Archives of Insect Biochemistry and Physiology 13:95-106 (1 990)

Venom Proteins of the Endoparasitic Wasp Chelonus Near Curvimaculatus: Characterization of the Major Components Davy Jones and Jacek Leluk Department of Entomology, University of Kentucky, Lexington, Kentucky

The venom apparatus of Chelonus near curvimaculatus (Braconidae) has a simple (type 2) morphology. Most of the venom is accumulated in a thin-walled venom reservoir at the distal end of the gland filament as a 10-17% protein solution. The best results for isolation of the proteins were obtained using 7.5% sucrose in phosphate buffer, pH 7.4. There are four major proteins, with respective M, values of 32,500, 47,000, 53,000, and 131,000. Of these, those of M, 32,500, 53,000, and 131,000 contain carbohydrate. Most of the venom proteins are acidic with p l values between 4.9 and 6.9. The venom does not show proteolytic activity corresponding to serine or thiol proteinases, nor does it show antitrypsin or antichymotrypsin activity. Using immunoblotting techniques, it was established that during parasitization of a single host egg (Trichoplusia ni) about 1/200 of a venom reservoir equivalent is injected. All major venom proteins have been found in stung T. ni eggs; thus, no detecta- ble changes in their molecular weight occur during injection or shortly after injection into the host.

Key words: parasitization, Trichoplusia ni, toxin

INTRODUCTION The venom proteins of vertebrates have been the subject of a large number

of studies concerning their physicochemical and biochemical characteristics as well as their biological function [1,2]. The venoms of many arthropods such as scorpions, spiders, and certain hymenopteran insects (social and solitary bees, ants, and wasps) are well characterized with respect to their paralyzing

Acknowledgments: The authors thank Drs. Grace Jones and Mietek Wozniak for their help during this study. We also acknowledge the technical assistance of Anita Click and the efforts of Surnedha Weeratunga and Bryan Gifford in maintaining the insect cultures. This study was supported in part by NIH grant GM-33995 and is published with the approval of the Director of the Kentucky Agricultural Experiment Station (88-7-158).

Received December 12,1988; accepted January 18,1989.

Address reprint requests to Davy Jones, Department of Entomology, University of Kentucky, Lexington, KY 40546.

Dr. Leluk i s on leave from the Institute of Biochemistry, University of Wroclaw, Poland.

96 Jones and Leluk

and cytolytic effects [3-51. However, little is known about the protein biochem- istry of the nonparalyzing venoms of endoparasitic wasps, because the report of the electrophoretic profile of Cotesiu congregutu venom proteins under de- naturing conditions 161 is the only analysis present in the published literature. The known venoms of Hymenoptera are characterized by the presence of highly abundant, low-molecular-weight, basic proteins that either interact with phospholipids to disrupt membranes (e.g., mellitin) or that act at the nerve synapse to paralyze the host [7,8]. Another common feature of some insect venom proteins is the exceptionally low stability of their activity, which has caused many difficulties during purification [9,10].

A nonparalyzing endoparasitic wasp (Chelonus near curvimaculatus) has been the object of study as a model of biochemical host-parasite interaction [11,12]. The host, stung during the egg stage, prematurely initiates larval-pupal meta- morphosis [ll-151. The female wasp injects material into the host during mi- position, which redirects host development [16]. This material includes a virus that replicates in the female reproductive tract [17] and venom from a venom gland. In order to study the role of the venom components in this host-parasite interaction, it is necessary first to identify and to characterize the venom com- ponents. The present report describes analysis of the venom proteins of C. near curvimaculatus; the first detailed biochemical characterization of venom proteins of an endoparasitic wasp on the basis of immunology, isoelectric point, two-dimensional electrophoresis, and glycosylation state. Also reported here is the first biochemical measurement of venom proteins injected and the time of first injection, i.e., during the oviposition process.

MATERIALS AND METHODS

Insects The endoparasitic wasps, Chelonus near curvimaculatus, were reared under

conditions 14:lO h light:dark, at 28°C [13]. The host insects, Tric~zoplusia ni (Huebner), were reared under the same conditions as described previously [13].

Chemicals 1251 Labeled antirabbit IgG from goat serum was prepared as described else-

where [MI. Dye reagent for protein concentration assay was obtained from Bio-Rad Laboratories (Rockville Centre, NY). Acetic acid, formaldehyde, glutaraldehyde, glycerol, hydrochloric acid, potassium phosphate monobasic, sodium chloride, and sodium phosphate dibasic were from Fisher Scientific Co. (Pittsburgh, PA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), including bovine serum albumin no. A-7030, alpha- chymotrypsin from bovine pancreas Type 11, papain Type IV, soybean trypsin inhibitor Type I-S, and bovine pancreatic trypsin Type 111.

Dissection of Venom Glands The venom glands of C. near curvimaculutus were dissected into solutions

varying in the type of buffer and sucrose concentration used (see Results). Female wasps first were anesthetized by cooling at 4"C, and dissected glands were washed several times by sequential transfer to fresh dissection solutions.

Chelonus Near Curvimaculatus Venom Proteins 97

The venom reservoirs then were broken with tweezers and the contents squeezed out. The contents of 20-40 venom reservoirs (each female has one reservoir) collected in such manner were immediatly frozen at - 70°C.

Protein Concentration Assay Protein concentration was determined spectrophotometrically using Bio-Rad

dye reagent for microassay (1-10 pg protein). The standard curve was based on bovine serum albumin.

Molecular Weight Estimation

polyacylamide). Molecular mass markers (14.3-205 kDa) were from Sigma.

Proteolytic Activity Proteolytic activity was estimated using synthetic substrates: TAME for

trypsin-like esterolytic activity and BTEE for chymotrypsin-like activity. As a positive control, esterolytic activity of 2.5 pg bovine trypsin and 2 pg alpha- chymotrypsin, respectively, were determined under similar conditions.

For detecting albuminolytic activity 30 ~1 containing 40 kg of bovine serum albumin and venom from ten reservoirs or 1 pg of proteinase (positive control for albumin digestion) in 0.1 M Tris-HC1 buffer, pH 8.0 or 0.1 M phosphate buffer, pH 6.5, with 10 mM mercaptoethanol were incubated at room temper- ature for 6 h. The reaction was stopped with an equal volume of 2x loading buffer for SDS-PAGE (containing 4% SDS) and boiled for 5 min. As a control, albumin incubated in the same conditions and venom or proteinase without albumin also were loaded. For a positive control, trypsin at pH 8.0 and papain at pH 6.5 with reducing agent were used.

Antiproteinase Activity Antitrypsin and antichymotrypsin activity were estimated using TAME and

BTEE as substrates, respectively. The procedures were identical to those used for trypsin-like and chymotrypsin-like activity, with one additional step. Prior to the esterolytic incubation, the proteinase solution was preincubated for 30 min at room temperature with 20 venom reservoir equivalents.

Preparation of Antibodies Against C. Near curvimaculatus Venom Proteins Three hypodermic injections of venom proteins were made into a rabbit at

3-month intervals. Each injection consisted of about 150 pg of venom proteins isolated from 250 venom glands (in 7.5% sucrose in 0.1 M phosphate buffer, pH 7.4). The first injection was made with Freunds complete adjuvant and the remaining two with incomplete adjuvant. Ten days after the third injec- tion, the serum was collected.

Electrophoretic Methods SDS-PAGE was done on 20 x 20 cm slab gels according to Laemmli [19]. IEF

was carried out in 5% polyacrylamide gels at a wide pH range (3.5-9.5) as

Molecular weights of proteins were estimated following SDS-PAGE" (10%

'Abbreviations used: BTEE = N-benxoyl-L-tyrosine ethyl ester; IEF = isoelectric focusing; kDa = kilodalton; PB = phosphate buffer; SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis; TAME = N-alpha-P-tosyl-L-arginine methyl ester.

98 Jones and Leluk

described [20]. Proteins in SDS-polyacrylamide gels were silver stained as described [21]. Alternatively, separated proteins were stained for carbohydrates. They were electrophoretically transferred to nitrocellulose, and the sheet was air-dried and then incubated with Con A-peroxidase solution (0.01%) for 30 min in 25 mM Tris-HC1 buffer, pH 7.6, containing 1 mM MgC12, 1 mM CaC12, 0.5M NaC1, and 0.02% NaN3.

Immunoblotting Immunoblotting was carried out according to procedures described else-

where [22]. After electrophoretic transfer to nitrocellulose, the sheet was incu- bated in blocking solution containing 20% horse serum and 5% bovine albumin in buffer (20 mM Tris-HC1, pH 7.5 with 0.9% NaC1). The sheet was incubated with antivenom rabbit serum (1:50 dilution) in the same buffer (2% solution) overnight at room temperature. After washing out the excess rabbit IgG with the same buffer now containing 0.2% SDS, 0.5% Triton X-100, and 0.5% milk powder, the sheet was placed in lZ5I-antirabbit IgG in blocking solution (2 x lo7 cpm in 20ml) for 4 h at room temperature. At the end, excess lZ5I-antirabbit IgG was washed off, and the nitrocellulose sheet was air-dried and exposed to Kodak x-ray film at - 70°C for 2 to 7 days.

Carbohydrate Staining Staining with concanavalin A/horseradish peroxidase was as described [23].

RESULTS Chelonus Near curuimaculatus Venom Apparatus

The venom apparatus of C. near cumimaculatus has the morphology of type 2 venom gland [24,25]. The shape of the thin-walled reservoir is very depen- dent on the hypotonic or hypertonic environment. In an isotonic solution, the short and long dimensions of the reservoir are approximately 0.18-0.2 mm and 0.2-0.3 mm. From these the volume of venom reservoir (as oval in shape) is calculated to be approximately 4.85 nl. The amount of protein in a single reservoir was measured to be in the range of 0.5-0.8 pg or a concentration of 10.3-16.5%. Such a high concentration of protein explains the very viscous appearance of the venom reservoir contents seen upon their liberation into dissecting buffer.

Optimal Conditions for Venom Protein Isolation When 50 mM ammonium bicarbonate was used for isolation, four major

and several minor protein bands were observed. A better result was obtained when dissection and isolation were carried out in PB, pH 7.4. When venom glands were held in ammonium bicarbonate for longer than 15 min, part of the contents of the venom became insoluble. This phenomenon also occurred when PB was used, but only after a much longer time. The best protein pattern was obtained from the sample dissolved in 7.5% sucrose in PB. The major protein bands were most intense in this sample, and there appeared several minor bands not detectable in samples of much lower or much higher sucrose con- centration. On this basis, in subsequent studies, the glands were dissected and venom was isolated in 7.5% sucrose in PB (pH 7.4). The increase in the

Chelonus Near Curvimaculatus Venom Proteins 99

number of minor bands with this buffer was not due to proteolytic degrada- tion, because inclusion of protease inhibitors such as 2 mM PMSF, iodoaceta- mide, and EDTA had no influence on the protein profile. The protein profiles of venom reservoir contents vs. the entire venom gland were similar (not s h own).

Partial Characterization of Chelonus Near curvimaculatus Venom Proteins The average 2 S.E. molecular masses of the four major protein were 131.3 ?

6.0,53 ? 7.1,47.2 % 6.4, and 32.5 +- 3.5 kDa (n = 6). No highly abundant, low molecular proteins were observed (Fig. 1). A number of venom proteins con- tained carbohydrates. Among the staining signals obtained were several cor- responding to major venom proteins, i.e., those of M, 131.3, 53, and 32.5. The 53 kDa glycoprotein appeared to be relatively richer in sugar than the others. No sugar residues were detectable on proteins of low molecular weight. Preparations from female wasps of different ages as well as from mated and unmated females showed very similar protein profiles, and no consistent dif- ferences were detected between these groups (Fig. 2).

Upon silver staining, the 32.5 kDa protein gave a different color than the other major bands, suggesting distinct differences in amino acid composition between the smallest major protein and the others (26). Isoelectric focusing and two-dimensional electrophoresis (Fig. 3) showed that most of the venom

Mr

116 -

66 - 45-

24 - 18- 14-

2

134.2

56.5

35.1

Fig. 1. Carbohydrate staining of Chelonus near curvimaculatus venom proteins after SDS- PAGE. Each lane received the amount of protein in five venom reservoirs. 1, venom proteins stained with silver; 2, venom proteins stained for mannose with a concanaval in A-horseradish peroxidase probe.

100 Jones and Leluk

1 2 3

Fig. 2. SDS-PACE of the Chelonus near curvimaculatos venom isolated from mated and unmated female wasps of different ages. 1, venom from mated 3-4 day old females; 2, venom from 1 day old unmated females; 3) venom from 5 day old unmated females.

proteins were acidic (pH range, 4.9-6.9). The most abundant basic protein appeared to be the protein with M, 47,000. Although acidic proteins were detected with M, below 30,000, no low-molecular-weight basic proteins were detected.

Proteolytic and Antiproteinase Activity of the Venom No trypsin-like or chymotrypsin-like activity of the venom was detected.

Also, the venom did not exhibit either antitrypsin or antichymotrypsin activ- ity. The protein substrate, bovine serum albumin, was not degraded by venom extracts at either pH 8.0 or pH 6.5. Therefore, the venom of this species lacks neutral or alkaline proteinases as well as inhibitors of typsin-like or chymo- trypsin-like enzymes.

Amount of the Venom Injected Into Host Eggs During Parasitization The venom proteins of as little as 0.01 venom reservoir equivalents were

detectable on immunoblots, i.e., 5-8 ng of total venom protein (Fig. 4). Use of this antiserum permitted detection of venom proteins in stung host eggs (Fig. 5). All major venom proteins are injected into T. ni eggs simultaneously (Fig. 5). They are present in the host eggs within 2 s of initiation of stinging, whereas complete oviposition takes 15-20 s. Moreover, no major size changes of these venom proteins occur during transportation to the host egg, as all detectable

Chelonus Near Curvimaculatus Venom Proteins 101

Fig. 3. Two-dimensional e imaculatus venom proteins isolated from 20 venom reservoirs. The first dimension (shown on top margin) was isoelectric focusing at wide pH range 2-11). The second dimension was SDS-PAGE (10% polyacrylamide). Proteins were visualized by silver stain.

Fig. 4. Western blot of C. near curvimaculatus venom proteins after SDS-PAGE. 1, venom amount equal to the volume of one venom reservoir; 2, venom amount equal to 0.1 volumes of the venom reservoir; 3, venom amount equal to 0.01 volumes of the venom reservoir. The probe was a polyclonal antibody preparation made against total venom apparatus proteins.

102 Jones and Leluk

Fig. 5. lmrnunoblot detection of the venom proteins in parasitized T. nieggs after2 s of sting- ing (1/10 of time required for the complete stinging process), by immunoblotting of proteins after SDS-PAGE. 1, T: ni nonstung egg proteins from 25 eggs; 2, proteins from 25 T. ni eggs stung for 2 s before interruption; 3, proteins from 25 Z ni eggs stung completely; 4, C. near curvimacuhtus venom proteins from one gland. The probe was a polyclonal antibody prepa- ration made against total venom apparatus proteins.

venom bands from parasitized egg preparations had the same mobilities as the proteins of the venom itself. Especially distinguishable were those major proteins that contained carbohydrate components (32.5, 53, and 131.3 kDa). In addition to signals for these antigens unique to stung eggs, several signals common to stung and nonstung eggs were observed. Further experiments showed that these signals were due to cross-reaction with the secondary anti- body. In some immunoblot experiments, the venom in stung eggs and differ- ent amounts of venom were run on the same gel. Comparative analysis of the intensity of signals from stung eggs vs. the intensity from different amounts of the venom made it possible to calculate that the female wasp injects about 1/200 volume of the venom reservoir during each oviposition, i.e., 2.5-4 ng of proteins in 24 pl.

DISCUSSION

Larvae developing from T. ni eggs parasitized by the endoparasitic wasp C. near cuvvimaculatus initiate precocious metamorphosis 10 days later during what is normally the penultimate larval stadium [14,15]. Data on host-parasite inter- actions involving larval parasites indicate that such constituents as venom, calyx fluid, and virus present in the ovaries of adult parasitoid females have an important role in the immunosuppression of parasitized host and other effects [27-301.

The eastern hemisphere endoparasitic wasp C. near curvimaculatus has a

Chelonus Near Curvimaculatus Venom Proteins 103

type 2 venom apparatus, which is similar in morphology to the venom appa- ratus of Western hemisphere Chelorz insularis [24,25]. The venom reservoir con- tains a highly concentrated protein solution (10-17%), but its small volume (4.85 nl) is a serious limiting factor for collecting large amounts of venom proteins.

There are four major proteins present in C. near curvimaculatus venom (131, 53, 47, and 32.5 kDa). There are no highly abundant low-molecular-weight proteins below 20 kDa, and no basic proteins were detected below 30 kDa. This situation distinguishes the venom of Chelonus from the other reported wasp, bee, and ant venoms [31]. The well-characterized insect venoms, such as honey- bee venom 141, contain highly basic proteins of strong neurotoxic and cyto- lytic activity (phospholipase AZ, lysophospholipase, mellitin, apamin, peptide 401, secapin). Isoelectrofocusing of Chelonus near curvimaculatus venom showed no highly abundant protein of pI37.0. These data suggest that the abundant proteins of Chelonus venom do not share conserved charge and molecular weight properties from their primary sequence with the abundant venom proteins of previously studied Hymenoptera. Recent comparative studies support this inter- pretation [31]. This aspect is relevant in view of other data that suggest some vertebrate venom proteins of one function may have evolved from others of a different function [l]. The differences observed in Chelonus venom and those of previously studied Hymenoptera may reflect behavioral and developmen- tal differences among insect species. Because parasitization by C. near curvi- maculatus is carried out on host eggs, paralyzing properties are not required, which may explain the lack of this type of neurotoxic protein in its venom. Also, the stinging apparatus of nonparalyzing, endoparasitic wasps is not adapted for defensive purposes; therefore, the venom would not need pain- producing and cytolytic proteins.

Another feature that distinguishes Chelonus venom from the known venoms of other insects is its glycoprotein composition. Venoms of the higher Hyme- noptera are characterized by the presence of glycoproteins of M,S30 kDa [31]. Because carbohydrate moieties are important in protein targeting and func- tion [31], different functional or regulatory mechanisms may apply to Chelonus venom, as compared with previously examined Hymenoptera.

Another important consideration is the relationship of major venom pro- teins among themselves. The molecular weights of three smaller abundant proteins (32.5,47.2, and 53 kDa) add up to the approximate value of the molec- ular weight of the largest abundant venom protein. One simple explanation is that cleavage of the single large precursor gives rise to the three products, and these accumulate in the reservoir (which itself does not appear to have protease activity). In recent years it has become apparent that many proteins arise as components of a parent precursor molecule. Processive cleavage then liberates the individual active components. If these three major proteins in Chelonus venom were just cleavage products of a common precursor, then in the simplest situation they would appear in equimolar ratio. However, elec- trophoretic results show that 32.5 kDa glycoprotein is present in a much larger quantity than the 47.2 and 53 kDa proteins. The color of silver-stained major bands also shows a difference between the 32.5 kDa band and other major proteins, suggesting that the amino acid composition of the smaller protein is

104 Jones and Leluk

different from the others. Also, the addition of proteinase inhibitors to dis- secting solution did not change the ratio. The major venom proteins are thus probably not related to each other in this simple way.

With respect to regulation and processing of the individual proteins, there are no differences in the relative abundances of the major proteins in venom isolated from the venom reservoir and those isolated from the entire venom gland. This situation indicates against processing of the large protein to the major smaller ones during transport to and from the reservoir. It was also shown that the relative abundance of the venom proteins is not dependent on the age or mating of the female wasps. In at least the honey bee the composition of the venom changes as the female ages [4].

Antibodies have been used successfully for the biochemical and physiologi- cal characterization of the snake, scorpion, honey-bee, and other venom pro- teins [2]. Using Western blotting techniques, the amount of venom proteins equal to 0.01 of venom reservoir equivalents, i.e., 5-8 ng of protein mixture, can be detected. These proteins can be detected in parasitized T. ni eggs with- out preliminary purification (Fig. 4). Several host egg proteins crossreacted with the particular goat antirabbit secondary antibodies used. Fortunately, this situation did not interfere with use of the antiserum to address questions on the entry of venom into the host, amount of venom injected, etc., as several distinctly reacting venom proteins have a different mobility than those from the crossreacting proteins in host eggs.

It was determined that during a single oviposition, about 1/200 venom res- ervoir equivalents is injected. Thus, a parasitized T. ni egg has received about 24 pl containing 2.5-4 ng of venom proteins. In comparison, paralyzing para- sitic wasps of the genus Brucon inject about 1/30 of total'reservoir equivalents in a single injection, i.e., 0.3-0.4 nl of paralyzing venom [9]. The complete oviposition process of Chelonus near curvirnuculafus takes 15-20 s, but within first 2 s (1/10 of oviposition period), the venom proteins are detectable in the host egg.

In summary, this study has provided the first biochemical data on venoms from an endoparasitic wasp concerning the amount of venom protein stored in the venom gland, the quality and quantity injected during oviposition, venom in the reservoir vs. in the venom apparatus as a whole, and on the time dur- ing oviposition at which the venom begins to be injected. Recent studies have shown that the venom in Chelonus near curvirnuculutus functions to permit the survival of the parasite in the host [33,34]. The study of this model system may also provide new leads to the practical use of venom-derived agents in the control of agriculturally important insects [14,30,31].

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