c

b d Fig. 4. Fluorescence micrographs showing localization of FITC-labelled wheat germ lectin in the gut of larvae of Lucilia cuprina. (a) Peritrophic membrane incubated with 25 ~M FITC-labelted wheat germ ]ectin for I hr at room temperature followed by extensive washing; (b) as for (a) except that 50 mg/ml N-acetyl glucosamine was added with the FITC-labelled wheat germ lectin (control): (c) gut epithelial ccl]s incubated with FlTCqabelled wheat germ lectin as described in (a); (d) gut epithelial cells incubated with FITC-labelled wheat germ lectin and N-acetyl glucosamine as described in (b). All scales are 50/am.

a partial blockage of the pores of the PM; and third, the direct binding of specific tectins to the midgut epithelial cells. This binding could affect various functions of the cell membrane. However, there is no obvious evidence of disruption of these cells.

The first two mechanisms of action could cause a restriction in the nutrients available to the digestive cells and a subsequent general starvation effect in the larva. This conclusion is consistent with the observation that ingested wheat germ lectin, Con A or lentil lectin cause no obvious damage to the larvae and that larvae can have their weights reduced by up to 80-90% by these lectins before there is substantial

mortality. The feeding deterrence caused by the ingested lectins, if mediated by gustation, may be due to the binding oftectins to glycoproteins situated on dendrites of chemoreceptor neurones near the mouthparts of the larvae. A consequent disruption of the normal func- tioning of these neurones may give rise to abnormal sensory inpm to the central nervous system, resulting in a partial inhibition of feeding. The apparent blockage of the pores of the PM by ingested lectins may not be a simple event. The ]arge amount of undefined material localized on the gut lumen side of the PM after a larva has fed on the growth medium containing any of the lectins Con A, lentil lectin or wheat germ

Fig. 5. Transmission electron micrograph of the gut of larvae of Lucilia cuprina after ingestion of gold particles. (a) Control (no lectin) showing location of ingested gold particles (7.54-0.7 nm diameter; small arrows) in the gut of the larvae. ENPS, endoperitrophic space; ECPS, ectoperitmphic space; MV, microvilli of gut epithelial cells; PM, peritrophic membrane. (b) Location of gold particles in larvae of Lucilia cuprina after ingestion of a meal containing gold particles and 50/~M wheat germ lectin in the synthetic diet medium. B, bacteria. The two large arrows show the thickness of a layer lying along the peritrophic membrane at that point. The scales in both electron micrographs are 500 nm.

lectin suggests that the binding of these lectins to the PM induces the aggregation here of ingested material (probably protein).

The effects of the lectins on the PM clearly did not prevent some penetration of colloidal gold to the ECPS. The absence of large (15 nm) gold particles and bacteria from the ECPS in these cases suggests that breakage of the PM did not occur. It is therefore like- ly that there are areas of PM which are not affected by ingested lectins, perhaps because of a combination of rapid replacement of PM and irregular ingestion of lectin-containing diet owing to the demonstrated effects of the lectins on food intake. Alternatively, sugar residues for which these lectins are specific may be sparse or absent in restricted areas of the PM (Peters, 1992), resulting in less effective reduction of permeability in these areas,

The effects of the specific lectins Con A, lentil lectin and wheat germ lectin on the growth of L. cuprina larvae may serve as a model for immunological control of larvae of L. cuprina. It may be possible to vaccinate sheep with glycoproteins from the PM of larvae of L. cuprina. Larvae which then feed on these vaccinated sheep will ingest antibodies mimick- ing lectins that bind to PM glycoproteins, possibly reducing the permeability of (or even damaging) the PM. This effect would then lead to a reduced availability of nutrients to the larvae and a consequent general starvation effect similar to that apparently caused by

some ingested lectins. Preliminary experiments of this type have indicated that such an approach may be feasi- ble (East et al., 1993; Willadsen et al., 1993), although its practical utility remains to be demonstrated.

Acknowledgements

We thank the L. W. Bett Trust and the Australian Wool Research and Development Corporation for the sup- port of this research.

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