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The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca HymenopteraIchneumonidae involves Ca+2-dependent cell death pathways.

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A r t i c l e
THE MODE OF ACTION
OF VENOM FROM THE
ENDOPARASITIC WASP
Pimpla hypochondriaca
(HYMENOPTERA:
ICHNEUMONIDAE) INVOLVES
CA12-DEPENDENT CELL DEATH
PATHWAYS
David B. Rivers
Department of Biology, Loyola College in Maryland, Baltimore,
Maryland
M. Paulina Dani and Elaine H. Richards
Central Science Laboratory, Sand Hutton, York, United Kingdom
The endoparasitoid Pimpla hypochondriaca injects venom during
oviposition to condition its lepidopteran hosts. Venom is a complex
mixture of proteins and polypeptides, many of which have been identified
as enzymes, including phenoloxidase, endopeptidase, aminopeptidase,
hydrolase, and angiotensin-converting enzyme. Constituents of the
venom have been shown to possess cytolytic and paralytic activity, but the
modes of action of factor(s) responsible for exerting such effects have not
been deciphered. In this study, we examined the mode of action of isolated
venom using cultured cells (BTI-TN-5B1-4). A series of blockage and
inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti-calreticulin
antibodies. Monolayers exposed to venom alone were highly susceptible
with more than 84.672.3% dead within 15 min. Susceptible cells
Grant sponsors: Loyola College (Baltimore, MD); Pesticide Safety Directorate (Defra, UK).
Abbreviations: AM, acetoxymethyl ester; DPBS, Dulbecco’s phosphate buffered saline; ER, endoplasmic
reticulum; FBS, fetal bovine serum; LC99, lethal concentration needed to kill 100% of population;
DCm, mitochondrial membrane potential; PO, phenoloxidase; PTU, phenylthiourea
Correspondence to: Dr. David B. Rivers, Department of Biology, Loyola College in Maryland, 4501 N.
Charles Street, Baltimore, MD 21210. E-mail: drivers@loyola.edu
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 71, No. 3, 173–190 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20314
174
Archives of Insect Biochemistry and Physiology, July 2009
displayed a retraction of cytoplasmic extensions, rounding, and swelling
prior to lysis in more than half (55.771.7%) of the dying cells. Within
15 min of exposure to venom, cells displayed qualitative increases in
[Ca12]i as evidenced by staining with the calcium-sensitive probe fluo-4
AM, and mitochondrial membrane potential (DCm ) was undetectable by
5 min post-treatment with venom. These venom-mediated changes
occurred regardless of whether an external source of calcium was present,
or whether venom was pre-treated with PTU. In contrast, venom toxicity
was attenuated by treatment with anti-calreticulin antibodies. Not only
did fewer cells die when exposed to antibody-treated venom but also cell
swelling diminished and no increases in intracellular calcium were
detected. A possible mode of action for the venom is discussed.
& 2009 Wiley Periodicals, Inc.
Keywords: parasitoid; intracellular calcium; apoptosis; oncosis; venom
protein
INTRODUCTION
Parasitic wasps that invade the body cavity of their hosts face an onslaught of challenges
to survive. Following oviposition by endoparasitic species, the eggs and resulting larvae
become potential targets of the host immune system, which can mount cellular and
humoral defense responses aimed at eliminating and/or killing the invading parasitoids
(Beckage, 1998; Strand and Pech, 1995). Hemocytic encapsulation followed by
phenoloxidase-induced intoxication will be the final demise of a parasitic organism
entering the hemocoel of most arthropods, barring some form of protection.
Endoparasitic wasps do not leave their eggs defenseless, as most combat the host’s
internal environment by injecting fluids derived from the reproductive system
(e.g., venom and calyx fluid) of the adult female at the time eggs are deposited into
the host hemocoel (Beckage, 1998; Shelby and Webb, 1999). Within the fluids are an
array of regulatory factors, which often include viruses, virus-like particles, proteins,
and peptides that function to help subdue and condition the host for the successful
development of wasp progeny (Asgari et al., 2003; Ergin et al., 2006; Moreau and
Guillot, 2005; Parkinson and Weaver, 1999; Strand and Pech, 1995; Zhang et al., 2006).
Regardless of the source or type of regulatory factor, the effect on the host usually
is exerted in the induction of paralysis (temporary or permanent), suppression of host
immune responses, alterations in physiology, biochemistry, and behavior, and/or
initiation of developmental arrest in the host insect (Beard, 1963; Coudron, 1991;
Rivers et al., 2002; Strand and Pech, 1995; Whitfield and Asgari, 2003). Depending on
the wasp species, multiple regulatory agents may be needed to work synergistically or
additively to manipulate the host, or, as is the case with some parasitoids, only venom is
required. Despite the considerable attention given to deciphering the roles of venoms,
viruses, and other agents in host-endoparasitoid systems, only a rudimentary
understanding of the modes of action or molecular target sites is available. Thus,
how the regulatory factors induce changes in the host is not well known and is in need
of further investigation.
Pimpla hypochondriaca Retzius (Hymenoptera: Ichneumonidae) is a solitary pupal
endoparasitoid that relies on venom intoxication to subdue its lepidopteran hosts. The
Archives of Insect Biochemistry and Physiology
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175
wasp has a well-developed venom system, and the venom itself appears to be a
complex mixture of proteins and peptides (Parkinson et al., 2003, 2004; Richards and
Dani, 2008). Among the venom proteins are several enzymes, including phenoloxidase(s) (Parkinson and Weaver, 1999), endopeptidase, aminopeptidase, hydrolase,
and angiotensin-converting enzyme (Dani et al., 2003, 2005), as well as a low molecular
weight, heat-stable inhibitor of phenoloxidase (Parkinson and Weaver, 1999).
Sequence analyses of a cDNA library prepared from venom glands of P. hypochondriaca
suggest that a serine protease, a reprolysin-like metalloprotease, and trehalase are
also present in venom (Parkinson et al., 2002a,b, 2003). Functionally, venom
is immunosuppressive toward submissive hosts, inducing an inhibition of host
hemocyte aggregation/recruitment and a suppression of the encapsulation response
(Richards and Parkinson, 2000; Parkinson et al., 2002a; Richards and Dani, 2008).
Venom constituents have been shown to possess cytolytic and paralytic activity
(Parkinson et al., 2002a; Parkinson and Weaver, 1999; Richards and Parkinson, 2000),
both in vivo and in vitro, and isolated venom displays weak antimicrobial activity (Dani
et al., 2003).
Venom of P. hypochondriaca is the best characterized of all parasitic wasp venoms
analyzed to date in terms of composition and identification of venom genes (Parkinson
et al., 2002a,b, 2003, 2004; Richards and Dani, 2008). However, information is lacking
on how any of the proteins operate at the cellular level or the interactions that occur
between venom proteins and receptors on target tissues. Recent observations
(Richards and Dani, 2007) suggest that crude venom triggers multiple signaling
pathways that can result in cytolysis by an oncotic mechanism, or alternatively cells
display morphological characteristics of apoptosis. The precise factors determining
which death pathways are manipulated by venom have not been fully deciphered, but
recent observations suggest that cell fate following venom exposure may be linked to
venom phenoloxidase activity (Richards and Dani, unpublished data). A key role for
this enzyme in host manipulation has also been suggested for the ectoparasitoid
Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae), a wasp that also appears to
trigger multiple death pathways following envenomation, including both oncotic and
apoptotic mechanisms (Rivers and Brogan, 2008). Rivers and Brogan (2008) have
argued that venom from N. vitripennis contains a calreticulin-like protein that
appears to stimulate mobilization of intracellular calcium, and that the mode of
action of calreticulin is tied to the activity of venom phenoloxidase. A calreticulin-like
protein has also been identified in venom of the endoparasitoid Cotesia rubecula
(Marshall) (Hymenoptera: Braconidae), but the function of the protein has been
speculated to be associated with a disruption of hemocyte adhesion and not to interfere
with host phenoloxidase activity (Zhang et al., 2006). In this study, we examined
the mode of action of isolated venom from P. hypochondriaca using an in vitro
approach with cultured cells (BTI-TN-5B1-4) from Trichoplusia ni Hübner
(Lepidoptera: Noctuidae). To determine if venom from this wasp elicits cell death in a
manner similar to that of N. vitripennis venom, a series of blockage and inhibition assays
were performed using a potent inhibitor (phenylthiourea, PTU) of venom
phenoloxidase and anti-calreticulin antibodies (generated against calreticulin isolated
from Galleria mellonella L. [Lepidoptera: Pyralidae]). Measurements of intracellular
calcium levels were also performed to determine whether this endoparasitoid’s venom
stimulates mobilization of intracellular calcium and/or increases influx from
extracellular sources. A possible mode of action involving both phenoloxidase and
calreticulin is discussed.
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MATERIALS AND METHODS
Insect Rearing
A laboratory colony of P. hypochondriaca was maintained on pupae of the tomato moth,
Lacanobia oleracea L. (Lepidoptera: Noctuidae) as previously described (Richards and
Parkinson, 2000). The host was reared under long-day conditions (201C, LD 16:8 h) at 65%
relative humidity on an artificial diet according to the methods of Corbitt et al. (1996).
Isolation of Venom
Venom was collected from P. hypochondriaca as described previously (Parkinson and
Weaver, 1999). Venom sacs were dissected from adult females, rinsed in sterile
Dulbecco’s phosphate buffered saline (DPBS), and then transferred to a microcentrifuge tube containing DPBS. The venom sacs were ruptured using forceps and the
tissue debris discarded. Samples were then homogenized by passing through pipette
tips of decreasing bore sizes, then centrifuged at 10,000g for 1 min at room
temperature, and the resulting supernatant lyophilized. Venom was reconstituted in
DPBS before use and stored at 701C.
Cell Culture
Cells (BTI-TN-5B1-4, also called High FiveTM) derived from minced embryos of T. ni
(Davies et al., 1993) were purchased from Invitrogen (San Diego, CA) and grown in
TC-100 insect media (Sigma Chemical Co. St. Louis, MO) containing 10% fetal bovine
serum (FBS) (Sigma) at 271C.
Venom Assays
BTI-TN-5B1-4 cells were counted with a hemacytometer and seeded (2 103 cells/
well) into 96-well plates (Falcon) with 100 ml TC-100 containing 10% FBS. Cells were
grown at 271C for 2–3 days to confluency. Cell monolayers were washed with PBS
(pH 7.4) by removing spent culture media, adding 100 ml PBS, and then gently rocking
the plate for 10–20 sec before discarding the buffer. After the wash, 100 ml TC-100 with
10% FBS was added to each well. Cell viability was assessed with trypan blue dye
(Sigma) exclusion staining (final concentration was 0.04%) as previously described
(Rivers et al., 1993). Changes in cell morphology were observed using a Spot Insight
Firewire color digital camera (Diagnostic Instruments) mounted on a phase contrast
inverted microscope (Nikon Eclipse TE-300) and connected to a Macintosh Power PC
G5 computer (Apple). Changes in cell shape and membrane integrity were determined
following the criteria of Himeno et al. (1985) and Trump and Berezesky (1995).
In a parallel set of experiments, venom assays were repeated using media (Hank’s
balanced salt solution lacking calcium, Sigma) without calcium. Changes in cell
morphology and viability were assessed as described above.
Effects of Phenoloxidase on Venom Toxicity
Venom from P. hypochondriaca has been shown to possess phenoloxidase (PO) activity
(Parkinson and Weaver, 1999) and three genes coding for pro-phenoloxidase-related
proteins have been isolated from a cDNA library constructed from venom glands
(Parkinson et al., 2001). Despite the presence of PO in venom, what role(s), if any, the
Archives of Insect Biochemistry and Physiology
Mode of Action of Venom from P. hypochondriaca
177
enzyme plays in the intoxication pathway is not known. In an attempt to decipher PO’s
potential involvement in the induction of venom-mediated cell death, venom assays
were performed using BTI-TN-5B1-4 cells. Venom PO was blocked by incubating
crude venom with 10 mM phenylthiourea (PTU) for 1 h at 271C. Observations of
changes in cell morphology and viability were made at 0, 0.5, 1, 2, 3, and 24 h.
Effects of Calreticulin on Venom Toxicity
Recent observations (Richards and Dani, unpublished data) suggest that crude venom
triggers multiple signaling pathways. Venom phenoloxidase triggers cytotoxicity in
cultured insect cells but not the cytolysis that has been observed with some cell types
(Parkinson and Weaver, 1999), which indicates that some other venom constituent(s)
stimulates one or more other death pathways. To investigate the possibility that venom
from P. hypochondriaca manipulates similar pathways as venom from N. vitripennis,
venom was pre-treated with antibodies (polyclonals) generated against total
calreticulin protein isolated from Galleria mellonella. Polyclonal antiserum
(1:1,000–1:50,000) was incubated with venom (LC99 dose 5 0.12 mg/ ml) for 1 h at
271C prior to being added to confluent cell monolayers in 96-well plates. Observations
of changes in cell morphology and viability were made at 0, 0.5, 1, 2, 3, and 24 h.
Measurement of Intracellular Ca12 and Mitochondrial Membrane Potential (DWm )
Qualitative changes in cytosolic free Ca12 were measured using the Ca12-sensitive
probe fluo-4 acetoxymethyl (AM) esterTM (Invitrogen) as described previously (Rivers
et al., 2005). Confluent monolayers of BTI-TN-5B1-4 cells grown on glass cover slips
were exposed to crude wasp venom pre-treated with PTU or anti-calreticulin, and
then incubated at 271C. Following the incubation period, the media was removed and
100 ml fresh TC-100 containing 10% FBS and 5 mM fluo-4 AM was added. Monolayers
were then placed in the dark for 30–45 min at 271C. The cells were fixed in 4%
phosphate-buffered (PBS) formalin, washed 2 with 50 mM phosphate-buffered
saline (pH 7.4), and then 10 ml TC-100 with 10% FBS added to the cover slip.
Excitation of fluo-4 was accomplished using DAPI/FITC excitation filters (DAPI
excitation at 400–433 nm and FITC excitation at 478–500 nm) and emitted light
monitored at 535 nm. Cell-derived fluorescent images were visualized using a
100 /1.30 oil objective and a Spot Insight Firewire color digital camera mounted
on a phase contrast inverted microscope (Nikon Eclipse TE-300) and connected
to Macintosh Power PC G5 (Apple). Images were captured with Spot software
(v. 4.5; Sterling Heights, MI) and stored for subsequent analysis using IPLab software
(v. 3.2; Fairfax, VA).
Changes in mitochondrial membrane potential (DCm) induced by wasp venom
were monitored using the fluorescent dye rhodamine 123 as previously described
(Rivers et al., 2005). Excitation of rhodamine 123 (5 mM in 50 mM PBS, pH 7.4) was
achieved using a rhodamine excitation filter (excitation at 507 nm) with an emission
maximum of 560 nm. Images were captured and analyzed as detailed above for
calcium fluorescence.
Statistical Analyses
Means were compared using one- and two-way analysis of variance (ANOVA) and
Student Newman-Keul’s multiple comparisons tests using StatView statistical software
(v. 5.01, a 5 0.05). Percentage data was arcsine transformed prior to analysis.
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RESULTS
Venom Toxicity
When BTI-TN-5B1-4 cells were grown in TC-100 media supplemented with FBS, the
cells formed a confluent monolayer within 2–3 days at 271C. Individual cells attached
to the surface of the culture plates and formed cytoplasmic extensions (pseudopodia)
that gave the cells a bipolar or fibroblast-like morphology. Nearly all of the cells that
were untreated or incubated with venom isolation buffer (referred to as saline)
remained viable (96.771.9 and 95.372.2 [X7SEM] % for untreated and salinetreated cells, respectively; F 5 0.28, P40.05) at all time points tested for up to 24 h as
shown by trypan blue dye exclusion staining and image analysis (Fig. 1). All of these
cells also maintained the fibroblast-like or bipolar morphology, with the exception of
cells that underwent mitosis: pre-mitotic cells retracted the pseudopodia and rounded
up but remained viable (Fig. 2A–C).
In contrast to untreated or saline-treated cells, monolayers exposed to venom
from P. hypochondriaca were highly susceptible to the wasp venom as more than
84.672.3% (n 5 12,365 cells) of the cells died by 15 min post-treatment (Fig. 1).
Despite this rapid death of the majority of cells, an additional 2–3-h incubation period
was required to induce death in the remaining venom-treated cells (Fig. 1).
Morphologically, venom-treated cells began to retract cytoplasmic extensions within
10–15 min after exposure to an LC99 dose of venom (Fig. 2D), cells became round, and
more than half (55.771.7%, n 5 20,046 cells) became swollen by 30 min posttreatment (Fig. 2D, E). Nuclei of these cells were large, distinct, and appeared centrally
positioned, even in cells that appeared to be dying or were dead (Fig. 2D, F). By
30 min, more than 5273.1% (n 5 9,349 cells) of the venom-treated cells appeared to
100
Cell viability (X ± SEM) %
80
60
Untreated
Saline
Venom
40
20
0
0
50
100
150
200
Time (minutes)
Figure 1. Cytotoxicity of venom from Pimpla hypochondriaca toward monolayers of BTI-TN-5B1-4 cells.
Archives of Insect Biochemistry and Physiology
Mode of Action of Venom from P. hypochondriaca
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Figure 2. Morphological changes in cell monolayers (BTI-TN-5B1-4) exposed to (A–C) saline or (D–F)
venom from Pimpla hypochondriaca. Observations were made at (A, D) 15, (B, E) 30, and (C, F) 60 min. Images
were made using a 40 objective. Scale bar 5 5.0 mm.
have died due to lysis (Fig. 2E), and by 60 min, 71.972.4% (n 5 13,906 cells) had lysed
(Fig. 2F). The remaining dead cells died by a non-lytic means.
Venom-Mediated Changes in [Ca12]i and Mitochondrial Membrane Potential
In order to determine if the venom-mediated changes in cell morphology and viability
were linked to a disruption of calcium homeostasis, qualitative changes in free
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Figure 3. Changes in intracellular calcium levels in cell monolayers (BTI-TN-5B1-4) following exposure to
(A, C) saline or (B, D) venom from Pimpla hypochondriaca at (A, B) 15 min and (C, D) 60 min post-treatment.
Images were made using a 40 objective. Scale bar 5 6.0 mm.
intracellular calcium levels were monitored by staining with the fluorescent probe fluo4 AM. Within 15 min after exposure to wasp venom, BTI-TN-5B1-4 cells displayed
qualitative increases in cytosolic calcium concentrations by comparison to salinetreated cells (Fig. 3A,B). This incubation period appeared to represent the peak in
[Ca12]i increases as the intensity of the fluorescent signal did not rise with longer
incubation times, and by 60 min post-treatment, calcium fluorescence in venomtreated cells was dramatically reduced in comparison to earlier time points (Fig. 3D).
To investigate the possible relationship between Ca12 rearrangement and
mitochondria (Petit et al., 1996; Rivers et al., 2005), cell monolayers were stained
with rhodamine 123, a dye whose fluorescent signal intensity correlates with
membrane potential of mitochondria. Cells stained with this dye 5 min after incubation
with venom demonstrated a nearly undetectable fluorescent signal, indicative of a very
low mitochondrial membrane potential (DCm). In fact, the fluorescent signal was so
low that no fluorescence was observed in captured images in venom-treated cells at any
time point examined throughout the 24 h after treatment (data not shown). In
contrast, untreated and saline-treated cells stained with rhodamine 123 displayed a
strong signal at all time points examined (data not shown).
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Effects of Extracellular Calcium on Venom Toxicity
Increases in intracellular calcium in cells exposed to venom can be the result of
enhanced influx from extracellular sources. To examine the possibility that venom
from P. hypochondriaca increases uptake of calcium, cell monolayers were incubated in
calcium-free media (Hank’s balanced salts solution) with an LC99 dose of wasp venom.
For comparisons, cells were maintained in insect culture media (TC-100) containing
calcium with venom. Regardless of whether an extracellular source of calcium was
present, BTI-TN-5B1-4 cells were highly susceptible to venom from P. hypochondriaca
(Fig. 4). In fact, no significant differences (F3, 36,781 5 1.56, P40.05) in cell viability
were detected at any time point between cells incubated in calcium-free versus
calcium-containing media (Fig. 4). By contrast, nearly all untreated and saline-treated
cells were viable throughout all time points tested.
Venom-treated cells in either type of media displayed the typical retraction of
cytoplasmic extensions, rounding, and swelling characteristic of cells susceptible to the
cytotoxic action of this wasp venom (Fig. 5C, D). Despite the similarities, far more cells
(75.673.1%, n 5 8,452 cells) appeared swollen, earlier in the intoxication pathway
when incubated in media containing calcium than those in calcium-free media
(51.772.2%, n 5 9,905 cells) (Fig. 5C, D). Cells incubated with saline or that were
untreated (data not shown) maintained the bipolar or fibroblast-like appearance
typical of adherent, spread cells, and did not display morphological differences when
incubated in either type of media (Fig. 5A, B).
When venom-treated cells were incubated with the calcium-sensitive probe fluo-4
AM, increased fluorescence was detected in the cytosol of cells cultured in calciumcontaining media within 15 min (Fig. 6C). However, by 60 min after treatment,
the intensity of the fluorescent signal was greatly diminished. In contrast, fewer
100
Cell viability (X ± SEM) %
80
60
Untreated
Saline
Venom-w/calcium media
Venom-calcium-free media
40
20
0
0
50
100
150
200
Time (minutes)
Figure 4. Cytotoxocity of venom from Pimpla hypochondriaca toward BTI-TN-5B1-4 cells in the presence or
absence of extracellular calcium.
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Figure 5. Morphological changes in cell monolayers (BTI-TN-5B1-4) exposed to (A, B) saline or (C, D)
venom from Pimpla hypochondriaca, in media (A, C) containing extracellular calcium, or media (B, D) lacking
an extracellular calcium source. Observations were made at 15 min post-treatment. SC 5 swollen cells;
CP 5 cytoplasmic extensions. Images were made using a 40 objective. Scale bar 5 3.0 mm.
venom-treated cells in calcium-free media displayed increases in cytosolic [Ca12]i by
15 min after exposure to wasp venom (Fig. 6D). As with cells in calcium-containing
media, fluorescence was reduced to almost undetectable levels 60 min after treatment
of cells incubated in calcium-free media with venom. Saline-treated cells remained
relatively unchanged in terms of the intensity of the fluorescent signal for the duration
of the 3-h test period, regardless of whether extracellular calcium was present (data
not shown).
Effects of Phenoloxidase and Calreticulin on Venom Toxicity
Venom from P. hypochondriaca is known to possess PO activity, which appears to be
attributed to one or more proteins in the venom (Parkinson et al., 2002a; Parkinson
and Weaver, 1999). As expected (Parkinson and Weaver, 1999), pre-treatment of
venom with the potent PO inhibitor PTU did not substantially reduce venom toxicity
toward BTI-TN-5B1-4 cells (Fig. 7). Fewer cells died (F3, 25,670 5 72.9, Po0.01) when
venom was treated with PTU than with crude venom alone. However, the effect of the
enzyme inhibitor was modest as most cells rounded, swelled, and died by lysis
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Figure 6. Changes in intracellular calcium in cell monolayers (BTI-TN-5B1-4) exposed to (A, B) saline or
(C, D) venom from Pimpla hypochondriaca, in media (A, C) containing extracellular calcium, or media (B, D)
lacking an extracellular calcium source. Observations were made at 15 min post-treatment. Images were
made using a 40 objective. Scale bar 5 5.5 mm.
(compare Fig. 8C with B). The remaining cells that died did so by a mechanism
independent of cytolysis.
In a parallel set of experiments, venom was pre-treated with anti-calreticulin
antibodies prior to exposure to cell monolayers. This experiment tested the hypothesis
that crude venom induces changes in intracellular calcium through the action of
calreticulin. If correct, pre-treatment of venom with antibodies should reduce or
completely attenuate cytotoxic activity toward BTI-TN-5B1-4 cells. To investigate this
possibility, filtered polyclonal antiserum (1:1,000–1:50,000) was incubated with venom
for 1 h at 271C prior to being added to confluent cell monolayers in 96-well plates.
Preliminary observations revealed that antibody titers higher than 1:10,000 were
necessary to reduce venom activity toward cell monolayers, and titers Z1:3,000
yielded maximum inhibition of venom toxicity. Therefore, all further assays used a
1:3,000 antibody titer. When isolated crude venom was pre-treated with anticalreticulin polyclonal antibodies for 1 h before exposure to T. ni cells, cell viability
was far higher (F13, 39.872 5 123.4, Po0.01) than when cells were exposed to crude
venom alone or venom pre-treated with PTU (Fig. 7). Few of these cells displayed any
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100
Cell viabilityX( ± SEM) %
80
60
40
Saline
Anti-cal
PTU
Venom
Venom + PTU
Venom + Anti-cal
20
0
0
50
100
150
200
Time (minutes)
Figure 7. Cytotoxicity of venom from Pimpla hypochondriaca toward BTI-TN-5B1-4 cells pre-treated for 1 h
with either phenylthiourea (PTU, 10 mM) or anti-calreticulin antibodies (1:3,000) prior to addition to cells.
evidence of the swelling of plasma membranes that was typical of the cytotoxicity
associated with venom alone or PTU-treated venom (Fig. 8B–D). Cells that did die
when exposed to antibody-treated venom did not lyse and displayed blebs along the
outside of the plasma membranes. Most of the dying cells also formed vacuoles
throughout the cytosol and sometimes nucleus prior to death (Fig. 8D), a feature
rarely observed in cells incubated with crude venom alone or venom pre-treated with
PTU (Fig. 8B, C).
Measurements of intracellular calcium in cells treated with venom alone, or venom
incubated with either PTU or anti-calreticulin antibodies were performed using the
calcium fluorescent probe fluo-4 AM. Venom alone and venom pre-treated with PTU
induced increases in intracellular calcium within 15 min of treatment (Fig. 9B, C),
although PTU-treated venom evoked only modest changes in cytosolic [Ca12]i (Fig. 9C)
by comparison to venom alone (Fig. 9B). In contrast, exposure of cells to venom
pre-treated with antibody or saline alone did not produce elevations in intracellular
calcium levels as evidenced by the intensity of the fluorescent signal (Fig. 9A, D).
DISCUSSION
During parasitic attack of lepidopteran hosts, the solitary endoparasitic wasp
P. hypochondriaca injects venom into host pupae as part of the adult female’s oviposition
program (Fitton et al., 1988; Parkinson and Weaver, 1999). The wasp lacks
polydnaviruses or virus-like particles, so conditioning of the host depends exclusively
on the action of the complex, proteinaceous venom (Parkinson et al., 2004). Venom is
composed of numerous enzymes and other proteins and polypeptides that either
alone or through protein–protein interactions exert their effect through paralytic and
cytotoxic pathways (Dani et al., 2003, 2005; Parkinson and Weaver, 1999), culminating
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Figure 8. Morphological changes in cell monolayers (BTI-TN-5B1-4) exposed to (A) saline, (B) venom
from Pimpla hypochondriaca, (C) wasp venom pre-treated for 1 h with phenylthiourea (10 mM), or (D) wasp
venom pre-treated for 1 h with anti-calreticulin antibodies (1:3,000). Observations were made at 15 min posttreatment. Images were made using a 40 objective. Scale bar 5 4.3 mm.
in a range of host changes. Much of our work has been done using hemocytes obtained
from L. oleracea larvae. Although this is a host species, it is not the usual stage of host
(this would be L. oleracea pupae; getting sufficient numbers of hemocytes from the
pupae for large numbers of complex assays is extremely difficult). However, the host
disturbances are the same, including immunosuppression, altered hemocyte activity,
and eventual host death (Dani et al., 2004; Richards and Parkinson, 2000; Richards
and Dani, 2007). How venom elicits these alterations has not been determined, but it
does appear that at least two pathways are manipulated to induce death: one that
involves the constitutively active venom PO (Parkinson and Weaver, 1999) and another
that triggers cytotoxicity independent of lysis (Richards and Dani, 2007) and that may
depend on the action of calreticulin.
The anti-hemocytic and immunosuppressive properties of venom from
P. hypochondriaca appear to be independent of active venom PO (Parkinson et al.,
2001; Richards and Parkinson, 2000). Richards and Dani (2007) have suggested that
venom evokes at least one death pathway that leads to cytotoxicity by an apoptotic
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Archives of Insect Biochemistry and Physiology, July 2009
Figure 9. Changes in intracellular calcium in cell monolayers (BTI-TN-5B1-4) exposed to (A) saline, (B)
venom from Pimpla hypochondriaca, (C) wasp venom pre-treated for 1 h with phenylthiourea (10 mM), or (D)
wasp venom pre-treated for 1 h with anti-calreticulin antibodies (1:3,000). Observations were made at 15 min
post-treatment. Images were made using a 40 objective. Scale bar 5 5.6 mm.
mechanism. In cultured hemocytes from L. oleracea, exposure to sufficiently high doses
of venom induces blebbing, some vacuole formation, and a loss of cell volume prior to
death (Richards and Dani, 2007). Staining of these cells with FITC-labelled phallodin
demonstrated that the cytoskeleton collapsed around the nucleus, but that nondiscriminate disintegration of the cell scaffolding did not occur (Richards and Dani,
2007). Further staining of DNA fragments and plasma membrane phosphatidyl serine
from venom-treated hemocytes (using Frag-EL and annexin-V assays, respectively)
revealed that apoptosis appeared to be the mechanism of cell death, at least for some of
the cells, but this was dependent on the dose of venom used and length of exposure
(Richards and Dani, 2007). Consistent with these findings were observations in this
study that vacuole and bleb formation occurred when crude venom was pre-treated
with polyclonal anti-calreticulin antibodies prior to incubation with T. ni cells, but not
when venom was treated with PTU. Antibody treatment of venom also inhibited the
swelling of susceptible cells that was evident with crude venom alone or venom
pre-treated with PTU. Collectively, these observations argue that venom from
P. hypochondriaca triggers apoptotic pathways leading to cell death in some cell types.
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Mode of Action of Venom from P. hypochondriaca
187
Surprisingly, however, this study argues that active PO in venom triggers apoptosis in
cultured insect cells, whereas in previous studies using primary cultured hemocytes,
apoptotic death appeared independent of venom PO (Richards and Dani, 2007).
Clearly, further study is needed to clear up these seemingly contradictory observations
and to determine whether venom-induced apoptosis is tissue or cell type–specific.
A second pathway leading to cell death elicited by wasp venom was also apparent.
When venom was pre-treated with the PO inhibitor PTU, susceptible cells swelled and
lysed. In contrast, when anti-calreticulin antibodies were used to pre-treat venom,
swelling and lysis were abolished, as were increases in venom-induced intracellular
calcium as detected by fluorescent microscopy. Cellular swelling followed by cytolysis is
typical of an oncotic lytic mechanism of cell death (Manjo and Joris, 1995; Trump and
Berezesky, 1995). This mechanism relies on an osmotic imbalance between the cytosol
and extracellular environment, which would be expected if venom disrupted
membrane integrity via pore formation or opening of existing channels. In this
scenario, increased calcium influx from extracellular fluids through ‘‘leaky’’ plasma
membranes could potentially explain the collapse of the cytoskeleton in hemocytes
exposed to venom (Richards and Dani, 2007) since cytoskeletal-digesting enzymes are
commonly activated by increased [Ca12]i (Gomperts et al., 2002; Troyer et al., 1986).
However, when venom assays were performed in media lacking calcium, the same type
of venom-induced alterations were observed, including increases in free Ca12 levels,
thus suggesting that elevations in cytosolic [Ca12]i were due to mobilization from
intracellular stores (Rivers et al., 2005). This oncotic pathway may also be responsible
for the flaccid paralysis that results from injection of venom into natural and factitious
hosts (Parkinson and Weaver, 1999), since swelling and lysis at neuromuscular
junctions has a paralytic effect in many insect species (Zdarek et al., 1987).
A calreticulin-like protein appears to be the constituent in venom from
P. hypochondriaca that is responsible for modifying cellular calcium homeostasis based
on the functional assays performed in this study. Calreticulin is a ubiquitous Ca12binding protein that modulates calcium levels in both endoplasmic reticulum (ER) and
mitochondria, and, hence, once in the intracellular environment, this protein could
conceivably stimulate the venom-induced mobilization of intracellular calcium, which
in turn would trigger numerous cellular changes including movement of the
cytoskeletal filaments, swelling, and death by oncosis and apoptosis (Michalak et al.,
1999). For these series of events to occur, calreticulin would need to gain entry into
susceptible cells since the protein cannot diffuse across plasma membranes (Gomperts
et al., 2002). Clearly an avenue for entry is possible since venom triggers cellular
swelling, but it most likely does not involve venom PO aiding in transport across the
cellular membranes as previously argued for N. vitripennis (Rivers and Brogan, 2008)
since PO inhibitors do not abolish venom toxicity. Work is presently underway to
isolate and characterize the venom protein(s) responsible for activating the death
pathway associated with swelling and lysis.
The possible intracellular storage site of calcium release is most likely
mitochondria. In this study, mitochondrial membrane potential was reduced to
undetectable levels within 5 min after venom treatment, an event that would eliminate
the electrical gradient necessary for ATP synthesis and eventually allow unregulated
calcium efflux from mitochondria into the cytosol (Petit et al., 1996). Involvement of
mitochondria does not preclude the possibility that venom stimulates, either directly
or indirectly, calcium release from other storage sites. The main intracellular calcium
store is the ER, but the mitochondria are very efficient at taking up and releasing Ca12,
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Archives of Insect Biochemistry and Physiology, July 2009
and are generally positioned in the cytosol near the ER to help coordinate calcium
signals and modulate calcium homeostasis across both organelles. To achieve
coordinated control over Ca12 levels and movement within the cell, mitochondria
and ER depend on transport and storage proteins to maintain tight regulation of Ca12
concentrations within their lumens. Calreticulin serves as one of the major proteins
regulating Ca12 in both organelles and has been shown to act as a signal that can
trigger cell death via both apoptotic and oncotic mechanisms (Arnaudeau et al., 2002;
Petit et al., 1996). Consequently, there are several lines of circumstantial evidence that
point to a key role for a calreticulin-like or similar calcium modulating protein in the
venom intoxication pathway leading to one or more mechanisms of cell death.
Additional studies are needed to determine what role, if any, mitochondria and/or
calreticulin have in the cell death induced by venom from P. hypochondria.
ACKNOWLEDGMENTS
The authors thank Dr. Bok Luel Lee (College of Pharmacy, Pusan National University,
Pusan, South Korea) for kindly providing polyclonal anti-calreticulin antibodies from
G. mellonella. We also thank Loyola College in Maryland for a faculty development
grant to D.B.R, and the Pesticide Safety Directorate (Defra, UK) for funding to E.H.R.
and P.D.
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