Possible involvement of the prophenoloxidase system of the locust, Locusta migratoria, in antimicrobial activity

JOURNAL

OF INVERTEBRATE

PATHOLOGY

56, 31-38 (1990)

Possible involvement of the Prophenoloxidase System of the Locust, Locusta migratoria, in Antimicrobial Activity ANDREW F. ROWLEY,'JAYNE

L. BROOKMAN,*AND

NORMAN A. RATCLIFFE

Biomedical and Physiological Research Group, School of Biological Sciences, University College of Swansea, Singleton Park, Swansea SA2 8PP, United Kingdom Received May 9, 1989; accepted October 11, 1989 The possible role of the prophenoloxidase system of Locusta migratoria in the killing/inhibition of growth of yeast, Saccharomyces cerevisiae, and several species of bacteria was investigated. Hemocyte lysate supematants and whole blood homogenates were used as a source of the prophenoloxidase cascade for the assays. The only bacterium to show any inhibition of growth was Micrococcus luteus, but this was probably brought about by lysozyme, which was found to be present in both the hemocyte lysate supematants and blood homogenates. Some bacteria also grew better in the whole blood homogenates than control reaction mixtures. In contrast, incubation of whole blood homogenates with S. cerevisiae produced marked killing/inhibition of growth. Both the antiyeast effect and prophenoloxidase activation were similarly diminished in the presence of the serine protease inhibitor, phenylmethysulfonyl fluoride, suggesting that the antimicrobial factor(s) may have been generated by either the prophenoloxidase cascade or a related enzyme system. 8 1990 Academic Press, Inc. KEY WORDS: Locusta migratoria; Saccharomyces cerevisiae; antimicrobial activity; prophenoloxidase system; lysozyme; Micrococcus luteus.

INTRODUCTION

A major end product of the prop0 system is the pigment melanin but other factors generated may act as recognition molecules during phagocytosis (Smith and Soderhall, 1983; Ratcliffe et al., 1984; Leonard et al., 1985; Brookman et al., 1988) and nodule formation (Brookman et al., 1989b). As melanin and its toxic intermediates, including aromatic quinones, have been shown to be antifungal and antibacterial (Kuo and Alexander, 1967; Soderhall and Ajaxon, 1982; St. Leger et al., 1988) the prop0 system may also function to kill potentially pathogenic microorganisms. Soderhall and Smith (1986) and Leonard (1985) have described preliminary findings on the antibacterial activity of hemocyte lysate supernatant (HLS) preparations as a source of active proP0, but the evidence for the participation of the prop0 cascade in microbial killing is mostly still circumstantial. In the present study, experiments have been carried out to determine the existence of any antimicrobial activity in the hemocytes of the locust, Locusta migratoria and more specifically if the prop0 system is ca-

Little is known concerning the nature and functioning of naturally occurring antimicrobial factors in insects and other invertebrates. Recently, however, it has been suggested that the prophenoloxidase (proP0) system may be involved in the generation of such factors (e.g., Soderhall and Smith, 1986; Ratcliffe and Rowley, 1987). This system is activated by a range of foreign materials including whole microorganisms, microbial cell wall components such as LPS, P-1,3-glucans and peptidoglycan, as well as by treatment with proteases, heat, and detergents (Siiderhall and Smith, 1986; Rowley et al., 1986; Brookman et al., 1989a). During activation, prop0 is converted to the active enzyme, phenoloxidase (PO), and this conversion is mediated by at least one serine protease (Ashida, 1981; Siiderhall, 1981). ’ To whom correspondence should be addressed. * Present address: Department of Biochemistry, Queens Medical Centre, University of Nottingham, Nottingham, UK. 31

0022-2011/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

32

ROWLEY,

BROOKMAN,

AND

buffer (cacS, as cacS-400 but with only 20 mM CaCl,). Phosphate-buffered saline (PBS), pH 7.3, was prepared and sterilized by autoclaving. Trypsin (EC 3.4.21.4, Type III from bovine pancreas, 11,600 units mgg ‘; Sigma Chemical Co. Ltd) was made upasalmgml-’ solution in cat or cacS. All solutions were stored at - 20°C in small aliquots and were not refrozen after use. L-Dihydroxyphenylalanine (L-DOPA, Sigma) was prepared as a saturated solution in double distilled water and used at a final working concentration of 1.5 mg ml- ‘. Bacteria. The bacteria used to test for antibacterial activity of the locust HLS preparations were Micrococcus luteus (NCIB 9278,35”(J); Staphylococcus aureus (Oxford H, NCIB 6571, 35°C); Klebsiella pneumoniae (capsulate strain NCIB 8805 and noncapsulate strain, NCIB 9261,35”(Z); Escherichia coli strains D3 1 and D2 1 (35°C; kind gifts of Professor H. G. Boman and Drs. A. D. Russell and J. R. Fur-r, respectively); Bacillus subtilis (NCIB 3610, 3O”C), B. thuringiensis (30°C) and Pseudomonas aeruginosa (30°C). Incubation temperatures are given for each species in parentheses. Whole blood homogenate preparations were only tested against seven of the above species (see Results, Table 1). Preparation of HLS. HLS preparations were prepared as described previously

pable of inactivating/inhibiting the growth of the yeast, Saccharomyces cerevisiae and a range of bacteria. In L. migratoria, the hemocytes have been shown to be the main hemocoelic repository for the prop0 system (Dularay and Lackie, 1985; Brookman et al., 1989a) and so HLS preparations and whole blood homogenates have been employed as a source of this enzyme cascade. MATERIALS

AND METHODS

Insects. Seven to fourteen-day-old adult male and female L. migratoria were used for all experiments and reared as described previously (Brookman et al., 1989a). Chemicals and solutions. Bacteria were grown in nutrient broth or on nutrient agar (D&o). Yeast were cultured in Y-M broth (Difco) or on Y-M agar plates (Difco). An anticoagulant was used during insect bleeding and this consisted of 100 mM glucose, 10 mM EDTA, 30 mM trisodium citrate, 26 mM citric acid, pH 4.6, adjusted to 400 mOsm kg-’ with NaCl. Sodium cacodylate buffer (cat; 0.01 M sodium cacodylate, pH 7, with 10 mM CaCl,) for the HLS experiments was prepared using pyrogen-free water and filter sterilized (0.2 km pore size). Two cacodylate sucrose buffers were also prepared: the HLS buffer (cacS-400; 0.01 M sodium cacodylate, 40 mM CaCl,, 0.27 M sucrose, pH 7,400 mOsm kg- ‘) and the homogenate TABLE ANTIBACTERIAL

ASSAY USING

WHOLE

RATCLIFFE

1

BLOOD

HOMOGENATES OF BACTERIA

FROM

Locusta

AGAINST

Plate counts Bacteria tested Staphylococcus aureus Klebsiella pneumoniae 8805 K. pneumoniae 9261 Bacillus subtilis B. thuringiensis Pseudomonas aeruginosa Escherichia coli

Bacteria + cacS 174.9 272.7 286.5 292.1 249.9 324.1 256.3

2 7.2” ? 5.1 IL 4.3 2 3.4 k 7.2 2 13.4 ” 5.4

A,,, values

Bacteria + homogenate 242.2 344.1 351.5 344.0 315.2 399.2 26’7.8

a Mean values t SE, n = 12 from three experiments. b Mean values _t SE, n = 6 from two experiments. * Significantly greater than cacS control, P c 0.05.

A RANGE

* f + + k + 2

13.8* 7.9* 9X* 7.4* 11.4* 11.8* 10.7

Bacteria + cacS 0.159 0.180 0.225 0.212 0.254 0.161 0.192

-+ 0.005b + 0.005 + 0.012 2 0.008 2 0.005 k 0.006 + 0.004

Bacteria + homogenate 0.222 0.182 0.259 0.253 0.291 0.215 0.240

2 o.oot3* 2 0.031 +- 0.015* 2 0.008* 2 O.OOS* + 0.004* 2 0.010*

ANTIMICROBIAL

ACTIVITY

(Brookman et al., 1989a). Briefly, locusts were chilled at -20°C for l&15 min and injected intrahemocoelicahy with 0.3-0.6 ml ice-cold anticoagulant. This anticoagulant was left to circulate for ca. 1 min. The insects were then bled by piercing the arthrodial membrane at the base of a walking leg and the hemolymph was quickly collected into a syringe containing 0.4 ml of ice-cold anticoagulant. The contents were discharged into a plastic centrifuge tube containing a further 0.5 ml of ice-cold anticoagulant and centrifuged (8OOg, 5 min, 4”C), the plasma was removed, and the cell pellet was washed with 3 x 0.5 ml cacS-400 buffer and resuspended in 0.2-&5 ml cat buffer. Pellets from six to eight insects were pooled, homogenized in a Dounce homogenizer, and centrifuged (lO,OOOg, 15 min) and the supernatant (HLS) was used as a source of proP0. Only HLS preparations were employed where the prop0 system had not been spontaneously activated during bleeding and where the protein concentration varied no more than 210%. The protein concentration of the HLS was determined by the Bradford (1976) method using bovine serum albumin as a standard. Preparation of whole blood homogenates. Two to four locusts were chilled, injected with homogenate buffer (cacS with 20 mM CaCl,) instead of anticoagulant solution, and bled as above. The diluted blood was homogenized and used immediately. Antibacterial assay procedure. Bacteria were grown-up overnight, washed twice with PBS then once with cat buffer, and adjusted to 1 x lo6 ml-‘. For the HLS experiments, the following mixtures were incubated in triplicate: (1) 25 ~1 HLS, 25 ~1 cat, and 100 t.~l bacteria; (2) 25 pl HLS, 25 l.~l trypsin (to give maximal prop0 activation in the HLS; 1 mg ml-‘) and 100 ~1 bacteria; (3) 25 pl cat, 25 l.~l cat, and 100 t.~l bacteria; and (4) 25 ~1 cat, 25 ~1 trypsin (1 mg ml-‘), and 100 ~1 bacteria. For the homogenate experiments, the following mixtures were also incubated in triplicate: (1) 50 (~1blood homogenate and 100 p.1bacteria

IN

LOCUSTS

33

and (2) 50 p,l cacS and 100 ~1 bacteria. Mixtures were incubated for 1 hr at 3O”C, and then 20-pl aliquots were removed from each tube and diluted with 2 ml PBS. This solution was plated out in lOO-~1 aliquots onto nutrient agar plates which were grownup for ca. 18 hr and counted. Four milliliters of nutrient broth was also added to the remaining original incubation mixtures which were incubated at the appropriate temperature until growth was seen (usually 8-16 hr). The growth of the broth cultures was then determined by measuring their A,,,. To confirm the activity of prop0 in the HLS and homogenates, these preparations were tested at time = 0 for activation with trypsin (1 mg ml- ‘) as described previously (Brookman et al., 1989a). Phenoloxidase activity was calculated as A A, min -’ mg-’ protein of the HLS (given as umts, U, where 18 A,, mm* 4m3-1 protein is equivalent to III), correcting for the dilution of the original reaction mixture. At the same time, the HLS and whole blood homogenate preparations were also tested for activation of prop0 by the bacterial cultures (at 1 x lo6 ml-’ in cat buffer). HLS preparations were assayed for antibacterial activity against M. iuteus in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF, 1 mM). Assay for lytic factors and lysozyme. The locust samples were tested for a direct lytic effect using 500 p,l of dense (AsYO ca. 0.5) bacterial suspension of all species and 100 p,l HLS or homogenate. The absorbance of the solutions at 570 nm was monitored over 1 hr and compared with control samples with no HLS or homogenate. Homogenate and HLS samples were also assayed for lysozyme using M. luteus cell wall extract (Sigma). One hundred microliters of HLS or homogenate was incubated with 500 ~1 turbid cell wall solution (Ads0 ca. 0.5) at RT. The change in absorbance at 450 nm with time measured and the lysozyme activity calculated by expressing the rate of change m AdSO in terms of total protein in the aliquot, compared with a curve constructed

34

ROWLEY,

BROOKMAN,

with egg white lysozyme (Sigma) as standard. Antiyeast assay. A haploid S. cerevisiae strain was incubated with whole blood homogenate to determine whether the prop0 cascade had any effect on the growth of yeast. Yeast were cultured overnight at 28°C with rotation (200 rpm) and washed in cacS, and the suspension was adjusted to ca. 1 x lo7 ml-’ and subsequently dispensed in l-ml aliquots. The concentration of yeast present at time 0 was determined using a Neubauer hemocytometer. One hundred microliters of test/control sample was added to each 1 ml yeast culture. In initial experiments, the following sampleyeast mixtures were incubated in triplicate at 28°C for up to 5 hr: (1) 1 ml yeast and 100 p.1 cacS; (2) 1 ml yeast and 100 pl homogenate; and (3) 1 ml yeast and 100 ~1 heatinactivated (100°C 10 min) homogenate. In subsequent experiments, designed to determine the effect of the protease inhibitor, PMSF, on prop0 activation and antiyeast activity, reaction mixtures consisting of (1) 1 ml yeast and 200 pl cacS; (2) 1 ml yeast, 100 p,l homogenate and 100 ~1 cacS; (3) 1 ml yeast, 100 l.~l homogenate, and 100 pJ PMSF (1 mrvr); and (4) 1 ml yeast, 100 ~1 cacS, and 100 p.1 PMSF (1 mM) were incubated for up to 2 hr at 28°C with constant rotation. In both sets of experiments, 25pl samples were withdrawn at regular intervals and the concentration of yeast was determined by hemocytometer counting. The homogenate used was also tested for prop0 activation with the yeast culture (1 x lo7 ml-‘), using 25p,l aliquots of each component (see Table 3) in the reaction mix plus 25 ~1 L-DOPA in flat-bottomed 96-well plates (Sterilin). The production of dopachrome and other intermediate products was monitored at 492 nm at 2- and 5-hr time intervals using a BioRad 2550 plate reader. Statistical analysis. Data were compared using a paired Student t test and the twoway analysis of variance method (ANOVA). The level of significance was set at P G 0.05 or P =z 0.001.

AND

RATCLIFFE

RESULTS

Antibacterial experiments. There was no significant decrease in the growth of S. aureus, K. pneumoniae strains 880519261, E. coli strains D21/D31, B. thuringiensis, B. cereus, B. subtilis, or P. aeruginosa in the presence of HLS from L. migratoria (P G 0.23, results not shown). The whole blood homogenates also showed no antibacterial activity against any bacteria tested (Table 1). In many cases, the homogenate gave significant enhancement of growth compared with cacS controls. For example, S. aureus showed an increase in growth of ca. 40% when incubated with homogenate rather than cacS alone (P 6 0.001, Table 1). The HLS preparations did, however, give a significant reduction of growth when tested against M. luteus (P < 0.001, Table 2). For example, incubation with HLS reduced the growth as determined by A,,,, from 0.608 +: 0.023 in cat controls to only 0.055 + 0.008 (mean ? SE, Table 2). This antibacterial activity was not diminished by the presence of the serine protease inhibitor PMSF in HLS preparations, as neither the plate counts nor the A,,, reading were significantly different from the HLS and bacteria without PMSF (P > 0.58, Table 2). A spectrophotometric assay using M. futeus cell walls demonstrated the presence of lysozyme-like activity in the HLS preparations from Locusta with values ranging from 0.73 to 0.85 units mgg’ protein. The HLS preparations did not show any direct lytic activity (results not shown) other than that directed against M. luteus, which was probably due to lysozyme. Trypsin activation of the HLS preparations during the incubation period did not result in any significant differences in bacterial growth/killing due to prop0 activation (results not shown except for M. luteus, Table 2), although the presence of trypsin alone did affect the growth of one bacterial species, S. aureus, giving a significant reduction in viability (P < 0.001, results not shown). All bacterial species gave

ANTIMICROBIAL

ANTIBACTERIAL

AND

ACTIVITY

IN

35

TABLE 2 P~~OPHENOLOXIDASEACTIVATION ASSAYS USING THE SAME HLS PREPARATIONS Locusta WITH Micrococcus luteus Antibacterial

bacteria trypsin + bacteria + cat + trypsin trypsin

+ 4.9’,’ -c 3.9” * 7.7 f 9.0 NA NA 38.9 f 3.7

bacteria + PMSF

Phenoloxidase activity (,‘)a

A 570

32.1 38.5 383.5 382.6

FROM

assay

Plate counts

Sample tested HLS + HLS + Bacteria Bacteria HLS + HLS HLS +

LOCUSTS

0.055 0.064 0.608 0.667

f 0.008’ f 0.003’ f 0.023 k 0.017 NA NA 0.054 f 0.002

6.2 + 0.6dpe 177.0 + 6.6’ NA NA 177.6 k 3.8’ 1.0 2 0.2 -

Note. NA, not applicable. a PO activity expressed as U, where 1 U is 1 A A, rnin-’ mgg ’ protein. b Mean values 2 SE, n = 6-12 each from 10 locusts in two experiments. ’ Significantly lower than bacteria and bacteria + trypsin controls, P < 0.001. d Mean values k SE, n = 6 from two experiments. e Significantly greater than HLS control, P 6 0.001.

significant levels of prop0 activation in the HLS assay compared with HLS only controls (P < 0.001, Table 2). There was no significant difference, however, in the activation of prop0 by bacteria plus trypsin compared with activation by trypsin alone (P > 0.09, Table 2 and results not shown for other bacteria). Antiyeast experiments. In initial experiments, incubation of HLS with S. cerevi-

siae showed no inhibition in the growth of yeast or prop0 activation compared with the controls (data not shown). Incubation of S. cerevisiae with locust blood homogenates, however, gave a significant reduction in growth compared with cacS controls and heat-inactivated homogenate (P < 0.05 for 1 and 2 hr and P < 0.001 for 3,4, and 5 hr incubation times, Fig. 1). Growth of yeast in the presence of heat-inactivated

TABLE EFFECT

3

PROTEASE INHIBITOR, PMSF, ON THE REDUCTION IN THE GROWTH OF cerevisiae BY PROPO ACTIVATION, IN Locusta WHOLE BLOOD HOMOGENATES

OF THE SERINE

Saccharomyces

Yeast concentration (X 10’ cells ml-‘)

prop0 Activation

(U’)

Sample tested

1 hr

2hr

lhr

2 hr

Yeast + cacS Yeast + PMSF Yeast + homogenate Yeast + homogenate + PMSF 5. Homogenate

1.62 f 0.05” 1.51 k 0.04 0.90 * O.Olb

2.48 f 0.04” 2.24 f 0.04 1.07 + 0.03b

NA NA 15.97 + 0.18”,d

NA NA 24.36 f 0.55”~~

1.12 * o.02c NA

1.68 + 0.04” NA

11.78 + 0.27e 4.34 f 0.20

15.03 + 0.22e 4.66 f 0.06

1. 2. 3. 4.

Note. NA, not applicable. D Mean f SE, n = 8 for yeast cell counts, n = 4 for prop0 activation assay, in both cases from four locusts in two experiments run in triplicate. b Significantly lower than cacS control (1) and PMSF experimental (4), P s 0.001. c Significantly lower than PMSF only control (2), P G 0.001. d PO activity significantly greater than homogenate only control (5) and PMSF experimental (4), P S 0.05. E PO activity significantly greater than homogenate only control (5), P S 0.001.

36

ROWLEY,

BROOKMAN,

AND

RATCLIFFE

Table 3. The yeast plus homogenate mixtures showed significantly lower growth of yeast than the mixtures containing PMSF alone (P < 0.001, Table 3). The PMSFcontaining samples, however, still showed significantly reduced growth compared with yeast plus cacS controls (P < 0.001, Table 3). Yeast activated prop0 in the blood homogenates and this activation was highly significant compared with the homogenatealone control (P < 0.001, Table 3). The presence of PMSF significantly reduced the level of prop0 formed in response to the yeast but did not totally inhibit its activation (P < 0.001, Table 3). DISCUSSION

;

2

3

i

5

INCLBATION TIME (hr)

FIG. 1. Effect of Locustn whole blood homogenates on the growth of Saccharomyces cerevisiae. Each point is a mean value f SE, n = 12 from nine locusts in three experiments. (* Significantly lower than cacS control (P < 0.05) and heated homogenate control (P G 0.001). (**) Significantly lower than cacS and heated homogenate controls (P G 0.001).

homogenate was slightly higher than in the cacS controls at l-4 hr, but this difference was not significant (P > 0.06). After 5 hr of incubation, the cacS control appeared to have significantly higher growth than the heated homogenate samples, but again these differences were not significant (P = 0.27). In simultaneous experiments with the same blood homogenates, PO levels generated were 3.2 ? 0.1 and 5.9 + 0.3 U for the homogenate plus cacS controls, measured at 2 and 5 hr, respectively. For the yeast plus homogenate samples, PO levels were significantly higher at 5 hr (8.8 + 0.7 U; P < 0.001; mean values + SE, n = 8 from two experiments). Incubation of the homogenate with yeast in the presence of PMSF partially removed the inhibitory effect on growth as shown in

The present experiments failed to demonstrate any antibacterial activity, other than that caused by lysozyme, in HLS preparations from Locusta. This is in contrast to the initial reports of bacterial killing by HLS preparations from Curcinus muenas (Soderhall and Smith, 1986) and Bluberus cruniifer (Leonard, 1985). The presence of lysozyme in the HLS of Locustu is, however, in agreement with the report of Zachary and Hoffmann (1984) for the same species. Locustu whole blood homogenate preparations also showed no antibacterial activity against the bacterial species tested despite slight prop0 activation. Indeed, in many cases the homogenate acted as a nutrient source and led to enhanced bacterial division. The fact that whole blood preparations failed to induce killing also makes the absence of plasma factors an unlikely reason for the failure of the HLS experiments to demonstrate killing. Hemolymph from many insect species has been found to exert either little or no natural bactericidal effect (other than that directly attributable to lysozyme) in, for example, Surcophugu peregrinu (Natori, 1977), Gulleriu mellonellu (Chadwick et al., 1982), and Bombyx mori (Morishima et al., 1988). The failure to demonstrate antibacterial

ANTIMICROBIAL

ACTIVITY

activity in the present study could be due to the use of a suboptimal assay system. Possible improvements could be to expose the bacteria to the HLUhomogenate samples for longer periods. Longer incubation times can cause practical problems, however, due to the considerable amount of growth possible by terrestrial bacteria in this time period. Reducing the numbers of bacteria, or increasing the amount of HLS/ homogenate, would perhaps more accurately reflect the natural situation in vivo. Such experiments would further help decide whether the prop0 cascade components simply do not have any antibacterial potential, as is suggested by the failure to detect activity in nonimmune hemolymph from many different species (see above), or whether it is masked due to suboptimal assay conditions. Saccharomyces cerevisiae was shown in the present study to activate the prop0 system in whole blood homogenates. This is not surprising as zymosan, a mixture of p-1,3-, and @l,dglucans, used as a standard activator for prop0 assays, is prepared from the cell walls of this yeast. Zymosan has been shown to activate the prop0 cascade in a number of insects including the closely related locust, Schistocerca gregaria (Dularay and Lackie, 1985). The demonstration of an apparent antiyeast activity of the prop0 cascade is in agreement with previously observed in in vivo and in vitro reactions to fungi, especially in crustaceans and dipterans. For example, Siiderh%ll and Ajaxon (1982) showed that activated PO in a HLS from the crayfish, Astacus astacus, was able to produce fungicidal quinones which inhibited the mycelial growth of the fungal hyphae. Gotz and Vey (1974) also demonstrated that humoral encapsulation and the associated melanization of Aspergillus niger by Chironomus luridis larvae also suppressed mycelial development, despite a reasonable level of germination within the capsule. In addition, Butt et al. (1988) reported humoral encapsulation with melanization of three different

IN

37

LOCUSTS

species of fungi, but this process was only effective in containing the slow growing species. The inhibition of growth of S. cerevisiae was also sensitive to the presence of PMSF, a serine protease inhibitor, and this correspondingly reduced the prop0 activation as determined in a separate assay for the generation of PO. The concurrence of prop0 activation with the antiyeast effect, together with the lack of activity in heated whole blood preparations, suggests that components of the prop0 activating system or the active PO may be at least partially responsible for the observed antiyeast activity. Further experiments are required, however, to determine whether the activity is due to PO-generated compounds, as shown by Siiderhall and Ajaxon (1982), or other unrelated factors present in the homogenate that are similarly inhibited by PMSF. ACKNOWLEDGMENTS We are grateful to the Science and Engineering Research Council (Grant GR/D.21684) for financial support and to Mr. L. O’Brien for rearing the insects.

REFERENCES M. 1981. A cane sugar factor suppressing activation of phenoloxidase in hemolymph of the silk-

ASHIDA,

worm. Bombyx BRADFORD, M.

mori.

Insect

Biochem.,

11, 57-65.

M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

Anal. Biochem., 72, 248-254. BROOKMAN, J. L., RATCLIFFE,

N. A., AND ROWLEY,

A. F. 1988. Optimization of a monolayer phagocytosis assay and its application for studying the role of the prophenoloxidase system in the wax moth, Galleria mellonella. .I. Insect BROOKMAN, J. L., RATCLIFFE,

Physiol., 34, 337-345. N. A., AND ROWLEY,

A. F., 1989a. Studies on the activation of the prophenoloxidase system of insects by bacterial cell wall components. Insect Biochem., 19, 47-57. BROOKMAN,

J. L., ROWLEY,

A. F., AND RATCLIFFE,

N. A. 1989b. Studies on nodule formation in locusts following injection of microbial products. J. Znvertebr. Pathol., 53, 315-323. BUTT, T. M., S., HUMBER,

WRAIGHT, S. P., GALAINI-WRAIGHT, R. A., ROBERTS D. W., AND SOPER,

R. S. 1988. Humoral encapsulation of the fungus Ervnia radicans _,._... -.. ~~(Entomouhthorales) \~~~ . ,- bv the -notato

38

ROWLEY,

BROOKMAN,

leaf hopper Empoasca fabae (Homoptera:Cicadellidae). .Z. Invert&r. Pathol., 52, 49-56. CHADWICK, J. S., DEVERNO, P. J., CHUNG, K. L., AND ASTON, W. P. 1982. Effects of hemolymph from immune and non-immune larvae of Galleria mellonella on the ultrastructure of Pseudomonas aeruginosa.

Dev.

Comp.

Zmmunol.,

6, 433-440.

DULARAY, B., AND LACKIE, A. M. 1985. Hemocytic encapsulation and the prophenoloxidase-activating pathway in the locust Schistocerca gregaria Forsk. Insect

Biochem.,

15, 827-834.

G~Tz, P., AND VEY, A. 1974. Humoral encapsulation in Diptera (Insecta): defence reactions of Chironomus larvae against fungi. Parasitology, 68, l-13. Kuo, M. J., AND ALEXANDER, M. 1967. Inhibition of the lysis of fungi by melanins. J. Bacterial., 94,624629.

LEONARD, C. M. 1985. “Studies on the Role of the Prophenoloxidase Activating System in Non-self Recognition in Insect Immune Defences.” Ph.D. thesis, University of Wales. LEONARD, C. M., RATCLIFFE, N. A., AND ROWLEY, A. F. 1985. The role of prophenoloxidase activation in non-self recognition and phagocytosis by insect blood cells. J. Insect Physiol., 31, 789-799. MORISHIMA, I., SUGINAKA, S., BOUGAKI, T., INOUE, M., AND UENO, T. 1988. Induction and partial characterization of antibacterial proteins in the haemolymph of the silkworm, Bombyx mori. Agric. Biol.

Chem.,

52, 929-934.

NATORI, S. 1977. Bactericidal substance induced in the haemolymph of Sarcophaga peregrina larvae. J. Insect Physiol., 23, 1169-1173. RATCLIFFE, N. A., AND ROWLEY, A. F. 1987. Insect responses to parasites and other pathogens. In “Immunology, Immunoprophylaxis and Immunother-

AND

RATCLIFFE

apy of Parasitic Infections” (E. J. L. Soulsby, Ed.), pp. 271-332. CRC Press, Boca Raton, Florida. RATCLIFFE, N. A., LEONARD, C. M., AND ROWLEY, A. F. 1984. Prophenoloxidase activation: Nonself recognition and cell cooperation in insect immunity. Science,

266, 557-559.

ROWLEY, A. F., RATCLIFFE, N. A., LEONARD, C. M., RICHARDS, E. H., AND RENWRANTZ, L. R. 1986. Humoral recognition factors in insects with particular reference to agglutinins and the prophenoloxidase system. In “Hemocytic and Humoral Immunity in Arthropods” (A. P. Gupta, Ed.), pp. 381406. Wiley, New York. SMITH, V. J., AND S~DERHXLL, K. 1983. p-1,3-Glucan activation of crustacean hemocytes in vitro and in vivo. Biol. Bull. Mar. Lab. Woods Hole, 164, 299-314. S~DERH~LL, K. 1981. Fungal cell wall p-1,3-glucans induce clotting and phenoloxidase attachment to foreign surfaces of crayfish hemocyte lysate. Dev. Comp.

Zmmunol.,

5, 565-573.

SODERHALL, K., AND AJAXON, R. 1982. Effect of quinones and melanin on mycelial growth of Aphanomyces spp. and extracellular protease of Aphanomyces astaci, a parasite of crayfish. J. Znvertebr. Pathol., 39, l&l1 1. SODERHKLL, K., AND SMITH, V. J. 1986. Phenoloxidase activating cascade as a recognition and defense system in arthropods. In “Hemocytic and Humoral Immunity in Arthropods” (A. P. Gupta, Ed.), pp. 251-285. Wiley, New York. ST. LEGER, R. J., COOPER, R. M., AND CHARNLEY, A. K. 1988. The effect of melanization of Manduca sexta cuticle on growth and infection by Metarhizium

anisopliae.

J. Znvertebr.

Pathol.,

52, 459-470.

ZACHARY, D., AND HOFFMANN, J. A. 1984. Lysozyme is stored in the granules of certain haemocyte types in Locusta. J. Insect. Physiol., 30, 405411.