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Venom proteins of the endoparasitic wasp Chelonus Near CurvimaculatusCharacterization of the major components.

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Archives of Insect Biochemistry and Physiology 13:95-106 (1990)
Venom Proteins of the Endoparasitic Wasp
Chelonus Near Curvimaculatus:
Characterization of the Major Components
Davy Jonesand 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 i s 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, p H 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 detectable changes in their molecular weight occur during injection or shortly after
injection into the host.
Key words: parasitization, Trichoplusiani, 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 January18,1989.
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
Jonesand Leluk
and cytolytic effects [3-51. However, little is known about the protein biochemistry of the nonparalyzing venoms of endoparasitic wasps, because the report
of the electrophoretic profile of Cotesiu congregutu venom proteins under denaturing 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 metamorphosis [ll-151. The female wasp injects material into the host during miposition, 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 components. The present report describes analysis of the venom proteins of C.
near curvimaculatus; the first detailed biochemical characterization of venom
proteins of an endoparasiticwasp on the basis of immunology, isoelectricpoint,
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~zoplusiani
(Huebner),were reared under the same conditions as described previously [13].
Chemicals
1251 Labeled antirabbit IgG from goat serum was prepared as described elsewhere [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, alphachymotrypsin 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 CurvimaculatusVenom 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 concentrationwas determined spectrophotometricallyusing Bio-Rad
dye reagent for microassay (1-10 pg protein). The standard curve was based
on bovine serum albumin.
Molecular Weight Estimation
Molecular weights of proteins were estimated following SDS-PAGE" (10%
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 alphachymotrypsin, 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 temperature 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-likeactivity, 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 injection, 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
'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
Jonesand Leluk
described [20]. Proteins in SDS-polyacrylamide gels were silver stained as
described [21].Alternatively, separated proteins were stained for carbohydrates.
They were electrophoreticallytransferred 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-HC1buffer, pH 7.6, containing 1mM MgC12, 1 mM CaC12,
0.5M NaC1, and 0.02% NaN3.
Immunoblotting
Immunoblotting was carried out according to procedures described elsewhere [22]. After electrophoretic transfer to nitrocellulose, the sheet was incubated 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-antirabbitIgG 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 dependent 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 concentration. 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 CurvimaculatusVenom Proteins
99
number of minor bands with this buffer was not due to proteolytic degradation, because inclusion of protease inhibitors such as 2 mM PMSF, iodoacetamide, and EDTA had no influence on the protein profile. The protein profiles
of venom reservoir contents vs. the entire venom gland were similar (not
sh 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 corresponding 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 differences 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
2
Mr
116
-
66
-
4524
134.2
56.5
35.1
-
1814-
Fig. 1. Carbohydrate staining of Chelonus near curvimaculatus venom proteins after SDSPAGE. 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
Jonesand Leluk
1
2
3
Fig. 2. SDS-PACE of the Chelonus near curvimaculatos venom isolatedfrom 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 activity. 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 chymotrypsin-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
Jonesand Leluk
Fig. 5. lmrnunoblot detection of the venom proteins in parasitized T. nieggs after2 s of stinging (1/10of 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 preparation 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 antibody. In some immunoblot experiments, the venom in stung eggs and different 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 interactions 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 apparatus of Western hemisphere Chelorz insularis [24,25]. The venom reservoir contains 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-characterizedinsect venoms, such as honeybee venom 141, contain highly basic proteins of strong neurotoxic and cytolytic activity (phospholipase AZ,lysophospholipase, mellitin, apamin, peptide
401, secapin). Isoelectrofocusingof 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 interpretation [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 developmental differences among insect species. Because parasitization by C. near curvimaculatus 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 painproducing and cytolytic proteins.
Another feature that distinguishes Chelonus venom from the known venoms
of other insects is its glycoprotein composition. Venoms of the higher Hymenoptera are characterized by the presence of glycoproteins of M,S30 kDa [31].
Because carbohydrate moieties are important in protein targeting and function [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 proteins 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 molecular 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, electrophoretic results show that 32.5 kDa glycoproteinis 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
Jonesand Leluk
different from the others. Also, the addition of proteinase inhibitors to dissecting 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 physiological characterization of the snake, scorpion, honey-bee, and other venom proteins [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 without 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 reservoir 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 parasitic 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 during 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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