Bidchimica et'Biophysica Acta, 1035 (1990) 37-43 37 Elsevier

BBAGEN 23327

Isolation and characterization of the 32.5 kDa protein from the venom of an endoparasitic wasp

T i m T a y l o r a n d D a v y J o n e s

Department of Entomology, University of Kentucky, Lexington, KY (U.S.A.)

(Received 12 October 1989) (Revised manuscript received 6 February 1990)

Key words: Epitope mapping; Peptide mapping; Venom protein

The major venom proteins from the endoparasitic wasp were analyzed for distribution in the venom gland. A 32.5 kDa protein was purified from the venom gland of the Chelonus near curvimaculatus wasp. The protein accounts for about 25% of the total protein content of the venom and each gland contains 3 -6 pmol of this component. The protein is acidic in nature and anion-exchange chromatography facilitated the purification of the protein to apparent homogeneity. On testing the purified protein by in vivo bioassay, it was found to elicit an effect comparable with the complete venom. The protein does not appear to have any disulfide bonds of major structural importance exposed under SDS-denaturing conditions. Products of chemical partial digest of the purified protein at the methionyl residues by cyanogen bromide were analyzed by SDS-PAGE. The 27.6 kDa fragment retained an epitope to an antibody raised against total Chelonus venom proteins, whereas no epitopes were detected for 4.9 and 0.6 kDa fragments.

Introduction

Although the study of endoparasitic wasp-host inter- action at the biochemical level is useful as a model for parasite regulation of host physiology and gene expres- sion, relatively little high-resolution study has been made of the biological role and biochemical mechanism of action of the non-paralyzing venoms characteristic of these wasps [1-3]. Despite the intense interest shown in characterization of paralyzing venoms from vertebrates [4] and arthropods [5], thorough biochemical charac- terization of the non-paralyzing venoms from endo- parasitic wasps has been sadly neglected until recently [6,7], and no venom protein from an endoparasitic wasp has ever been purified.

Our object of study has been the endoparasitic wasp Chelonus near curvimaculatus and in this paper we report the characterization of a purified, major compo- nent of the venom from the adult female wasp. During oviposition, the Chelonus wasp injects material into the host, Trichoplusia ni, this is necessary to enable the parasite to escape encapsulation by hemocytes (part of the host immune response) [8]. Leluk et al. [9] showed

that injection of Chelonus venom is necessary for para- site survival in the host, and that if it is injected late in host embryonic development, it is degraded very quickly and the parasite does not survive [10]. There is also immunological conservation between proteins in Chelonus venom and proteins in the venom of the phylogenetically higher honey-bee [7].

Hymenopteran venoms from biologically active bee and wasp venoms characteristically possess a highly abundant protein which induces a primary biological effect of the venom on the target. For example, honey- bee venom has been intensively studied as a model for nonparasitic hymenoptera, and the mellitin protein in its venom, which disrupts cell membranes in the target tissue, constitutes nearly 50% of the total protein in the bee venom reservoir [11]. In the biologically active venom from the Chelonus wasp, there is a highly abun- dant protein of 32.5 kDa [6] and in the present study we have intensively characterized this protein. To our knowledge, this is the first study on a venom protein of an endoparasitic wasp.

Materials and Methods

Abbreviations: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinyledene difluoride; CNBr, cyano- gen bromide.

Correspondence: D. Jones, Department of Entomology, University of Kentucky, Lexington, KY 40546, U.S.A.

Insects The endoparasitic wasps Chelonus near curvimacula-

tus were cultivated under a daily cycle of 14 h of fight followed by 10 h of darkness at 28°C, on the host Trichoplusia ni.

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

38

Chemicals Dye reagent for protein concentration assay was

obtained from Bio-Rad laboratories. All other chem- icals were obtained from Sigma.

Electrophoretic analysis of venom proteins Venom glands were removed from several adult

female wasps and were either dissected into their sep- arate components (filament, synthesis gland and venom reservoir) or retained in their complete form. The glands or the regions were manually homogenized and the soluble proteins were extracted into 10 mM sodium phosphate (pH 7.4) containing 0.9% (w/v) NaC1 and 7.5% (w/v) sucrose (PBS). Crude venom extract was loaded onto a slab gel (15 x 15 cm, 1.5 mm thick) containing 10% acrylamide and subjected to SDS-PAGE according to Laemmli [12]. SDS-PAGE of proteins was carried out under reducing conditions, using fl-mer- captoethanol, unless otherwise stated. After electro- phoresis, the gels were either stained with silver [13], or alternatively, the fractionated proteins were subjected to sequence analysis (below). Gels were scanned using an LKB Ultroscan XL enhanced laser densitometer.

Amino acid composition and N-terminal primary structure determination

Proteins fractionated by SDS-PAGE were soaked in transfer buffer (10 mM 3-(cyclohexylamino)- l - propanesulfonic acid (pH 11) containing 10% (v/v) methanol). The proteins were transferred to PVDF membranes for staining with Coomassie blue, according to Matsudaira [14]. PVDF membranes (Immobilon Transfer) 0.45/~M pore size were obtained from Milli- pore. After transfer and staining the filters were air dried. The band at 32.5 kDa was excised from the membrane, and the protein was subjected to precolumn- PTC amino acid analysis on a Waters Pico-tag amino acid analysis system. The N-terminal sequence of the protein was determined on a model 475 Applied Biosys- tems gas-phase sequence with a 120A PTH analyzer, with data analysis by a 900A module. P C / G e n e Nucleic Acid and Protein Sequence Analysis Software System (Biosearch) was used to analyze the protein sequences obtained.

Purification of venom proteins Venom glands were dissected from Chelonus wasps

and stored in PBS at - 7 0 ° C until use. The venom glands were manually homogenized in PBS and centri- fuged (1200 × g, 5 min). The supernatant was removed and a 100 ~tl sample was applied to a Pharmacia Mono- Q anion-exchange column (0.5 x 5 cm) equilibrated with 10 mM L-histidine (pH 6). Proteins were eluted over 30 rain, using a linear gradient of 0 to 1.0 molar NaC1 in 10 mM L-histidine (pH 6.0). HPLC separation of venom proteins (protein extract from 400 venom glands was

used for each HPLC run) was performed on a Beckman 126 programmable solvent module, with a 166 program- mable variable-wavelength detector and accompanying software. Detection was at 280 nm.

Protein concentration assay To determine the percentage recovery, protein de-

terminations were carried out on the crude venom gland extract and on the fractions (containing the 32.5 kDa protein) collected during HPLC separation of the ve- nom proteins. The protein concentration was de- termined spectrophotometrically using Bio-Rad dye re- agent for protein micro-assay (1-20 /~g protein). The standard curve was based on bovine serum albumin.

Bioassay of chelonus venom proteins T. ni eggs were manually injected with 100 nl PBS,

crude Chelonus venom gland extract in PBS, or purified 32.5 kDa Chelonus venom protein in PBS. Needles for injection were made using a Kopf Instruments 720 Vertical Pipette Puller. It is known that during oviposi- tion the Chelonus wasp injects 2.5-4.0 ng of venom protein into the host [6], so the venom protein manually injected into each egg was adjusted to yield this same concentration. After injection, the eggs were incubated for at least 48 h at 24°C. The eggs were then stung naturally and were further incubated at 24°C until the host larvae had hatched. The larvae were then reared under a dally cycle of 14 h of light followed by 10 h of darkness at 28°C for approx. 2 weeks, after which time it was possible to determine the parasitized condition of the larvae (pseudo-parasitized, in which the parasite died or truly parasitized in which the parasite survived).

SDS-PA GE of a purified venom protein in the presence and absence of fl-mercaptoethanol

Aliquots of the purified 32.5 kDa protein were sub- jected to SDS-PAGE (10% acrylamide). Prior to electro- phoresis, samples were mixed with equal amounts of sample buffer [12], with or without added /3-mercapto- ethanol, then immersed in a boiling water bath for 2 min. After SDS-PAGE the proteins were silver stained, as described by Morrissey [13].

Chemical digestion with cyanogen bromide (CNBr) and separation of peptides

The method for peptide m/lpping of proteins after partial cleavage was essentially that of Zingde et al. [15]. Crude venom gland extract from approx. 100 adult female Chelonus wasps was subjected to SDS-PAGE (10% acrylamide). Upon which, the gel was stained with 0.2% (w/v) Coomassie blue in 50% (v/v) methanol, 10% (v/v) acetic acid and then destained in 50% (v/v) methanol and 10% (v/v) acetic acid until the protein bands were visible. The band at 32.5 kDa (containing approx. 12 /~g of protein) was excised and placed in an

Eppendorf tube. The gel piece was dried and then subjected to indirect cleavage with CNBr. Formic acid (2 ml, 70% v/v) containing 100 mg of CNBr was placed in a 30 mi screw-top jar. The jar was sealed and equilibrated with acidic CNBr vapor for 30 rain. An Eppendorf tube containing the gel piece was then placed inside the equilibrated jar. In a separate jar the protein was exposed to vapor from 70% formic acid only, as a control. The jars were kept at room temperature (25 ° C) for 6 h, at which time the tubes were removed. The gel piece was suspended in 0.3 ml of sample buffer [12] and the buffer was changed twice over the course of 30 min. The gel piece was finally suspended in 0.5 ml of sample buffer and the tube immersed for 2 min in a boiling water bath. The gel piece was then loaded directly into the well of an SDS-polyacrylamide gradient gel (10-20% for separation of the peptides. Peptides were visualized by silver staining. Peptide fragmentation mapping was performed on five independent samples.

Immunoblotting of separated peptides Immunoblotting was carried out essentially accord-

ing to procedures described elsewhere [16]. After elec- trophoretic transfer of the proteins to nitrocellulose, the sheet was incubated in 20 mM Tris-HC1 (pH 7.5) con- taining 0.9% (w/v) NaC1, 20% (v/v) horse serum and 5% (w/v) bovine serum albumin (blocking solution). The sheet was incubated with anti-venom rabbit serum (1 : 100 dilution) in 2% (v/v) blocking solution at room temperature overnight. The anti-venom antibody was raised against all Chelonus near curvimaculatus venom proteins as described by Leluk and Jones [10]. After washing out the excess rabbit IgG with 20 mM Tris-HC1 (pH 7.5) containing 0.9% (w/v) NaC1, 0.2% (w/v) SDS, 0.5% (v/v) Triton X-100 and 0.5% (w/v) milk powder, the sheet was placed in 125I-labelled antirabbit IgG in blocking solution for 4 h at room temperature. 125I- labelled antirabbit IgG from goat serum was prepared as described elsewhere [17]. Finally, the excess 125I-anti- rabbit IgG was washed off the nitrocellulose, and the sheet was then air dried and exposed to Kodak XAR5 X-ray film at - 7 0 °C for 1 to 2 days.

Results

Distribution of venom proteins in the venom apparatus The protein profile of the venom reservoir was simi-

lar to that of the entire venom gland (Fig. 1), as previously reported by Jones and Leluk [6]. By densito- metric analysis, the 32.5 kDa protein appears to be the most abundant, contributing 25% of the protein extract from the entire venom gland. Several proteins of 97, 77, 52 and 37.5 kDa were present in the profile of the entire venom gland (Fig. 1), which were not present in the electrophoretic pattern from the venom reservoir. How- ever, they were present in the protein pattern from the

Mr x 1(-

16(

39

97

66

43

,q-

31-

ql-

21-

a b c d

Fig. 1. SDS-PAGE of venom protein extracted from: (a) the complete Chelonus wasp venom gland, 5 venom glands, 2.5 /lg total protein added; (b) the filament, 30 filaments, 0.5/xg total protein added; (c) the synthesis gland, 30 synthesis glands, 1 ~tg total protein added; and (d) the venom reservoir, 10 venom reservoirs, 4.5 /tg total pro-

tein added.

filament and the synthesis gland (Fig. 1). All proteins present in the venom reservoir were present in the filament and synthesis gland (Fig. 1, arrows). Visual comparison of the venom synthesis and storage regions of the gland (Fig. 1), shows that the vast majority of the 32.5 kDa component resides in the venom reservoir. This is also the case for the three other major compo- nents (molecular mass, 47, 53 and 131 kDa) of the Chelonus venom.

Amino acid composition The results of amino acid analysis are shown in

Table I. The protein has a high glutamic acid/glutamine and aspartic acid/asparagine content, which is con- sistent with the acidic nature of the protein observed previously by Jones and Leluk [6].

Primary structure determination The 20 residue N-terminal amino acid sequence of

the 32.5 kDa Chelonus venom protein shown in Fig. 2 was deduced from two separate determinations. The mean repetitive yield from the sequencer was 89%. No similar sequences were found in the PC-Gene protein bank. Jones and Leluk [6] observed, by conconavalin- A/horseradish peroxidase, that the 32.5 kDa protein from Chelonus venom was weakly glycosylated, but we

40

TABLE I

Amino acid composition of the Chelonus venom protein ( M r 32 500)

The assay was carded out with approx. 16 pmol of protein and tryptophan was assayed by a spectrophotometric method [23]. One cysteine residue has also been found by sequencing.

Amino acid 7o res/mol No. res/mol

Asp/Asn 12.9 38 Glu/Gln 17.3 51 Ser 17.6 52 Gly 11.2 33 His 0.3 1 Arg 3.7 11 Thr 3.7 11 Ala 5.1 15 Pro 2.4 7 Tyr 0.3 1 Trp 0.3 1 Val 7.5 22 Met 0.7 2 lie 2.4 7 Leu 4.1 12 Phe 2.0 6 Lys 8.8 26 Totai

d id no t f ind tha t the 32.5 k D a p ro t e in exh ib i ted any m a j o r b ind ing to the lect in on passage of c rude v e n o m th rough a c o n c o n a v a l i n - A af f in i ty co lumn (not shown). Also, the sequence mark ing a po ten t i a l site of N-g lyco- sy la t ion (Asn-Xaa-Thr (Ser ) ) does no t occur in the N - te rmina l sequence.

Purification of the 32.5 k Da protein The amino ac id compos i t i on and d a t a f rom isoelec-

tr ic focusing [6] es tabl ish that the 32.5 k D a c o m p o n e n t of Chelonus venom is an acidic pro te in . We, therefore, used an ion-exchange c h r o m a t o g r a p h y of c rude venom g land ext ract as a m e t h o d of pur i f ica t ion . Fig. 3 shows the abso rbance prof i le of the an ion-exchange sepa ra t ion of venom prote ins . F r ac t i ons A, B and C (Fig. 3) con ta ined the 32.5 k D a p ro t e in and y ie lded 30 /~g (measured b y the B io -Rad p ro t e in assay) of a p p a r e n t l y pu re p ro te in (Fig. 4). The mate r i a l was cons ide red to be pu re if i t exh ib i ted a single b a n d b y silver s ta in ing af ter S D S - P A G E .

Bioactioity o f Chelonus venom proteins The basis of the assay is tha t the venom (if in jec ted

on day 1) carr ies ou t its func t ion in the subsequent 48 h per iod, as af ter this t ime r a p i d deg rada t i on of the venom p ro t e in occurs [6]. In fact, if hosts a re s tung on d a y 3, then an average of 50% of the paras i tes succumb

l 5 10 15 20 Ile-Phe-Ser-Phe-Asp-Asp-Leu-Val-Cys-Pro-Ser-Val-Thr-Ser-LeuoArg-Val-Asn-Val-Glu

Fig. 2. N-terminal amino acid sequence of the 32.5 kDa protein from Chelonus venom.

A B C .15-- v v v

-~ J o -

~ .05

0 I I I I 0 2 . 0 4 . 0 6 . 0 8 . 0 10.0

VOLUME ELUTED ImL} Fig. 3. Anion-exchange chromatography of crude venom gland extract from Chelonus wasps. The protein was monitored at 280 nm. A total of 200-300/~g of protein was loaded onto the column for fractiona- tion. The flow rate was 0.7 ml/min and 0.35 ml fractions were collected. Fractions A, B and C contained the 32.5 kDa protein

(Fig. 4).

to the i m m u n e response of the host, appa ren t ly because the in jec ted v e n o m is deg raded before i t can exert its effects [6,8]. In jec t ion of c rude venom signif icant ly in- crease the pa ras i t e survival ( P = 0.011), c o m p a r e d with eggs in jec ted wi th PBS on ly (Table II). The results also show tha t the pur i f i ed 32.5 k D a prote in , on its own, is

M r x 10-:'

97-

6 6 -

4 3 -

. e l -

31--

( 1 ) A B C D E

Fig. 4. SDS-PAGE (10%) of selected anion-exchange fractions of venom proteins. A 25 #1 aliquot from each of the fractions was loaded onto the gel, and protein bands were visualized by silver staining. Fractions A-C contained purified 32.5 kDa protein, while D and E are the two fractions eluting immediately after fraction C. Fractions A-C were combined and used as purified 32.5 kDa protein. (1) Whole

venom gland extract (10 venom glands, 5 #g total protein added).

Mrx l(33

97~

6 6 - -

4 3 ~

3 1 ~

21--

3 - -

~ 3 2 . 5

j27.6%_ -%27.0/--

- - 4 . 9 C

a b Fig. 5. An SDS-polyacrylamide gradient gel (10-20%) showing elec- trophoretic patterns of purified 32.5 kDa venom protein, (a) after exposure to formic acid vapors, (b) after exposure to vapor from CNBr in formic acid and (c) after exposure to CNBr but understained to allow visualization of the separated bands, The peptide bands were

visualized by silver staining.

MrXlO 3

9 7 - -

6 6 ~

4 3 m

31m

2 1 ~

1 4 - -

m

1 2 3 4

Fig. 6. An immunoblot of CNBr fragments from 32.5 kDa Chelonus venom protein. Proteins in lanes 1, 2 and 3 were separated on an SDS-polyacrylamide gradient gel (10-20%), prior to transfer to nitro- cellulose and probing with an antibody raised to total venom proteins. (1) Crude venom gland extract, 10 venom glands, 5/~g protein added; (2) purified 32.5 kDa protein exposed to formic acid vapor only, 20 venom glands, 2.5/xg protein added; and (3) purified protein exposed to formic acid and CNBr, 50 venom glands. (4) Silver stained gel of purified protein exposed to CNBr vapors. Long overexposure did not

show any signal for the 4.9 kDa fragment.

41

TABLE II

Bioassay of venom proteins from Chelonus near curvimaculatus for immunosuppressive activity

The percentages displayed are results from four separate bioassays. The number of 'truly' parasitized larvae resulting from T ni eggs injected with PBS (control) was compared with the number of 'truly' parasitized larvae resulting from eggs injected with crude venom extract or purified 32.5 kDa venom protein, n = total number of eggs injected which hatched.

Material injected Parasitized condition of T. ni

pseudo truly (%) (%)

None (injury only) (n = 54) 48 52

PBS (n = 27) 44 56

Crude venom (n = 49) 18 82 (P = 0.011)

Purified 33 kDa venom protein (n = 40) 17 83 (P = 0.0096)

c a pa b l e of s igni f icant ly increas ing pa ras i t e survival ( P --- 0.0096) to a level s imi lar to tha t of the crude venom.

S D S - P A G E in the presence and absence of fl-mercapto- ethanol

The presence or absence of a reduc ing agent in the L a e m m l i buf fe r du r ing S D S - P A G E of the 32.5 k D a v e n o m p ro t e in does not affect the p ro t e in ' s mig ra t ion rate. This resul t suggests tha t if the molecules con ta in d isul f ide l inkages, then they do no t de t ec t ab ly p reven t un fo ld ing of the p ro t e in under S D S - d e n a t u r i n g condi - t ions.

Peptide mapping after partial cleavage with CNBr The chemically cleaved p roduc t s ob t a ined under our

cond i t ions gave a r ep roduc ib l e e lec t rophore t ic p a t t e r n wi th two ma jo r f ragments of 27.6 and 27 kDa, and one m i n o r f r agmen t of 4.9 k D a (Fig. 5). The lack of a me th ion ine res idue in the N - t e r m i n a l sequence p rov ided ass ignment of the 4.9 k D a f ragment to the N - t e r m i n u s and the unobse rved 0.6 k D a f ragment to the C- terminus .

Immunoblotting of CNBr fragments Prob ing of the C N B r f ragments wi th an a n t i b o d y

ra ised agains t to ta l venom pro te ins showed tha t all ep i topes r ema in ing af ter f r agmen ta t ion are loca ted on the large f r agmen t of 27.6 k D a (Fig. 6). The reso lu t ion of the t echn ique was not suff ic ient ly high to de te rmine whe ther or no t the ep i topes were re ta ined on the frag- men t of 27 kDa . N o signal was de tec ted f rom the f r agmen t of 4.9 kDa .

Discussion

The s tudy of the b iochemis t ry of v e n o m pro te ins has p r o v i d e d cons ide rab le i n fo rma t ion of genera l in teres t in

42

a number of different fields. For example, honey-bee mellitin, the most exhaustively worked, has been the only model for nonparalyzing and nonparasitic hy- menoptera. Also, the biosynthesis of mellitin has been a useful model in analysis protein processing [18,19]. Fur- thermore, mellitin has been an important pharmacologi- cal probe in the field of cell membrane structure.

Theoretically, after synthesis of the Chelonus venom proteins, secretion should occur involving transfer across a cell membrane to reach the lumen of the storage reservoir. However, electrophoretic analysis of Chelonus venom proteins (Fig. 1) did not detect evidence of size processing from the synthesis site to the storage re- servoir within our limit of resolution (10 residues). Leluk et al. [10] have shown, with experiments using naturally injected venom, that it is necessary for survival of the endoparasite and the data reported here strongly suggest that the 32.5 kDa protein is the major contribu- tor to this effect. Leluk and Jones [9] demonstrated with the use of western blotting techniques that the major Chelonus venom proteins, after injection into day 1 T. ni eggs, do not undergo major cleavage and are stable for 48 h. However, the venom proteins were rapidly de- graded in day 3 eggs and inhibitor studies indicated it was due to the trypsin-like and serine proteinases found to be active in eggs of that stage. On this basis, it was postulated that the venom carries out its function in the initial 48 h period. From the amino acid analysis carried out on the 32.5 kDa protein (Table I), the high con- centration of lysine and arginine residues present in the molecule would make it highly susceptible to the action of such enzymes.

Amino acid analysis of the 32.5 kDa protein (Table I), shows that the molecule contains two methionine residues. It was expected, therefore, that CNBr cleavage would produce a fragmentation pattern of three com- plete and two partial fragments. The pattern obtained from CNBr cleavage studies (Fig. 5) shows the presence of two large fragments of approx. 27.6 and 27 kDa, so it is assumed that a complete fragment of 0.6 kDa also exists. Presumably, the reason for its non-appearance on the gel is either due to diffusion from the gel, during buffer equilibration, or that this small peptide is eluted from the gel during electrophoresis. A partial fragment of 31.9 kDa should also exist, but it is doubtful if such a fragment would be separated from the intact 32.5 kDa molecule in a 12-25% polyacrylamide gradient gel. The fact that a fragment of 4.9 kDa is present on the gel (Fig. 5) and a 5.5 kDa band is absent indicates that the 4.9 and 0.6 kDa fragments are not adjacent in the sequence and therefore must flank the 27 kDa fragment. It is likely that the 0.6 kDa fragment is the C-terminal portion, as N-terminal sequencing (to 20 residues) did not reveal the presence of methionine. On silver staining of the peptides at the elevated temperature of 42°C [13], the 4.9 kDa peptide band always produced an

orange colour (in contrast to the brown of higher molec- ular weight fragments). For basic proteins, this indicates the predominance of lysine residues over arginine. Iso- electric-focusing should confirm whether or not the peptide represents a basic region of what is overall an acidic 32.5 kDa protein.

In a study to immunologically compare hy- menopteran venoms, Leluk et al. [7] observed conserva- tion of epitopes from Chelonus venom proteins with honey-bee venom proteins (despite the differences in function between honey bee venom and Chelonus ve- nom; defense and parasite survival, respectively), using an antibody raised to Chelonus venom proteins. Nota- bly, honey-bee venom also contains the enzyme hy- aluronidase, which is of similar molecular weight (35 kDa) to that of the major component of Chelonus venom. In common with the 32.5 kDa Chelonus venom protein, hyaluronidase is an acidic protein with a high aspartate and glutamate content [20]. The function of hyaluronidase in venoms has long been ascribed to that of a spreading agent, enzymatically opening up passages through the host tissue matrix, through which other venom proteins can diffuse. Despite the apparent struct- ural similarities, immunological crossreactivity between the 32.5 kDa Chelonus venom protein and honey-bee hyaluronidase is not evident [7].

To our knowledge, this is the first time a protein from a parasitic wasp has been purified and sequence data obtained. It also represents the first identification and isolation of the biologically active component of a nonparalyzing wasp venom. These results are especially relevant in view of the increasing evidence that the venom in some parasitic wasps acts in concert with polydnaviruses to regulate immune response [21]. Bee and predatory wasp venom components have been pop- ular subjects of study for considerations on the im- munogenic properties and mode of action of venoms [7,22]. Since there are over 100 000 species of parasitic wasps and advances in the study of honey-bee venom have had implications for so many biochemical disci- plines, the present results are an important advance into a large, neglected area of protein biochemistry.

Acknowledgement

This study was supported, in part, by NIH 33995 and published with the approval of the director of the Kentucky Agricultural Experiment Station.

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