Content uploaded by Fevzi Uçkan
Author content
All content in this area was uploaded by Fevzi Uçkan on May 23, 2014
Content may be subject to copyright.
PHYSIOLOGY,BIOCHEMISTRY,AND TOXICOLOGY
Determination of Venom Components from the Endoparasitoid Wasp
Pimpla turionellae L. (Hymenoptera: Ichneumonidae)
F. UC¸ KAN,
1, 2
S. SI
˙NAN,
1
S¸. SAVAS¸C¸I,
3
AND E. ERGI
˙N
1
Ann. Entomol. Soc. Am. 97(4): 775Ð780 (2004)
ABSTRACT Venom from the endoparasitoid wasp Pimpla turionellae L. (Hymenoptera; Ichneu-
monidae) was isolated in pure form. Total protein determination indicated an average value of 0.04
g
protein per venom sac. The molecular weights of the venom components were estimated with
reference to molecular weight markers and reference proteins by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE). Analysis indicated that venom primarily contains proteins
with molecular weights between 20 and 106 kDa. The presence of melittin and apamin in wasp venom
was shown by SDS-PAGE and reversed-phase high-performance liquid chromatography (HPLC).
Infrared spectroscopic data conÞrmed the acidic nature of the venom and the presence of amines,
peptides, proteins, and enzymes in the venom. Venom noradrenaline was separated using thin-layer
chromatography and veriÞed by infrared spectroscopy.
KEY WORDS Pimpla turionellae, venom, electrophoresis, chromatography, infrared spectroscopy
THE RECENT STUDIES OF hymenopteran venoms have
profoundly affected the advances in modern biochem-
istry, pharmacology, and medicine (Soldatova et al.
1998, Konno et al. 2000). The characterization of
venom is also a rich source of information on the
physiological functions (Coudron et al. 2000, Parkin-
son et al. 2002a, Rivers et al. 2002), ecological inter-
actions, taxonomic, and phylogenetic studies (Leluk
et al. 1989, Doury et al. 1997) of parasitoids. The
composition of venom from Hymenoptera may vary
within groups or even species (Leluk et al. 1989, Skin-
ner et al. 1990, Sanchez et al. 1994). Studies on venom
composition and activity have been greatly focused on
those of Apis and Vespa species, which are of great
interest to humans (Abreu et al. 2000, Costa and Pala
2000). However, parasitic Hymenoptera venom has
recently become a valuable resource of natural sub-
stances that have promise in the construction of bio-
logical insecticides (Coudron and Brandt 1996). Par-
asitic wasps, which lay their eggs in or on their hosts,
are important regulators of insect pests. Adult female
parasitoids possess a stinging apparatus that is used to
inject maternally derived secretions into the hemo-
coel of their hosts during oviposition (Parkinson et al.
2002b). Several physiological traits of the host are
altered by female wasp secretions (polydnaviruses,
ovarian proteins, and venom) injected at oviposition
(Doury et al. 1997, Digilio et al. 2000). Venom secre-
tions modify the host physiology in various ways to
assist the development of the parasitoidÕs progeny
(Parkinson et al. 2002b). The presence or absence of
proteins in parasitoid venom may even indicate the
host stage for oviposition (Leluk et al. 1989). Venoms
from koinobiont species are frequently associated
with temporary paralysis and involved in the suppres-
sion of host movements during oviposition (Naka-
matsu et al. 2001). However, most of the idiobiont
parasitoids paralyze the host permanently, and thus
preserves it for a long time for the feeding and devel-
opment of the progeny larvae (Wharton 1993, Naka-
matsu and Tanaka 2003).
Pimpla turionellae L. (Hymenoptera: Ichneumonidae)
is an idiobiont endoparasitoid wasp that uses hosts
from an extremely wide range of lepidopteran species
(Kansu and Ug˘ur 1984). Little is known about the
composition of venom from this endoparasitoid, al-
though functionally its role in inhibiting neuromus-
cular transmission at the synaptic site (Kõlõnc¸er 1975)
and disabling hemocytes (Osman 1978) in host species
has been suggested. The structure of P. turionellae
venom apparatus and the primary chemical groups
present in the venom sac have been demonstrated
(Uc¸kan and Gu¨lel 1990, Uc¸kan 1999). The current
study aims to determine the venom sac components
and the molecular weights of peptides and proteins of
P. turionellae venom.
Materials and Methods
Insects. The solitary, pupal endoparasitoid P. turio-
nellae were reared on pupae of greater wax moth,
Galleria mellonella L. (Lepidoptera: Pyralidae). Adult
parasitoids were maintained at 25 ⫾2⬚C, 12:12 h (L:D)
photoperiod and fed with a 50% (vol:vol) honey so-
lution. Parasitoid females were also provided with host
1
Department of Biology, Faculty of Science-Art, Balõkesir Univer-
sity, Balõkesir 10100, Turkey.
2
E-mail: uckanf@balikesir.edu.tr.
3
Department of Chemistry, Faculty of Science-Art, Balõkesir Uni-
versity, Balõkesir 10100, Turkey.
0013-8746/04/0775Ð0780$04.00/0 䉷2004 Entomological Society of America
pupae (four pupae for 10 female wasps) once every 3 d
to meet their protein requirement (Kansu and Ug˘ur
1984).
Venom Extraction. The venom sacs of 15- to 20-d-
old female wasps were removed by grasping the ovi-
positor and pulling. The venom sac was collected by
grasping the duct leading to the ovipositor with Þnely
tipped forceps under a stereoscopic microscope and
placed in 100
l distilled water. The content of each
venom sac was removed by piercing and drained into
distilled water. The solution was centrifuged at
3,000 ⫻gfor 10 min to remove cell debris. For our
experiments, three tubes, each containing the con-
tents of 50 venom sacs and 100
l distilled water, were
made up to a Þnal volume of 200
l by adding distilled
water and adjusted to a Þnal concentration of 0.25
venom sac content per microliter (0.01
g/
l). The
tubes were kept at ⫺20⬚C for further investigations.
Total Protein Assay and Gel Electrophoresis. Total
protein determination was performed by the method
of Bradford (1976) using bovine serum albumin (BSA;
Sigma, St. Louis, MO) as the standard. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE; 20% acrylamide) was carried out with the
method described by Laemmli (1970). SDS-PAGE
(14 and 12.5%) was performed according to Shagger
and Von Jagow (1987) using Bio-Rad Mini Protean III
apparatus. The gel that contained 20% polyacrylamide
was stained with silver stain (Silver-stain kit, cat. no.
AG-25; Sigma). Fourteen and 12.5% polyacrylamide
gels were stained with Coomassive Blue (G250; Bio-
Rad, Richmond, VA). For electrophoresis, BSA (68
kDa),

-lactoglobulin (18.4), lysozyme (14.3), apro-
tinin (6.5), melittin (2.84), apamin (2.02), and brady-
kinin (1.06; Sigma) were used as reference proteins.
Bio-Rad low range molecular weight standards, phos-
phorylase B (106), BSA (81), ovalbumin (47.5), car-
bonic anhydrase (35.3), soybean trysin inhibitor
(28.2), and lysozyme (20.8), were used as molecular
weight markers. The molecular weights of the protein
bands were estimated with reference to molecular
weight markers and reference proteins (Sigma and
Bio-Rad).
High-Performance Liquid Chromatography. High-
performance liquid chromatography (HPLC) was
performed to conÞrm the presence of pure apamin
and melittin determined by SDS-PAGE to be present
in venom. Apamin and melittin (Sigma) were used as
standards. The contents of 20 venom sacs were placed
into 100
l distilled water, and the solution was chro-
matographed on a reverse-phase HPLC column
(AceIII C
18
, 12.5 cm by 4.0 mm i.d.; MAC-MOD An-
alytical; Chadds Ford, PA). The mobile phases were
(A) 0.1% TFA in acetonitrile:water (80:20) and (B)
0.1% TFA in water. Venom was separated by linear
gradient (5Ð 80%) using mobile phase A at 40 min. The
ßow-rate was maintained at 1.0 ml/min, and the elu-
tion was monitored at 280 nm.
Thin Layer Chromatography. Thin-layer chroma-
tography (TLC) was used to separate seratonin, do-
pamine, noradrenaline, and histamine (Sigma). 10 by
10 cm silica gel plates (generated from splitting pre-
parative 20 by 20 Silica gel 60 GF
254
into four pieces;
Merck, Damstadt, Germany) were used for the de-
velopment of samples. Each plate was loaded with 5
l
of venom and each standard solution and developed
with the following solvent systems: (A) ethanol-water-
ammonia, 100:12:16 (vol:vol); (B) methanol-chloroform,
1:1 (vol:vol) (Leonard 1972); and (C) trißuoracetic
acid-acetonitrile-
water, 1:95:5 (vol:vol). The plates were viewed under
UV light (254 nm) and photographed. Retardation
factor (R
f
) values were estimated.
Infrared Spectroscopy. The presence of peptides
and proteins determined by SDS-PAGE led us to ver-
ify this result by infrared spectroscopic analysis. For
infrared spectroscopy, 10
l venom solution was kept
on a watch glass at 30⬚C until its liquid phase (distilled
water and ethanol added for facilitating the evapora-
tion of water) was completely removed. The dry ma-
terial was homogeneously ground with potassium bro-
mide, and the infrared spectrum was shown.
The bands formed in TLC by reference noradren-
aline and the venom component that migrated at the
same position as standard noradrenaline were scraped
off, transferred into eppendorf tubes, and dissolved in
200
l ethanol. The solutions were centrifuged at
3,000 ⫻gfor 3 min to remove silica gel. The super-
natants were removed on to watch glasses and incu-
bated for 30 min at 30⬚C. After removing the liquid
phase (ethanol), the infrared spectrum was recorded
at room temperature and compared to verify the pres-
ence of noradrenaline. A Perkin-Elmer Spectrum
BX-II infrared spectrometer was used for all spectra
(Perkin-Elmer, BeaconsÞeld Buks, England).
Results and Discussion
Parasitoid venoms that cause paralysis of host spe-
cies are complex mixtures of low- and high-molecular
weight compounds such as amines, peptides, proteins,
and glycoproteins (Doury et al. 1997, Coudron et al.
2000). UV absorbance comparison of venom sac com-
ponents with standard protein solutions indicated an
average value of 0.04
g protein per venom sac. This
value is signiÞcantly lower than the average value of
180
g of protein per venom sac for P. hypochondriaca
(Retzius) (Parkinson and Weaver 1999). P. turionellae
females may use their venom for the purpose of re-
production and nutrition. The ability of a single female
wasp to paralyze a great number of host pupae to feed
or lay eggs indicates that a small fraction of the venom
sac contents may be sufÞcient to produce effective
paralysis in the host species.
SDS-PAGE proÞles indicated that P. turionellae
venom is composed of a highly complex mixture of
polypeptides (Fig. 1). Venom primarily consists of
components with molecular weights between 20 and
106 kDa (Fig. 1). However, there were also protein
bands higher or lower than this range. Studies have
revealed that social and some parasitoid Hymenoptera
venoms constitute low molecular weight proteins
(Leluk et al. 1989, Doury et al. 1997). Unlike the
venom of the other endoparasitoid species, Cotesia
776 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4
congregate (Say) (Beckage et al. 1987), Chelonus near
curvimaculatus (Jones and Leluk 1990), and Micro-
plitis demolitor Wilkinson (Strand et al. 1994), the
venom of P. turionellae contains several proteins
smaller than 20 kDa (Fig. 1). The venom of Eupelmus
orientalis (Crawford) has also been shown to contain
several proteins smaller than 15 kDa (Doury et al.
1997). These small peptides may generally be neuro-
toxins in higher Hymenoptera (Schmidt 1982), which
is consistent with the paralyzing function of P. turio-
nellae venom (Kõlõnc¸er 1975). Venom from an endo-
parasitoid wasp P. hypochondriaca was previously in-
vestigated, and proteins including phenoloxidases, a
laccase, and a serine protease were identiÞed (Par-
kinson et al. 2002b). Thus, several bands with molec-
ular weights around or higher than 106 kDa demon-
strate the presence of high molecular weight proteins
and enzymes in P. turionellae venom. Protein bands
with molecular weights ranging from 21 to 97 kDa have
been detected in venoms of other parasitic hym-
enopterans (Digilio et al. 2000, Parkinson et al. 2002a,
Nakamatsu and Tanaka 2003). The results of our SDS-
PAGE analysis are also in close agreement with those
reported by Parkinson et al. (2002b). Both P. turio-
nellae and P. hypochondriaca seem to contain a con-
siderably greater number of constituents in their ven-
oms than the venom from the egg parasitoid C. near
curvimaculatus (Jones and Leluk 1990) and the larval
endoparasitoid M. demolitor (Strand et al. 1994).
SDS-PAGE analysis (Fig. 1) indicated that venom
might contain melittin (2.84 kDa) and apamin (2.02
kDa). The presence of paralyzing proteins such as
apamin, melittin, and kinin in parasitoid wasp venom
has been suggested to occur in those species in which
oviposition occurs in an active host stage such as
P. turionellae (Leluk et al. 1989). However, we could
not detect the presence of bradykinin in venom.
Fig. 1. SDS-PAGE at 20% (AÐD), 14% (EÐG), and 12.5%
(HÐJ) (wt:vol) gel for standards (1 mg in 1 ml buffer
solution) and venom components (5
l venom solution).
(A)

-lactoglobulin (18.4), lysozyme (14.3), aprotinin (6.5);
(B, F, and I) venom; (C) apamin (2.02); (D) BSA (68);
(E and H) BSA (68), melittin (2.84), apamin (2.02), brady-
kinin (1.06); (G and J) Bio-Rad low range molecular weight
standards: phosphorylase B (106), BSA (81), ovalbumin
(47.5), carbonic anhydrase (35.3), soybean trysin inhibitor
(28.2), lysozyme (20.8).
Fig. 2. Fractionation of the venom sac content (4 venom sac equivalents) of P. turionellae and determination of the
presence of apamin and melittin (1
g/
l; Sigma) in venom sac content by reversed-phase HPLC using AceIII C
18
(12.5 cm
by 4.0 mm). Eluent A: 0.1% TFA in acetonitrile:water (80:20); eluent B: 0.1% TFA in water. Linear gradient: 5Ð80% A at 40
min. Injection volume: 20
l. Detection: 280 nm. Flow rate: 1.0 ml/min.
July 2004 UC¸KAN ET AL.: VENOM COMPONENT OF P. turionellae 777
HPLC analysis supported the presence of melittin and
apamin in the venom (Fig. 2).
Infrared spectrum of the venom is shown in Fig. 3,
and characteristic infrared bands are interpreted in
Table 1. The infrared absorption bands at 3,410, 1,648,
and 1,547 cm
⫺1
can be interpreted to indicate the
presence of secondary amine and amide groups and to
conÞrm the proteinous nature of the venom. How-
ever, the infrared band at 3,410 cm
⫺1
is dispropor-
tionally large and could be caused in part by traces of
ethanol. The vibration at 1,398 cm
⫺1
may be associated
with the acidic (carboxylic) nature of the venom
(Leonard 1972). The CÐH in-plane bending at
1,125 cm
⫺1
is characteristic of aromatic compounds
(Stuart 1997). The absorption band at 1,050 cm
⫺1
suggests that there may also be structures containing
phosphorous in the venom, possibly enzymes. The
CÐH bending at 618 cm
⫺1
is much more likely to be
oleÞnic C-H rock (characteristic of alkynes) (Stuart
1997). The venom sac components seem to lack a
carbohydrate moiety because there were no absorp-
tion bands at 3,600 (OH peak), 2,900 (CÐH stretch-
ing), and 1,700 (C⫽O stretching) cm
⫺1
, which are
basic characteristic infrared spectroscopic bands of
carbohydrates (Fig. 3; Table 1) (Fritz and Schenk
1979, Stuart 1997). Our results suggesting the acidic
and proteinous nature of the venom are in conformity
with those the investigation of Leonard (1972) from
sawßy larvae venom. Leluk et al. (1989) also con-
Þrmed the presence of acidic proteins in the venom of
an ichneumonid parasitoid, Chelonus sp. near curvi-
maculatus, with their isoelectrofocusing study.
The R
f
values from TLC analysis of venom and
standards on three different solvent systems are given
in Table 2. The best separation was achieved on trif-
luorasetic acid-acetonitrile-water (1:95:5 by vol.).
However, we could only detect the presence of nor-
adrenaline in P. turionellae venom (Fig. 4). The very
close R
f
values obtained for venom and noradrenaline
from three different solvent systems (Table 2) is a
Fig. 3. Infrared spectrum of P. turionellae venom (10
l venom solution).
Table 1. Characteristic infrared bands and their possible
assignments verifying the presence of peptides and proteins in
P. turionellae venom sac content
Frequency
(cm
⫺1
)Possible assignment
3410 N™H stretching, secondary amines
2361 CO
2
1648 C¢O stretching, amides
1547 N™H bending, secondary amines
1398 C™O™H in-plane bending, carbonyl compound
1125 C™H in-plane bending
1050 P™O™H bending
618 OleÞnic C™H rock
Ten microliters of venom solution (2.5 venom sac equivalents) was
used for infrared spectroscopic analysis.
Table 2. R
f
values from thin-layer chromatographic analysis of
venom and standard solutions developed with three different sol-
vent systems: ethanol-water-ammonia, 100:12:16 by vol; metha-
nol-chloroform, 1:1 (v/v); and trifluoracetic acid-acetonitrile-
water, 1:95:5 by vol
R
f
values
Development systems
Ethanol-water-
ammonia
Methanol-
chloroform
Trißuorasetic acid-
acetonitrile-water
Venom 0.77 0.79 0.66
Seratonin 0.59 0.63 0.70
Dopamine 0.71 0.72 0.64
Noradrenaline 0.78 0.78 0.66
Histamine 0.00 0.00 0.21
Each plate was loaded with 5
L of venom (1.25 venom sac equiv-
alents) and each standard solution.
778 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4
strong indication that noradrenaline is actually a com-
ponent of the venom. The widespread existence of
biogenic amines and catecholamines, which we used
as standards in our investigation, has also been shown
in the venom content of Apis and Vespa species (Owen
and Sloley 1988, Weisel-Eichler et al. 1999). Endo-
parasitic wasps, such as those from the genus Chelonus,
which parasitize host eggs, do not possess acutely toxic
venomous components (Leluk et al. 1989). However,
the signiÞcant quantity of noradrenaline detected by
TLC in our study indicates that noradrenaline is one
of the components of the pupal endoparasitoid
P. turionellae venom and therefore may be fairly ef-
fective in the paralysis of host pupae. Thus, analysis of
the parasitoid venom components may indicate the
correlation between the venom content of the wasp
and its host stage preference. P. turionellae can also
parasitize and lay eggs in larvae and prepupae of dif-
ferent host species (Kansu and Ug˘ur 1984). Therefore,
the presence of noradrenaline in venom is consistent
with the polyphagus nature of this wasp. It was also
previously reported that the paralysis of host pupae by
the parasitoid wasp could be explained by the pres-
ence of venomous contents inhibiting neuromuscular
transmission at the synaptic site (Kõlõnc¸er 1975). The
presence and functions of neurotoxic compounds in
parasitic wasp venom have also been investigated by
other studies (Visser et al. 1976, Skinner et al. 1990).
We also compared the infrared spectrum of stan-
dard noradrenaline and noradrenaline developed on
TLC (Fig. 4). Comparative infrared spectroscopic
analysis revealed that two spectra illustrated similar
peaks. The bands between 2,850 and 3,000 cm
⫺1
can
be attributed to the symmetric and asymmetric CÐH
stretching bands from aliphatic compounds like
noradrenaline. The strong bands observed near
2,360 cm
⫺1
might have resulted from atmospheric
absorption by CO
2
(Stuart 1997). The NH
2
bending
vibrations occurring near 1,600 cm
⫺1
indicate the al-
iphatic compound of noradrenaline (Stuart 1997). The
stretching and bending of CÐH and CÐHÐ0 between
1,100 and 1,400 cm
⫺1
can be attributed to the presence
of a benzene ring. The bands representing the out-
of-plane bending vibrations of substituted benzene
between 650 and 950 cm
⫺1
may also be an indicator of
a benzene ring in both compounds (Fig. 4) (Fritz and
Schenk 1979, Stuart 1997). The presence of noradren-
aline in wasp venom was consistent with the infrared
spectrum of noradrenaline standards. P. turionellae
venom has a wide range of proteins in molecular
weight and includes neurotoxic components such as
apamin, melittin, and noradrenaline. These observa-
tions are consistent with the polyphagus nature of
P. turionellae and thus the requirement for a venom
that has paralytic activity in several developmental
stages of a wide range of hosts.
Acknowledgments
We thank D. Rivers and two anonymous reviewers for
valuable comments and contributions on this manuscript.
This work was supported in part by the BAU ScientiÞc Re-
search Fund and Central Research Laboratory.
References Cited
Abreu, R.M.M., R.L.M. Silva de Moraes, and O. Malaspina.
2000. Histological aspects and protein content of Apis
mellifera L. worker venom glands: the effect of electrical
shocks in summer and winter. J. Venom Anim. Toxins. 6:
87Ð98.
Beckage, N. E., T. J. Templeton, B. D. Nielsen, D. I. Cook,
and D. B. Stoltz. 1987. Parasitism-induced hemolymph
polypeptides in Manduca sexta (L.) larvae parasitized by
the braconid wasp Cotesia congregate (Say). Insect
Biochem. 17: 439Ð455.
Fig. 4. Comparative infrared spectrum of standard noradrenaline (Sigma) and venom noradrenaline detected in TLC.
July 2004 UC¸KAN ET AL.: VENOM COMPONENT OF P. turionellae 779
Bradford, M. M. 1976. A rapid and sensitive method for the
quantiÞcation of microgram quantities of protein utilizing
the principles of protein-dye binding. Anal. Biochem. 72:
248Ð254.
Costa, H., and M. S. Pala. 2000. Agelotoxin: a phospholipase
A
2
from the venom of the neotropical social wasp cas-
sununga (Agelaio pallipes pallipes) (Hymenoptera-
Vespidae). Toxicon. 38: 1367Ð1379.
Coudron, T. A., and S. L. Brandt. 1996. Characteristics of a
developmental arrestant in the venom of the ectopara-
sitoid wasp Euplectrus comstockii. Toxicon. 34: 1431Ð1441.
Coudron, T. A., M.M.K. Wright, B. Puttler, S. L. Brandt, and
W. C. Rice. 2000. Effect of the ectoparasite Necremnus
breviramulus (Hymenoptera: Eulophidae) and its venom
on natural and factitious hosts. Ann. Entomol. Soc. Am.
93: 890Ð897.
Digilio, M. C., N. Isidoro, E. Tremblay, and F. Pennacchio.
2000. Host castration by Aphidius ervi venom proteins.
J. Insect Physiol. 46: 1041Ð1050.
Doury, G., Y. Bigot, and G. Periguet. 1997. Physiological
and biochemical analysis of factors in the female venom
gland and larval salivary secretions of the ectoparasitoid
wasp Eupelmus orientalis. J. Insect Physiol. 43: 69Ð81.
Fritz, J. S., and G. H. Schenk. 1979. Electronic absorption
spectra, ßuorescence and infrared spectroscopy, pp. 430 Ð
460. In J. S. Fritz and G. H. Schenk (eds.), Quantitative
analytical chemistry, Allyn and Bacon, Boston, MA.
Jones, D., and J. Leluk. 1990. Venom proteins of the endo-
parasitic wasp Chelonus near curvimaculatus: character-
ization of the major components. Arch. Insect Biochem.
Physiol. 13: 95Ð106.
Kansu, I
˙. A., and A. Ug˘ur. 1984. Pimpla turionellae (L.)
(Hym.-Ichneumonide) ile konukc¸usu bazõ lepidopter pu-
palarõ arasõndaki biyolojik ilis¸kiler u¨zerine aras¸tõrmalar.
Dog˘a Bilim Dergisi. 8: 160 Ð173.
Kılınc¸er, N. 1975. Untersuchungen u¨ber die ha¨mocyta¨re
Abwehrreaktion der puppe von Galleria mellonella L.
(Lep.) und u¨ber ihre Hemmung durch den puppen-
parasiten Pimpla turionellae L. (Hym; Ichneumonidae).
Z. Angew. Entomol. 78: 340Ð370.
Konno, K., M. Hisada, H. Naoki, Y. Itagaki, N. Kawai, A. Miwa,
T. Yasuhara, Y. Morimoto, and Y. Nakata. 2000. Struc-
ture and biological activities of eumenine mastoparan-
AF(EMP-AF), a new mast cell degranulating peptide in
the venom of the solitary wasp (Anterhynchium flavomar-
ginatum micado). Toxicon. 38: 1505Ð1515.
Laemmli, U. K. 1970. Cleavage of structual proteins during
the assembly of the head of bacteriophage T4. Nature
(Lond.). 227: 680Ð685.
Leluk, J., J. Scmidt, and D. Jones. 1989. Comparative studies
on the protein composition of hymenopteran venom res-
ervoirs. Toxicon. 27: 105Ð114.
Leonard, G. J. 1972. The isolation of a toxic factor from
sawßy (Lophyrotoma interrupta klug) larvae. Toxicon. 10:
597Ð603.
Nakamatsu, Y., Y. Gyotoku, and T. Tanaka. 2001. The en-
doparasitoid Cotesia kariyai (Ck) regulates the growth
and metabolic efÞciency of Pseudaletia separata larvae by
venom and Ck polydnavirus. J. Insect Physiol. 47: 573Ð584.
Nakamatsu, Y., and T. Tanaka. 2003. Venom of ectoparasi-
toid, Euplectrus sp. near plathypenae (Hymenoptera:
Eulophidae) regulates the physiological state of Pseuda-
letia separata (Lepidoptera: Noctuidae) host as a food
resource. J. Insect Physiol. 49: 149Ð159.
Osman, S. E. 1978. Die wirkung der sekrete der weiblichen
genitalanhangsdru¨sen von Pimpla turionellae L. (Hym.,
ichneumonidae) auf die hamocyten und die ein-
kapselungsreaktion von wirtspuppen. Z. Parasitenkd. 57:
89Ð100.
Owen, M. D., and B. D. Sloley. 1988. 5-Hydroxytriptamine
in the venom of the honey bee (Apis mellifera): variation
with season and with insect age. Toxicon. 26: 577Ð584.
Parkinson, N. M., and R. J. Weaver. 1999. Noxious compo-
nents of venom from pupa-speciÞc parasitoid Pimpla
hypocondriaca. J. Invertebr. Pathol. 73: 74Ð83.
Parkinson, N., C. Conyers, and I. Smith. 2002a. A venom
protein from the endoparasitoid wasp Pimpla hypo-
chondriaca is similar to snake venom reprolysin-type met-
alloproteases. J. Invertebr. Pathol. 79: 129Ð131.
Parkinson, N., E. H. Richards, C. Conyers, I. Smith, and J. P.
Edwards. 2002b. Analysis of venom consitituents from
the parasitoid wasp Pimpla hypochondriaca and cloning of
cDNA encoding a venom protein. Insect Biochem. Mol.
Biol. 32: 729Ð735.
Rivers, D. B., M. M. Rocco, and A. R. Frayha. 2002. Venom
from the ectoparasitic wasp Nasonia vitripennis increases
Na
⫹
inßux and activates phospholipase C and phospho-
lipase A
2
dependent signal transduction pathways in cul-
tured insect cells. Toxicon. 40: 9Ð21.
Sanchez, F., M. Blanca, A. Miranda, M. J. Carmona, J. Garcia,
J. Fernandez, M. J. Tarres, M. C. Rondon, and C. Juarez.
1994. Comparison of Vespula germanica venoms ob-
tained from different sources. Int. Arch. Allergy Immu-
nol. 104: 385Ð389.
Schmidt, J. O. 1982. Biochemistry of insect venoms. Annu.
Rev. Entomol. 27: 339Ð368.
Shagger, H., and G. Von Jagow. 1987. Tricine-sodium do-
decyl sulfate-polyacrylamide gel electrophoresis for the
separation of proteins in the range from 1 to 100 kDa.
Anal. Biochem. 166: 368Ð379.
Skinner, W. S., P. A. Dennis, and G. B. Quistad. 1990. Partial
characterization of toxins from Goniozus legneri
(Hymenoptera: Bethylidae). J. Econ. Entomol. 83: 733Ð
736.
Soldatova, L. N., R. Crameri, M. Grachl, D. M. Kemeny,
M. Schmidt, M. Weber, and U. R. Mueller. 1998. Supe-
rior biologic activity of the recombinant bee venom al-
lergen hyaluronidase. J. Allergy Clin. Immunol. 101: 651Ð
698.
Strand, M. R., J. A. Johnson, T. Noda, and B. A. Dover. 1994.
Development and partial characterization of monoclonal
antibodies to venom of the parasitoid Microplitis
demolitor. Arch. Insect Biochem. Physiol. 26: 123Ð136.
Stuart, B. 1997. Biological applications of infrared spectros-
copy. John Wiley & Sons, New York.
Uc¸kan, F. 1999. The morphology of the venom apparatus
and histology of venom gland of Pimpla turionellae
(Hym.; Ichneumonidae) females. Tr. J. Zool. 23: 461Ð 466.
Uc¸kan, F., and A. Gu¨lel. 1990. Endoparazitoit Pimpla
turionellae (Hymenoptera: Ichneumonidae) dis¸ilerinde
zehir aparatõnõn yapõsõ ve zehirin bas¸lõca kimyasal grubu-
nun tayini. X. Ulusal Biyoloji Kongresi, Erzurum.
Visser, B. J., W. Spanjer, H. De Klonia, T. Piek, C. Van Der
Meer, and A.C.M. Van Der Drift. 1976. Isolation and
some biochemical properties of a paralysing toxin from
the venom of the wasp Microbracon hebetor (Say).
Toxicon. 14: 357Ð370.
Weisel-Eichler, A., G. Haspel, and F. Libersat. 1999. Venom
of a parasitoid wasp induces prolonged grooming in the
cockroach. J. Exp. Biol. 202: 957Ð964.
Wharton, R. A. 1993. Bionomics of the braconidae. Annu.
Rev. Entomol. 38: 121Ð143.
Received 15 April 2003; accepted 2 December 2003.
780 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 4
