Vegetative development of Beauveria bassiana in the hemocoel of the beet armyworm, ... labeling tissue explants revealed that vegetative growth of B. bassiana ...
J. Insect Physiol. Vol. 42, No. 2, pp. 91-99, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/96 $15.00 + 0.00
0022-1910(95)00101-8
Pergamon
Effects of the Fungal Pathogen, Beauveria bassiana on Protein Biosynthesis of Infected Spodoptera exigua Larvae I. MAZET* Received
and D. G. BOUCIAS*t
12 May 1995; revised 21 July 1995
Vegetative development of Beauveria bassiana in the hemocoel of the beet armyworm, Spodoptera exigua did not cause significant alterations in the profile of hemolymph proteins. Pulselabeling tissue explants revealed that vegetative growth of B. bassiana hyphal bodies did not impact the biosynthetic capability of either the fat body or cuticle epidermis, both major sites of host protein synthesis. Even at the late stage of hyphal body development, when concentrations reached 3 x 10” fungal cells per ~1 of hemolymph, both fat body and cuticle explants were capable of synthesizing and secreting the majority of proteins detected in naive samples. Hemocyte cultures were found to secrete significantly fewer labeled proteins than either fat body or cuticle explants. Replication of hyphal bodies in hemocyte cultures established from infected larvae induced the synthesis and secretion of 3 peptides having M, of 31, 32 and 40 kDa. Interestingly, peptides having identical M,s were detected in infected cell free hemolymph samples. These peptides did not correspond to proteins induced in response to heat shock suggesting that they may represent in vivo produced fungal metabolites. At the very late vegetative growth stage, the hyphal bodies produced germ tubes which invaded host tissues. Unlike the hyphal bodies, the mycelial tissue invasion phase inhibited host protein biosynthesis and produced a range of exocellular peptides. Beauveria
bassiana
Insect
mycopathogens
Spodoptera
INTRODUCTION
exigua
Insect
proteins
Insect
disease
cells per microliter of hemolymph by 60 h after challenge (Hung and Boucias, 1992). These in vivo hyphal bodies, unlike the in vitro produced blastospores, lack a welldeveloped cell wall and do not possess the surface epitopes required for recognition by host phagocytic cells (Pendland et aE., 1993). During the vegetative hyphal body growth phase, this fungus like other insect mycopathogens, disrupts the biology of host hemocytes. Within 24 h after challenge, B. bassiana replication inhibits the host hemocytes from producing pseudopodia and spreading over an artificial substrate (Hung and Boucias, 1992; Hung et al., 1993). This inhibition immunosuppresses infected larvae and allows for the survival and development of nonpathogens (Hung et al., 1993). Soluble toxic components, released into the hemolymph, have been extracted from infected larvae and have been demonstrated to inhibit hemocyte spreading and to disrupt metamorphosis when injected into naive larvae (Mazet et al., 1994). In addition to inhibiting hemocyte spreading, the replicating hyphal bodies by 4840 h after challenge cause a depletion of the immunoresponsive granulocytes and plasmatocytes (Hung and Boucias, 1992). During the hyphal body phase of B. bassiana development, host larvae continue to feed, develop, and respond
The fungal pathogen, Beauveria bassiana is a causal agent of mycoses of many insect species. It is currently being developed as a microbial control agent against a spectrum of plant sucking, soil inhabiting and urban insect pests. Various applied programs have addressed the selection of virulent strains, production and formulation of conidial based products, and the evaluation of the persistence and stability in various habitats (McCoy et al., 1988). The majority of fundamental studies on this pathogen have been directed at elucidating the determinants which regulate penetration of host cuticle barriers (Charnley and St. Leger, 1991). Several years ago we initiated studies to examine the post penetration events. B. bassiana undergoes a biphasic (dimorphic) development in host S. exigua larvae. Fungal cells, injected into the hemocoels of late fifth instar larvae replicate as budding hyphal bodies, reaching concentrations of 3 x lo4
*University of Florida, Entomology and Nematology Department, Bldg. 970, Box 110620, Gainesville, FL 32611, U.S.A. tTo whom all correspondence should be addressed.
91
I. MAZET
92
and D. G. BOUCIAS
to external stimuli (Hung, 1993). No general paralysis or dysfunction in larval behavior is observed during the initial 48 h after challenge. However, a marked reduction in the feeding rate, and an inhibition in development is observed in infected larvae by 50-72 h after challenge. Naive last instar larvae consume large quantities of diet, enter the wandering stage, and construct pupal cells. Infected larvae cease feeding, become paralyzed, do not undergo metamorphosis and become moribund by 7884 h after challenge. At this late stage, hyphal bodies produce elongate germ tubes (Boucias, unpublished) which invade and colonize insect tissues. In other mycopathogens, the germ tubes produced from in vivo hyphal bodies have been shown to possess laminin-binding surface epitopes which mediate the binding to basal membrane components (Pendland et al., 1994). Due to the high numbers of hyphal bodies in the hemocoel, the invasion of tissues by hyphae leads to rapid death and mummification of infected larvae. The objective of this study was to examine the effects of B. bassiana development on the protein biosynthetic capability of host S. exigua larvae. At various intervals during the hyphal body and mycelial developmental phases, host tissues were dissected from infected larvae. The following 35S labeling studies were designed to provide data on the biosynthetic capabilities of various naive S. exigua tissues to measure the effects of the B. bassiana development on host protein synthesis, and to detect potential in vivo metabolites released by B. bassiana during mycosis. MATERIALS
AND METHODS
Experimental model Eggs of the beet armyworm, Spodopteru exigua were obtained at the USDA/ARS laboratory (Gainesville, FL, U.S.A.), and the larvae were reared on an artificial pinto bean diet at 25°C under a 16 h light:8 h dark photoperiod. Blastospore cultures of B. bassiana (UFI 5477) were produced by inoculating conidia into Sabouraud dextrose broth. Cultures were incubated at 25°C on a gyrotary shaker for 2 days, then filtered through sterilized Miracloth to remove mycelia. Blastospores were collected from filtrates by centrifugation and washed twice in 0.85% saline. Fungal cells were counted on a hemacytometer and diluted in sterile saline to a concentration of 1 x 1051~1.Five ~1 of this concentration (500 blastospores) were injected into the hemocoels of late fifth instar S. exigua larvae. Control larvae were injected with 5 ~1 of sterile 0.85% saline. Treated and control larvae were placed on artificial diet and incubated at 25°C. At 24, 48, 60 and 72 h after challenge representative sixth instar larvae were sacrificed and the hemolymph examined under phase contrast optics to insure that the B. bassiana was replicating in challenged larvae. The relative numbers of hyphal bodies, and the overall morphology of host hemocytes were used as indicators of disease development (see Hung and Boucias, 1992; Hung et al., 1993).
Explant preparation and labeling Fat body and cuticle epidermis were dissected from B. bassiana-injected and saline-injected S. exigua larvae at 24, 48, 60 and 72 h after injection. Initially, larvae were surface sterilized with 95% ethanol and rinsed twice with sterile H,O. Fat body and cuticle explants, cleaned of extraneous tissue, were rinsed in Grace’s insect medium to remove the nonadherent hyphal bodies present in the infected larval hemolymph. Fat body and cuticle epidermis dissected from 2 or 3 larvae were pooled and transferred to microcentrimge tubes containing 100 ~1 of Grace’s insect medium without methionine (Sigma Chem). Hemolymph samples were collected and pooled from surface-sterilized infected and healthy lavae at 24,48 and 60 h after injection. Hemolymph (200 pi/tube) was diluted 1: 1 with Grace’s insect medium without methionine containing several crystals of phenylthiourea (PTU) to prevent melanization. For the preparation of hemocyte culture supernates, the hemolymph was diluted 1: 1 with citrate-EDTA buffer. After centrifugation at 1000 rpm for 5 min, supematants were removed and hemocytes with affiliated hyphal bodies were resuspended in Grace’s insect medium without methionine + PTU (50 pi/tube). Fat body, cuticle explants and hemocyte cultures were supplemented with 40 /_&i of 35S protein labeling mix EXPRE (New England Nuclear, DuPont) and were labeled for 4 h at room temperature. Additional noninfected hemocyte cultures were heat-shocked at 37 and 43°C for 30 min in water baths and then labeled for an additional 1 h at heat-shock temperatures. Control hemocyte cultures were incubated and labeled for 1 h at 25°C. A total of three independent replicates were conducted for both the pulse labeling of infected vs healthy explants and for heat shock experiments. Protein extraction Fat body, cuticle explants and hemocyte cultures were centrifuged at 10,000 rpm for 5 min and supemates, free of residual tissue fragments or cells, were collected. Cells and/or tissue pieces, were washed once with Grace’s insect medium to remove residual culture fluid, and were homogenized on ice in 100 ~1 of 1% Triton X- 100, 50 mM Tri-HCl (pH 7.4), 0.14 M NaCl, 50 pg/ml PMSF, 0.5 pg/ml leupeptin. A model 300 Dismembrator (Fisher Scientific) fitted with a microprobe tip set at a power of 35% for 30 s was utilized to homogenize cell/tissue preparations. Proteins were precipitated from supemates and homogenates with 2 vol of ice cold acetone. The precipitates were incubated at -70°C for 20 min then centrifuged at 10,000 rpm for 5 min. Pellets, washed twice with 66% cold acetone, were resuspended at 4°C in 50 ~1 of 2% SDS, 50 mM Tris-HCl (pH 6.8) by sonication. Protein concentration in the lysates was determined with the microtiter plate BCA Protein Assay (Pierce) using bovine serum albumin as the standard. Radioisotope levels (CPM) in lysates were calculated with a Tracer Delta 300 liquid scintillation counter.
B. BASSIANA,
EFFECT
ON INSECT
Electrophoresis andfluorography Protein extracts from cell/tissue supernates and homogenates were electrophoresed on 12% SDS-PAGE minigels. Prior to electrophoresis, lysate samples were denatured with 5% 2-mercaptoethanol, treated at 100°C for 3 min and centrifuged at 10,000 rpm for 5 min to remove insoluble precipitates. Approximately 10 pg of total protein/sample was applied to each well. Low molecular weight standards (BioRad Corp) were run in each gel. Gels were stained with Coomassie blue R-250 (BioRad Corp), destained with MeOH/acetic acid and photographed. Gels were then treated with ENTENSIFY@ fluors according to manufacturer’s protocol (DuPont Corp). Labeled proteins were detected by fluorography performed at -70°C using high speed X-ray film (Kodak Corp). The M,s of the different bands in the Coomassie blue stained gels and on the autoradiographs were calculated using computer program of Johnson et al. (1987). Only the peptides which were detected in all 3 replicates of the infected samples versus their respective controls (only 60 h samples presented in Figs 224) were used in generating the data in Table 2. RESULTS SDS-PAGE of S. exigua hemolymph revealed the presence of numerous proteins (Fig. 1, lane a). The major peptides, detected with Coomassie blue R250 had M,s of 130, 78, 48, 29 and 19 kDa. During the last larval instar the abundance of the majority of the hemolymph peptides remained constant. However, as the larvae progressed through this instar, increasing levels of the 78 kDa peptide were observed in sera samples. Challenging late fifth instar larvae with B. bassiana by hemocoelic injection did not induced detectable alterations in the peptide profile of the initial plasma samples (Fig. 1, lane a). However, as the fungal hyphal bodies continued to develop within the hemocoel, several new peptides were detected
TABLE
1. Radiolabeling of the tissue explants of naive and B. basiana infected S. exigua during the last larval instar cpm x 10Vmg protein”
Sample
Supemate
Healthy tissue Fat body Cuticle epidermis Hemocytes B. bassiana infected Fat body Cuticle epidermis Hemocytes
Homogenate
44.5 + 16.1b 16.3 + 9.3 11.1 k3.7
3.8 + 3.8 3.4 * I .9 6.0 + 2.4
35.2 k 11.2 20.7 + 13.4 18.9 + 14.8
3.3 * 1.7 5.8 f 5.4 9.0 f 5.2
tissues
“Relative levels of protein in supemates and homogenates were calculated using BCA Protein Assay kit (Pierce). bAverage cpm f SD were compiled from 3 replicates at 24, 48 and 60 h intervals.
PROTEIN
BIOSYNTHESIS
93
in Coomassie blue R250 stained SDS-gels. By 48 h after challenge (Fig. 1, lanes, c, d) peptides with M, of 22, 3 1, 32, 36 and 40 kDa were observed in infected sera samples. Additionally, B. bassiana infection induced increases in the levels of peptides having a M, of 38 kDa and ~14 kDa bands (Fig. 1, lanes c-e). Radiolabeling tissue explants from naive larvae demonstrated that many of the proteins synthesized by the fat body and cuticle explants had M, corresponding to plasma proteins (Figs 2 and 3). Fat body explants had a high biosynthetic activity and released a number of 35Slabeled peptides having M, which corresponded to 8 of the 10 sera peptides identified in Fig. 1 (Table 2, Fig. 2). The major labeled peptides released from fat body explants included those having M,s of 17, 19, 25, 26 and 78 kDa whereas moderately expressed peptides had M,s of 27, 29, 34-38, and 4448 kDa (Fig. 2). The cuticle explants also produced and secreted a range of 35S-labeled peptides which had M,s similar to those detected in hemolymph and fat body supemate samples (Figs 2 and 3). The major proteins produced by cuticle explants contained the 12, 25,45 and 78 kDa peptides and the moderately expressed proteins included the 17, 19, 29, 34, 38 and 48 kDa peptides. Naive hemocyte cultures appeared to have a lower biosynthetic activity (Table 1) and did not secrete the high levels of peptides associated with the fat body explants (compare Fig. 4 to Fig. 2). The naive hemocyte cultures did synthesize and secrete a high molecular weight peptide which corresponded to the 130 kDa hemolymph protein. Naive hemocytes heat shocked at 37 and 43°C were induced to synthesize a set of six peptides having M,s of 23, 26, 30, 70, 78 and 95 kDa (Fig. 5). The 70 and 78 kDa heat shock proteins were the major peptides synthesized by cells exposed to high temperature. Comparison among the profiles of radiolabeled peptides of hemocytes incubated at 25, 37, and 43°C demonstrated that the 43°C heat shock treatment suppressed normal protein synthesis. Comparative analysis of fluorographs of SDS gels of 35S labeled samples of tissues explanted from naive and B. bassiana infected S. exigua demonstrated that the replication of hyphal bodies in the hemocoel caused only minor alterations in the peptide profiles (Table 2, Figs 2-4). Even at the late stage (60 h after challenge) of the fungal hyphal body development (3 x lo4 cells/$ hemolymph), both the cuticle and fat body explants were capable of synthesizing and secreting the majority of peptides detected in naive samples. For example, the production of the 78 kDa peptide by both infected and naive fat body explants increased throughout the initial 60 h after challenge. Minor changes between the peptide profiles of naive vs infected fat body explants were observed in the replicated 35S labeling experiments (Fig. 2, Table 2). A 32 kDa peptide was detected in both the supemate and homogenate samples of infected fat body explants. Additional peptides having M,s of 9, 26, and 58 kDa were detected in infected fat body homogenates. In
94
I. MAZET
and D. G. BOUCIAS
Infected
Healthy a
b
c
d
e
a
b
c
d
e
97.4 66.2
45.0 31.0 215 14.4
FIGURE 1. Electrophoretic patterns of denatured hemolymph sera samples from healthy vs infected larvae analysed in 12% SDS-PAGE gels stained with Coomassie blue R250. The numbers on the left margin denote molecular weights of standards. The numbers between the healthy and infected samples represent the calculated M, for resolved hemolymph peptides. The letters a, b, c, d, and e represent the 12, 24, 36, 48 and 60 h post-challenge samples. The (0) denotes peptides detected in infected hemolymph sera samples and the (+) denotes increased levels of peptides.
addition to these. new peptides, the presence of certain fat body proteins appeared to be enhanced or suppressed during the in vivo development of B. bassiana hyphal bodies. For example, the synthesis of the 17 and 26 kDa peptides, major fat body peptides, was repressed whereas the levels of the 9, 36 and 38 kDa peptides were higher in the infected fat body samples (Fig. 2, Table 2). Radiolabeling cuticle explanted from infected larvae produced results similar to those observed with fat body explants. Only minor changes in the types and levels of cuticle proteins were observed during the hyphal body developmental phase (Fig. 3, Table 2). Four new peptides (9, 26, 28 and 30 kDa) were detected in supernates and five peptides (9, 22, 40, 58 and 100 kDa) were observed in the homogenates of infected cuticle explants. Reduced levels of the 12 and 29 kDa peptides and enhanced levels of the 38 kDa peptide were observed in the infected cuticle explants. Electrophoretic analysis of the naive and infected hemocyte cultures demonstrated that both the host hemocytes and in vivo replicating hyphal bodies secreted only a few well resolved peptides (Fig. 4). By 48 h after challenge, the presence of replicating B. bassiana hyphal bodies induced the synthesis and secretion of 3 peptides having a M, of 3 1, 32 and 40 kDa (Table 2, Fig. 4). Additional peptides having a M, of 26, 58 and 100 kDa were detected in hemocyte homogenates at the late stage (60 h) of hyphal body development. The levels of several hemocyte peptides (12, 17, 24 and 98 kDa) were repressed by the in vivo developing hyphal bodies. At 72 h the hyphal bodies underwent a transition producing germ tubes which attached to and invaded the insect tissues. Pulse-labeling tissues explanted from
infected larvae during this late infection phase revealed marked alterations in the biosynthesis of host peptides (Fig. 6). In fat body explants the synthesis and release of various peptides including the major 78 kDa peptide were suppressed during the late tissue-invasive phase of this fungal disease (Fig. 6, lane D; Table 3). Four major peptides having a M, of 30, 3 1, 38 and 41 kDa were detected in the supemates of infected fat body explants (Fig. 6, lane D). Fat body explanted from naive insects at the 72 h sampling interval produced a labeled peptide profile similar to fat body explanted at early (2460 h) intervals. The homogenates of the late phase infected fat body samples produced a distinct peptide profile and contained many more peptides than comparable naive fat body samples (Fig. 6, lane B; Table 3). The tissue invasive phase of B. bassiana also disrupted the biosynthetic capabilities of the cuticle epidermis resulting in a marked suppression of several major host peptides (Fig. 6, lanes E-H). Interestingly, these infected cuticle epidermal explants produced various labeled peptides (9, 15, 24, 30, and 31 kDa) that had a M, similar to peptides associated with late phase infected fat body (Fig. 6, Table 3). DISCUSSION
SDS-PAGE gels of peptides from S. exigua hemolymph sampled during the last larval instar produced peptide profiles similar to those of other lepidopteran species (Ferkovich et al., 1983; Beckage and Kanost, 1993). The hemolymph peptides, >200, 130, 78-82 and 19 kDa bands, had M,s similar to the well-characterized subunits of the inset lipophorins and arylphorins (Kanost et al.,
B. BASSIANA, EFFECT ON INSECT PROTEIN BIOSYNTHESIS
6oh II
6Ob I
Ii
24b I
95
48h
6Oh
I
I
KDa
60h H
60h I
H
I
KDa
KDa
-
97.4
-66.2 -
45.0
-
31.0
FIGURE 2. Electrophoretic patterns of peptides of healthy (H) and infected (I) fat body tissues. Upper panel represents peptides from supemates of fat body explant cultures. The 2 left lanes (A) are the electrophoretic profiles of healthy and infected cultures (60 h) visualized with Coomassie blue R250. The next 4 lanes (B) represent the ?S labeled proteins detected by fluorography. The numbers on the left of the 60 h healthy (H) sample represent calculated M, of detected peptides. The lower panel represents peptides detected in SDS gels of homogenates of fat body tissues. The 2 left lanes (A) represent Coomassie blue R250 stained electrophoretic profiles of 60 h healthy and infected fat body homogenates. The 2 right lanes (B) represent identical samples which have been electrophoresed and processed by fluorography. In both panels the (a) denotes a peptide unique to the infected samples; the (+) denotes a peptide which is found in increased titers in infected samples; and the (-) denotes a peptide in which titer has been reduced by the presence of 3. bassiana. The numbers on the right and left margins of both upper and lower panels represent the migration pattern of the molecular weight markers. X-ray films were exposed for 2 days at 70°C.
1990). During the last larval instar of S. exigua, distinct increases were observed in the titer of the 78-82 kDa band (comigrating subunits of arylphorin and apolipophorin II). In other insects the titers of arylphorin, a major storage protein, is known to increase dramatically during the last instar. The presence and levels of the other
hemolymph proteins remained constant throughout the initial 60 h of the last larval instar. Radiolabeling experiments suggested that the S. exigua fat body and cuticle epidermis were the major sites for biosynthesis of hemolymph proteins. Prior research by Palli and Locke (1987, 1988) using a combination of radiolabeling and selected
96
I. MAZET and D. G. BOUCIAS
6Oh
6Oh
24h
48h
60h
H
I
I
I
I
H KDa
6Oh H
60 h I
KDa
H
I
KDa
FIGURE 3. Electrophoretic patterns of peptides detected in naive and infected cuticular epidermis explants. Upper panel represents the peptides detected in supemates of the cuticle explants. The 2 left lanes (A) are electrophoretic patterns of the healthy (H) and infected (I) cuticle epidermis supemates at 60 h after challenge visualized with Coomassie blue R250. The 4 right lanes (B) represent the 9 labeled supemate peptides produced by healthy (60 h) and infected (24, 48, 60 h) cuticle epidermis explants. The lower panel represents the peptides detected in the homogenates of the cuticle epidermis explants. The 2 left lanes (A) represent Coomassie blue R250 stained peptides of the healthy and infected tissues and the 2 right lanes (B) represent 35S-labeled peptides detected by fluorographic methods. See Fig. 2 for explanation of +, -, l, and the molecular weight markers. X-ray films were exposed for 2 days at 7O’C.
antibodies determined that the majority of hemolymph proteins of Culpodes ethhis (Hesperidae) were synthesized by these tissues. More recently, Sass et al. (1993, 1994), utilizing various immunocytochemical probes, determined that the insect cuticle epidermis is able to directionally route peptides into the cuticle and/or basally into the hemolymph. Vegetative development of B. bassiana hyphal bodies
in S. exigua larval hemocoels had no significant effects on hemolyph proteins. Throughout this hyphal body growth phase, the M,s and levels of peptides in the hemolymph samples were similar to those found in the naive hemolymph samples. Additionally, the supernate and homogenate preparations of infected fat body and cuticle epidermis produced peptide profiles in Coomassie blue stained SDS-PAGE gels that were indistinguishable from
B. BASSIANA.
EFFECT
ON INSECT
PROTEIN
BIOSYNTHESIS
97
60h I
KDa
KDa
H j
KDa
1u_.
*
663
I
KDa
1
-a /
97.4 -
60h
?
,“,. 6 ,, ,”‘r
-
FIGURE 4. Electrophoretic patterns of the peptides of healthy (H) and infected (I) hemocyte cultures. Upper panel represents the peptides from supemates of hemocyte cultures. The 2 left lanes (A) are the Coomassie blue R250 stained peptides of healthy and infected hemocyte cultures. The 4 right lanes (B) represent the ?3 labeled peptides detected in the healthy (60 h) and infected (24, 48, 60 h) supemates by fluorography. The lower panel represents the electrophoretic patterns of the peptides present in the homogenates of the healthy and infected hemocyte monolayer cultures. The 2 left lanes (A) represent Coomassie blue R250 stained peptides and the 2 right lanes (B) represent the ‘3 labeled peptides detected by fluorography. See Fig. 2 for explanation of 0, +, -, and the molecular weight markers. X-ray films were exposed for 4 days at 70°C.
the peptide profiles of naive tissues. Challenging S. exigua larvae with B. bassiana did not induce nor interfere with the synthesis of defense-related proteins which have been observed in S. exigua challenged with either bacteria or related cell components (Boucias et al., 1994). Finally, the replication of the exocellular hyphal bodies did not induce the dramatic changes in the host biosynthetic events which have been observed when lepidopteran larvae are challenged with certain intracellular pathogens or endoparasitoids (Beckage, 1993). For
example, the injection of certain insects with calyx fluid associated polydnavirus (PDV) preparations resulted in dramatic alterations in the biosynthesis of hemolymph proteins. The PDV from Campoletis sonorensis has been demonstrated to cause a translational block in the fat body synthesis of arylphorin resulting in detectable decreases in hemolymph titers within 24 h of challenge (Shelby and Webb, 1994). Furthermore, parasitization of host larvae by Cotesia congregatu results in a specific inhibition of certain peptides (arylphorin, prophenoloxi-
98
I. MAZET
and D. Cl. BOUCIAS
TABLE 2. Summary of the changes in the %S-labeled peptide profiles of tissues explanted from B. bassiuna infected S. exigua larvae during the hyphal body growth phase Supemates Fat body B9
