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Disruption of pupariation and eclosion behavior in the flesh fly Sarcophaga bullata Parker DipteraSarcophagidae by venom from the ectoparasitic wasp Nasonia vitripennis Walker HymenopteraPteromalidae.

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Rivers et al.
Archives of Insect Biochemistry and Physiology 57:78–91 (2004)
Disruption of Pupariation and Eclosion Behavior
in the Flesh Fly, Sarcophaga bullata Parker
(Diptera: Sarcophagidae), by Venom From the
Ectoparasitic Wasp Nasonia vitripennis (Walker)
(Hymenoptera: Pteromalidae)
David B. Rivers,1* Jan Zdarek,2 and David L. Denlinger3
The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned
muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected
into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but
puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in
venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom
caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom
posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone’s role in stimulating the release of anterior retraction factor and
puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected
into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior
were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in
extrication and post-eclosion behavior. Arch. Insect Biochem. Physiol. 57:78–91, 2004. © 2004 Wiley-Liss, Inc.
KEYWORDS: ectoparasitoid; wasp venom; mode of action; ecdysone; fly development
Non-aculeate parasitic wasps are a tremendously
diverse group, with over 600,000 described species and an estimated 400,000 more yet to be discovered (Whitfield, 1998). The venoms of these
Department of Biology, Loyola College in Maryland, Baltimore
Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Prague, Czech Republic
Department of Entomology, Ohio State University, Columbus
parasitic wasps are known to have a wide range of
effects on their hosts, including arrestment of development, alterations in growth and physiology,
suppression of immune responses, paralysis, and
death (Jervis and Copeland, 1996; Quicke, 1997).
Such biological and physiological diversity suggests
Abbreviations used: CNS = central nervous system; EtOH = ethanol; 20E = 20-hydroxyecdysone; LC = lethal concentration; LT = lethal time; VRE =
venom reservoir equivalent.
Contract grant sponsor: Czech Granting Agency; Contract grant number: 522/01/0501; Contract grant sponsor: USDA-NRI; Contract grant number: 98-353026659; Contract grant sponsor: USDA-NRICGP; Contract grant number: 2001-1005; Contract grant sponsor: Loyola College.
*Correspondence to: Dr. David Rivers, Department of Biology, Loyola College in Maryland, 4501 North Charles Street, Baltimore, MD 21210.
Received 14 January 2004; Accepted 12 May 2004
© 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.20015
Published online in Wiley InterScience (
Archives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
that these venoms contain a wealth of compounds
with unique chemistries and modes of action
(Jones and Coudron, 1993). Due primarily to limitations imposed by the small size of most parasitic wasps, research investigating the insecticidal
properties of these venoms has been limited.
Several species of wasps belonging to the family Pteromalidae produce venoms with high potency toward an array of pest insects. The most
intensively studied member of this family, Nasonia
vitripennis (Walker), has an elaborate venom system (Ratcliffe and King, 1967, 1969) that is central to successful parasitism (Rivers et al., 1999b).
A fly accepted as a suitable host is always injected
with venom prior to oviposition (Dawei and
Dingxi, 1987), with posterior body regions usually serving as the sites for the sting and egg deposition (Rivers, 1996). The venom inhibits host
cellular immune responses within 30 min after envenomation (Rivers et al, 2002), suppresses respiratory metabolism within a matter of hours (6–8
h) (Rivers and Denlinger, 1994a), elevates hemolymph and fat body lipid titers (Rivers and Denlinger, 1995; Rivers et al., 1998), and ultimately
induces death. In muscoid flies, N. vitripennis
venom can retard adult fly mobility within 1–2 h
when injected artificially, and the flies will succumb
to death in less than 24 h (Beard, 1964; Rivers et
al., 1993). The venom has been shown to be highly
active toward several life stages (wandering larvae,
pupae, pharate adults, adults) of flies from at least
4 families (Muscidae, Drosophilidae, Calliphoridae,
and Sarcophagidae) (Rivers et al., 1993). Additionally, in vitro assays using cultured insect cells have
revealed high dipteran specificity in terms of LT50s
and LC50s but virtually no mammalian cell sensitivity (Rivers et al., 1999a,b). Thus, venom from
N. vitripennis appears to be a rich source of toxins
displaying specificity toward dipterans.
The precise host tissue targeted by the wasp
venom and its mode of action has not been fully
revealed and is the focus of this study. Tensiometric recordings of pupariation and eclosion behavior of the flesh fly, Sarcophaga bullata Parker
(Diptera: Sarcophagidae) have been used previously as tools for monitoring immediate and protracted effects of several drugs, venoms, and
October 2004
insecticides (Zdarek and Fraenkel, 1987). An
eclosing flesh fly performs stereotypic extrication
behavior as long as it is confined in the puparium
or in the substrate (soil) in which the larva
pupariated (Reid et al., 1987a,b). This stereotypic
behavior can be monitored barographically or
tensiometrically (Zdarek et al., 1986; Zdarek and
Denlinger, 1992). The techniques can also be exploited for monitoring the effects of various neurotropic agents (drugs, poisons, insecticides) on
insect behavior (Zdarek and Denlinger, 1992). The
latency period, lethal time, and type of action can
be deduced from the records. In the present study,
we used these techniques for the study of the effects of crude venom from N. vitripennis.
Pupariation behavior is also a stereotypic activity that can be recorded barographically (Zdarek,
1985) and tensiometrically (Zdarek and Denlinger,
1991). As with eclosion, the barographic and tensiometric techniques can serve as tools for monitoring the pharmacological effects of noxious
compounds (Zdarek et al., 1987). Venom from N.
vitripennis has been shown to delay the onset of
pupariation in S. bullata and disrupt puparial morphology (Rivers et al., 1993). Thus, we employed
the barographic technique to probe the nature of
the irregular pupariation behavior.
We also explored the possibility that the venominduced delay in pupariation was a consequence
of a deficiency in ecdysteroids, the insect growth
hormone that is essential for puparium formation.
Venom from N. vitripennis has been shown previously to suppress development of nondiapausing
pupae and pharate adults of S. bullata in a manner consistent with an ecdysteroid deficiency, although, surprisingly, exogenous injections of
20-hydroxyecdysone (20E) did not restore fly development (Rivers and Denlinger, 1994a). Here we
show that 20E can rescue some elements of pupariation in venom-injected larvae, but the effect is
dose- and age-dependent.
Insect Rearing
N. vitripennis was maintained as a laboratory
colony on pupae and pharate adults of S. bullata
Rivers et al.
as previously described (Rivers and Denlinger,
1994a). Adults and larvae were reared under a LD
15:9 h light-dark cycle at 25°C. Twenty to thirty
females (3–7 days after emergence from host puparia) were placed in a Petri dish (15 × 100 mm)
with 50–75 nondiapausing pupae (4 days after
pupariation) of S. bullata and a 50% (v/v) honey
solution. After 24 h, the adult wasps were removed
and parasitized pupae maintained at 25°C, LD 15:9
h. Under these conditions, N. vitripennis develops
from egg to adult (eclosion) in 12 days.
A colony of S. bullata was reared according to
Denlinger (1972). Larvae and adults were fed pork
liver throughout development at 25°C with a lightdark cycle of LD 15:9 h. To synchronize fly development for assessing host age, third instar larvae
that had begun to wander from food (but prior to
crop emptying) were collected and placed in a
vented glass jar (1 liter) with 1–2 ml tap water.
Larvae were held under these conditions for 3 days
at 25°C with frequent (3–5 times/d) water changes.
This “wet” treatment temporarily inhibits the release of ecdysteroids until the larvae are placed in
dry conditions, thereby synchronizing pupariation
(Ohtaki, 1966).
Isolation of Crude Wasp Venom
Crude venom from N. vitripennis was isolated
from host-fed females in phosphate-isolation
buffer [10 mM sodium phosphate (pH 8.0), 0.9%
(w/v) NaCl, 15% (w/v) sucrose, 1 mM ethylenediamineteraacetic acid, and 1 mM phenylmethylsulfonyl fluoride] (Rivers et al., 1993) and stored
frozen at –70°C. Venom activity was confirmed in
vitro and in vivo with BTI-TN-5B1-4 cells and
young pharate adults (5 days after pupariation at
25°C) of S. bullata, respectively, as described previously (Rivers et al., 1993).
Venom Injection
Wandering larvae of S. bullata were injected with
venom in the pre-red spiracle (6 h prior to pupariation at 25°C), early-red spiracle (2 h prior to
pupariation) or late-red spiracle (30–60 min prior
to pupariation) stage of development. Larvae were
immobilized on ice for 5-10 min prior to injections, and injections were accomplished by means
of finely-drawn glass capillaries (Rivers et al.,
1993). Volumes of injected solutions ranged from
1–6 µl.
Newly emerged imagoes (≤5 min after emergence) were immobilized briefly (2–3 min) on ice
and then injected (1 µl/fly) with crude venom lateral to the dorsal midline of the abdomen using
finely pulled capillaries. Larvae and imagoes injected with phosphate isolation buffer served as
Effects on Puparium Morphology and Behavior
The effects of crude wasp venom on pupariation
morphology and behavior were investigated as described (Zdarek et al., 1987). The behavior of
venom-injected larvae following recovery from being placed on ice prior to injections (termed immediate effects) until the completion of tanning
was monitored continuously at 25°C. The size and
shape of puparia were also recorded with the following morphological features being scored (according to the criteria of Zdarek et al., 1987): (1)
ability to retract anterior segments (r), (2) ability
to contract longitudinally to a barrel shape (c), (3)
smoothing of the cuticle (s), and sclerotization and
tanning of puparia (t).
Effects on Denervated Musculature
To test the importance of the central nervous
system (CNS) on venom-induced changes in
pupariation behavior, pre-red spiracle or early-red
spiracle larvae were immobilized on ice and then
tightly ligated behind the fused CNS (Zdarek et
al., 1987). This procedure disconnected all nerves
leading from the fused CNS to the abdomen
(Zdarek and Fraenkel, 1972). Larvae were then removed from ice and after the anterior segments
resumed activity, crude venom was injected into
posterior body regions as described above. Ligated
larvae were then kept at 25°C, LD 15:9 and observations of cuticular tanning made at 24-h intervals for 4 days.
Archives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
Injection of 20-Hydroxyecdysone
Preliminary observations revealed an age-dependent inhibition of puparial tanning that suggested
a possible venom-evoked ecdysteroid deficiency. To
examine whether development in venom-injected
larvae could be rescued by exogenous ecdysteroids,
pre-red spiracle larvae were injected with 20hydroxyecdysone (20E) [5 µg/larva in 10% (w/v)
EtOH] using finely pulled glass capillaries. Since
the effect of wasp venom on ecdysteroid responsiveness was likely also time-dependent, fly larvae were injected with 20E at different time
intervals (0–120 min) post-venom injection. Larvae injected with 10% EtOH (1 µl/larva) were
used as controls. All larvae were maintained at
25°C, LD 15:9 h following injections and checked
at 24-h intervals for the puparium morphological
features described above.
siometric transducer (Grass Instruments, Quincy,
MA) into signal outputs that were amplified (Universal Precision Amplifier CA 110, Peckel Instruments, Rotterdam, The Netherlands) and recorded
(Line Recorder LZ 4620, Laboratorni pristroje,
Prague, Czech Republic).
To record the behavioral patterns characteristic
of obstacle removal (Zdarek et al., 1986), a puparium containing a pharate adult just about ready
to emerge was inserted into a plastic 1-ml pipette
tip and positioned in front of the force transducer.
Mechanical resistance of the sensor prevented the
imago from extricating itself from the puparium,
and a cotton plug positioned behind (posterior)
the puparium prevented backward movement.
Comparisons were made between saline-injected
and venom-injected imagoes. Injections were made
into the dorsal surface of the thorax using finely
drawn capillaries.
Effects on Hemolymph Pressure During Pupariation
Effects on Post-Eclosion Behavior
A barographic method of recording hemocoelic
pressure changes that reflect muscular activity and
cuticular transformation (Zdarek et al., 1979) was
used to continuously monitor hemolymph pressure in red spiracle stage (1–2 h prior to pupariation) larvae of S. bullata. Larvae were immobilized
on ice for 5–10 min and then injected with crude
wasp venom [1–6 venom reservoir equivalents
(VRE)/larva: 1 VRE = LC99 dose for larvae and pupae of S. bullata (Rivers et al., 1993)]. Immediately
following injection, the immobilized larvae were
catheterized with a hypodermic needle (23-G) connected to the hydraulic system of a pressure transducer. Three pressure records (barograms) were
made for each venom dose tested. Comparisons
were made to untreated and saline-injected larvae.
All recordings were performed at room temperature.
Effects on Extrication Behavior
Extrication behavior was studied using the tensiometric technique described previously (Zdarek
et al, 1986; Zdarek and Denlinger, 1992). Movement of the ptilinum was transformed by a tenOctober 2004
Flies ready to emerge from puparia were prepared as described by Zdarek et al. (1984). Immediately after emergence, adult flies were injected
with either saline (1 µl/fly) or crude venom (1 or
3 VRE/fly) from N. vitripennis and then placed in
Petri dishes (15 × 100 mm) lined with filter paper.
The onset and duration of several aspects of posteclosion behavior were monitored continuously at
room temperature and included: (1) walking (W),
(2) abdominal expansion (AE), (3) wing expansion (WE), (4) wing smoothing (WS), (5) tanning
(t), and (6) grooming (G) (Zdarek et al., 1984;
Zdarek and Denlinger, 1987). Post-eclosion behaviors were followed until adult flies resumed walking behavior (W2).
Statistical Analyses
Means were compared using 1-way and 2-way
analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons tests (alpha =
0.05; Sokal and Rohlf, 1969). Percentage data were
arcsine transformed prior to analysis. Data analyses were performed using StatView statistical software (v. 5.0.1, Abacus, Berkeley, CA).
Rivers et al.
Puparium Morphology and Behavior
Saline injection into larvae of S. bullata (regardless of age tested) had no obvious effects on the
onset of pupariation or puparium morphology
(Fig. 1A–C). In contrast, crude venom from N.
vitripennis induced a dose-dependent delay or inhibition of pupariation: the onset of pupariation in
larvae injected with 1 VRE crude venom was delayed by 1–2 days at 25°C, whereas higher concentrations (3 VRE) completely inhibited pupariation
in a high proportion of larvae injected in either the
pre-red spiracle (87.5%, n = 24) or early-red spiracle (49.9%, n = 27) stage of development (Table
1). The timing of pupariation initiation in late-red
spiracle larvae and the number of larvae that attempted to pupariate did not appear disrupted by
crude venom, regardless of the dose tested.
Larvae that were delayed in pupariation did
eventually pupariate but they did not form normal puparia. Puparial shape ranged from larva-like
to elongated and partially folded intersegmentally
(Fig. 1E,F). None of the puparia formed by venomtreated larvae properly contracted nor were they
smooth (Fig. 1D–F).
Pupariation behavior is characterized by a stereotypic chain of events that include gradual immobilization, retraction of anterior segments,
contraction longitudinally to a barrel shape, smoothing of the surface due to shrinkage of the cuticle,
and sclerotization and tanning (Zdarek et al.,
1986). Injection of saline into S. bullata in different stages of larval development did not alter these
normal elements of pupariation behavior (Table
1, Fig. 2). The same was not true for larvae injected
with crude venom as several aspects of pupariation
behavior were observed to be altered or absent entirely, and larval age did influence the effects
evoked by venom on pupariation behavior (Table
1). At low doses of venom (1 VRE/larva), most larvae tanned, but all behavioral events preceding sclerotization and tanning were altered in a manner
dependent on larval age (Table 1). Likewise, high
doses (3 VRE/larva) of venom suppressed or abol-
ished all behavioral events preceding sclerotization
and tanning. Only larvae injected in the late-red
spiracle stage showed tanning comparable to saline-injected flies (Table 1).
Pupariation behavior was also monitored by
continuous measurements of changes in hemocoelic pressure as pupariation proceeded. The entire activity appeared on a barogram as a unique
series of pressure pulses that reflect specific muscle
movements over time within different body segments. The muscle movements, and hence pressure pulses, are specific for the different stages of
the stereotypic behavior (Fig. 2A). Barographic
records revealed that all elements of pupariation
behavior of venom-injected larvae (early-red spiracle) were present, but were performed earlier in
relation to the onset of tanning in comparison to
saline-injected flies (Fig. 2). The immobilization
period that precedes puparial contractions was also
extended in duration (Fig. 2B). Puparial contractions in venom-injected larvae were patterned regularly, but the intensity of muscular efforts was
subdued and tanning was greatly delayed (Fig. 2B).
Denervated Musculature
To test the importance of the CNS for venominduced changes in pupariation behavior, pre-red
spiracle larvae were tightly ligated behind the fused
CNS and then injected with wasp venom (1 VRE)
in the posterior body half. Cuticular changes were
monitored for 4 days after ligations. All larvae
tanned in front of the ligation within 48 h. However, tanning posterior to the ligature did not occur in any larva ligated during the pre-red spiracle
Fig. 1. Morphogenetic effects of saline and crude venom
from Nasonia vitripennis on puparium morphology in 3
developmental stages of Sarcophaga bullata. Normal puparium formation occurred in S. bullata following saline
injection (1 µl/larva) in (A) pre-red spiracle, (B) early-red
spiracle, and (C) late-red spiracle larvae. Age-dependent
effects on puparium formation were observed in larvae
injected with crude wasp venom (1 VRE/larva = 1.35 µg
venom protein/larva): (D) pre-red spiracle, (E) early-red spiracle, and (F) late-red spiracle larvae. Thirty larvae were injected per treatment using pulled glass capillaries.
Archives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
October 2004
Figure 1.
Rivers et al.
TABLE 1. Venom-Induced Changes in Pupariation Behavior of Third Instar Larvae of Sarcophaga bullata*
Percent response (X ± SEM)
Experimental group
1 VRE/larva
3 VRE/larva
Immediate effects
Retraction of
anterior segments (r)
Contraction to
barrel shape (c)
Smoothing (s)
Sclerotization and
tanning (t)
96.7 ± 0.01a
96.7 ± 0.01a
96.7 ± 0.01a
96.7 ± 0.01a
13.0 ± 4.5b
64.5 ± 4.6c
13.3 ± 1.2b
8.7 ± 2.5c
55.6 ± 3.7d
13.3 ± 1.2b
98.5 ± 2.3a
91.3 ± 1.7ab
7.4 ± 0.8e
64.3 ± 1.5c
7.1 ± 2.6c
12.5 ± 2.4c
50.1 ± 4.2d
94.8 ± 3.8ab
*Wandering 3rd instar larvae of Sarcophaga bullata were injected lateral to the dorsal midline in the posterior 1/3 of the body with crude wasp venom (either1 VRE- or 3
VRE/larva) from Nasonia vitripennis using pulled glass capillaries (5 µl). Fly larvae were wet treated for 48 h to synchronize development for injections at 3 developmental stages: pre-red spiracle (6 h prior to pupariation at 25°C), early-red spiracle (2 h prior to pupariation), and late-red spiracle (30–60 min prior to pupariation). Injected
larvae were allowed to pupariate at room temperature, being monitored continuously until the end of tanning. Larvae injected with saline (1 µl/larva) and untreated larvae
served as controls. Percentage data was arcsine transformed prior to statistical analysis. Values in the same column followed by the same letter do not differ significantly
from each other at P = 0.05.
stage regardless of treatment (Fig. 3). When the
same experiment was repeated using early-red spiracle larvae, cuticular tanning was evident in front
and behind the ligature by 96 h for all larvae except those injected with crude venom (Fig. 3). In
those larvae, tanning never occurred and eventually (1–2 weeks) the posterior body regions slowly
became necrotic and desiccated.
Hormone Injections
At high doses (3 VRE/larva), wasp venom triggered an age-dependent inhibition of puparial tanning (Table 1) that suggested a possible ecdysteroid
deficiency. To examine whether tanning, as well as
other aspects of pupariation, in venom-injected larvae could be rescued by exogenous ecdysteroids,
pre-red spiracle larvae were injected with 20E at
different time intervals post-venom injection. Larvae treated with saline-only or saline and 20E displayed the typical events of pupariation with nearly
all becoming properly sclerotized and tanned
(Table 2). A similar trend was not evident for prered spiracle larvae injected with crude venom only,
as none of the flies (n = 55) retracted anterior segments, contracted longitudinally, or tanned (Table
2). Hormone treatment did restore some aspects
of pupariation in venom-injected larvae in a timedependent manner, but the responses were not as
fully expressed as in control flies. For example,
though a high percentage of venom-injected larvae tanned following injection of 20E (up to 60
min post-venom injection, Table 2), the cuticle was
not as intensely tanned as with control flies, tanning was incomplete or patchy over the surface of
puparia, and in many cases the cuticle remained
soft. By 15-min post-venom injection, 20E was not
capable of rescuing longitudinal contraction, and
by 2 h, >90% of the venom-treated larvae were entirely unresponsive to 20E (Table 2).
Extrication Behavior
In saline-injected flies, extrication behavior occurred with the same frequency and amplitude of
contractions for several hours (Fig. 4A) and gradually disappeared only after 11 h (n= 3). Imagoes
injected with 1 VRE of crude wasp venom at the
time of emergence performed regular extrication
behavior for 7.0 h (n = 5). Typically, only the last
hour of behavioral performance was less regular,
showing at first a decreased frequency of muscular
contractions before their rather sudden disappearance. The effects of venom on extrication behavior
Archives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
Fig. 2. Barographic recordings of pupariating larvae (red
spiracle stage) of Sarcophaga bullata that were injected (A)
with isolation buffer (1µl/larva) or (B) crude wasp venom
(1 VRE/larva) using pulled capillaries. T: beginning of tanning. The X-axis indicates time after recovery from immo-
bilization on ice (in minutes). Letters represent specific
pupariation events: i, immobilization of the larva; r, retraction of the anterior segments; s, shrinkage of cuticle;
t, tanning of the cuticle.
became much more pronounced with higher
(pharmacological) doses. For example, flies that
received 3 VRE performed extrication efforts for
only 1.2 h (n = 5) and typically the cycles were
progressively slower with decreasing amplitude of
pressure peaks (Fig. 4D). A further depression of
muscular activity was detected when 4 VRE were
injected (Fig. 4C) and 6 VRE completely immobilized adult flies within 15 min (Fig. 4B).
then followed by expansion behavior in the newlyeclosed imagoes. This latter set of behaviors (posteclosion or expansion) is necessary to inflate the
imago body for attainment of the final size and
shape of the adult. Untreated or saline-injected,
newly eclosed imagoes walked (W) rapidly for a
short period of time (Table 3), and then stopped
and remained stationary, unless disturbed, for the
remainder of the expansion period before resuming walking (W2) 133–137 min later (Table 3).
During the stationary period, flies pumped air into
the gut, elevating internal pressure to facilitate abdominal expansion followed shortly (9–11 min)
by wing expansion (Table 3). When the wings were
Post-Eclosion Behavior
The initial phase of eclosion in higher Diptera
involves extrication from the puparium, which is
October 2004
Rivers et al.
Fig. 3. Importance of the CNS for
the venom-induced changes in pupariation behavior of third instar larvae of Sarcophaga bullata. The ability
to tan in posterior body regions was
monitored in pre-red spiracle and
early-red spiracle 3rd instar larvae of
S. bullata that were ligated and then
injected either with saline (1 µl/larva)
or crude wasp venom (1 VRE/larva).
Ligations were performed at midbody (behind the CNS). Cuticular
tanning was assessed at 24, 48, and
96 h after manipulations. Twenty larvae were used for each treatment.
completely spread, the fly used the hind legs, alternating between the left and right leg, to smooth
the wings. Tanning of the adult cuticle occurred
approximately 57–60 min after the onset of expansion behavior, and was followed by grooming
of the entire adult body before walking resumed
(Table 3).
Low doses (1 VRE) of venom did not alter any
of the expansion activities prior to the initiation
of tanning, and induced only slight delays in the
induction of tanning, grooming, and resumption
of walking behavior in imagoes (Table 3). Higher
concentrations, however, disrupted all elements of
expansion behavior as flies injected with 3 VRE required significantly more time than controls before initiating each activity (Table 3). Despite these
venom-induced disturbances, all flies, regardless of
venom dose tested, were able to complete all aspects of post-eclosion behavior.
The mechanism of action of most parasitic wasp
venoms has not been studied sufficiently to reveal
the details of which tissues are targeted in the host
and how those tissues are injured to alter normal
functions (Piek and Spanjier, 1986; Quicke, 1997).
Venom from N. vitripennis has been the focus of
several recent studies (Rivers et al., 1999a,b, 2002)
that have examined the intoxication pathways associated with cell death using an in vitro approach
(Rivers et al., 1999a). In cultured cells, crude
venom from this ectoparasitic wasp appears to
evoke cell death by a colloid osmotic lytic mechaArchives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
TABLE 2. Injection of 20-Hydroxyecdysone Into Pre-Red Spiracle Larvae of Sarcophaga Bullata
Following Venom Injection*
Percent response (X ± SEM)
Retraction of
anterior segments (r)
Contraction to
barrel shape (c)
and tanning (t)
Saline + 20-E
Time of injection (min)
Venom + 20-E
Time of injection (min)
96.7 ± 3.5a
95.8 ± 2.3a
95.0 ± 1.4a
91.9 ± 2.5a
95.1 ± 1.4a
91.9 ± 2.5a
95.0 ± 1.4a
93.6 ± 3.1a
80.7 ± 4.1c
51.3 ± 3.6d
30.4 ± 1.6e
40.0 ± 2.7f
78.1 ± 1.1c
80.1 ± 2.2c
80.0 ± 3.4c
62.3 ± 1.4d
9.5 ± 2.0e
*Pre-red spiracle 3rd instar larvae of S. bullata were injected with either saline (µl/larva) or crude wasp venom
(1 VRE/larva) from Nasonia vitripennis. Larvae were then injected with 20-E (5 µg/larva) at various time intervals following saline or venom injection in an attempt to rescue development from the effects of venom. All
injections were performed lateral to the dorsal midline in posterior body regions using a 5-µl Hamilton syringe.
Treated larvae were kept at 25°C, L15:D9, and monitored at 24 h-intervals for 5 days. Percentage data was
arcsine transformed prior to analysis by ANOVA and Student-Newman-Keul’s multiple comparisons tests. Values in the same column followed by the same letter do not differ significantly from each other at P = 0.05.
nism (Knowles and Ellar, 1987) that involves the
release of intracellular calcium stores via the activation of phospholipase C and subsequent phosphoinositol turnover (Rivers et al., 2002). While
these investigations have provided details of the
venom’s action at the cellular level, it is unclear
whether the same venom effects are triggered in
the intact insect. Complicating efforts to decipher
this issue is a lack of understanding of which tissues are directly targeted by venom from N.
vitripennis and difficulty in working with a fly pupa
(and pharate adults), the stage of host development parasitized by the wasp. The pupal and
pharate adult stages develop within a puparium,
the walls of which result from the cuticle of the
last larval instar. During formation of the puparium, a high hemocoelic pressure is established
that results from longitudinal contraction of the
body and shrinkage of the cuticle, events that are
essential for proper morphology of the puparium
(Zdarek et al, 1979).
In this study, neuropharmacological evaluation
of crude wasp venom relied on the pupariation asOctober 2004
say described by Zdarek et al. (1987). During
pupariation, the fly larva performs specific temporal and spatial patterned movements of two muscular systems: (1) the segmental and intersegmental
somatic musculature, and (2) the retractors of the
anterior prespiracular segments. Longitudinal contraction of the body during pupariation is under
the control of the muscles of system 1 whereas system 2 causes withdrawal of the frontal segments
and eversion of the anterior spiracles (Zdarek et
al., 1987). The combined actions of these two
muscle systems comprise a stereotypic pupariation
behavior (Zdarek et al., 1979) that, if altered by
noxious compounds, can yield information about
specific target sites and receptors. The use of this
evaluation approach in the present study revealed
that venom from N. vitripennis delays or inhibits
the ability of fly larvae to contract into barrelshaped puparia and subsequently tan, but these
effects are dependent on venom concentration and
larval age. It has been hypothesized that formation of a normal-shaped puparium is dependent
on patterned muscular contractions during the
Rivers et al.
Fig. 4. Tensiometric recordings of ptilinum movements
of eclosing imagoes of Sarcophaga bullata after injection
of (A) isolation buffer (1 µl), (B) 6, (C) 4, and (D) 3
venom reservoir equivalents of crude wasp venom from
Nasonia vitripennis at the beginning of extrication behavior. The vertical bar indicates 0.5-mm displacement of the
tensiometric sensor and is a measure of ptilinum movement during extrication.
contraction phase of pupariation (Zdarek et al.,
1979) and that if the patterns are disrupted, distorted puparia are formed (Zdarek et al., 1987).
If true, then the morphological and behavioral effects evoked by venom from N. vitripennis would
be the direct result of disruption of the patterned
muscular contractions of peripheral neuromuscular that are under the control of muscle system 1.
This system is, in turn, under the control of the
motor neurons of the CNS. There are several lines
of evidence that suggest that venom alters pupariation both via the CNS and through somatic
1. Barograms of venom-injected larvae showed
that all the normal elements of pupariation behavior were present, but the timing of events
had been altered in relation to the onset of tanning, a feature consistent with modification of
temporal organization of central motor programs (Zdarek et al., 1987);
2. Ligated larvae did not respond to venom injection into posterior body regions, suggesting no
direct stimulatory effects on muscles or neuromuscular junctions (Zdarek et al., 1987; Zdarek
and Denlinger, 1992);
3. Injection of exogenous ecdysteroid could restore
Archives of Insect Biochemistry and Physiology
Action of Venom From N. vitripennis
TABLE 3. Quantitative Aspects of Post-Eclosion Behavior in Venom Injected Adults of Sarcophaga bullata
Time distribution (X min ± SEM)
Experimental group
Walking (W)
Expansion (AE)
Expansion (WE)
Smoothing (WS)
Tanning (T)
Grooming (G)
Walking (W2)
13.7 ± 2.1a
22.2 ± 3.4a
33.4 ± 2.6a
40.5 ± 3.6a
59.4 ± 0.9a
110.8 ± 3.1a
137.5 ± 2.7a
15.0 ± 1.9a
25.6 ± 2.1a
34.6 ± 3.0a
36.9 ± 2.8a
57.8 ± 1.2a
106.4 ± 3.5a
133.6 ± 2.6a
1 VRE/fly
14.2 ± 2.4a
17.9 ± 3.4a
30.8 ± 1.1a
34.6 ± 2.9a
66.3 ± 1.5b
120.1 ± 2.8b
150.4 ± 3.9b
3 VRE/fly
25.0 ± 3.2b
30.5 ± 1.6b
55.0 ± 0.7b
55.9 ± 0.6b
58.8 ± 2.0a
127.9 ± 2.8c
147.5 ± 2.5b
*Adult flies emerging from puparia were injected with either saline (1 µl/fly) or crude venom (1 or 3 VRE/fly) from Nasonia vitripennis. All injections were performed
lateral to the dorsal midline in the posterior half of abdomens using pulled glass capillaries. Behavioral observations were made at room temperature (23–25°C) until
adult flies initiated resumed walking (W2) behavior. Values in the same column followed by the same letter do not differ significantly from each other at P = 0.05.
some elements of pupariation behavior in
venom-injected larvae, consistent with ecdysone’s role in stimulating release of anterior retraction factor (ARF) and puparium tanning
factor (PTF), two factors supposedly released
from the CNS to regulate pupariation (Fraenkel
et al., 1972; Seligman et al., 1977).
Recent observations revealed that precocious
shrinkage of the cuticle stimulated by a neuropeptide pyrokinin can cause a nearly perfect shape of
the puparium even without the specific “contraction” patterns of muscular activity (Zdarek et al.,
2000). Thus, the distorted puparia of venom-injected larvae may have been formed due to both
depressed muscular function and inhibited ability
of the cuticle to shrink. In fact, barograms revealed
a delay in the onset of shrinkage of the cuticle that
underlies the process of smoothing. This asynchrony of behavior and cuticular processes (i.e.,
shrinkage, loss of elasticity, phenolic tanning) can
account for the abnormal shape of the puparium
since only timely orchestration of neuromuscular
and cuticular processes can produce morphologically normal puparia (Zdarek and Fraenkel, 1972;
Zdarek et al., 1979). This does not preclude the
possibility that other specific gene expression
events required for puparium formation have been
altered by the venom.
The fact that exogenous ecdysteroid could rescue some aspects of pupariation in venom-injected larvae of S. bullata implies that venom may
have blocked ARF and/or PTF release, which is
October 2004
dependent on ecdysteroids (Fraenkel et al., 1972;
Fraenkel, 1975), and can be inhibited by specific
transcriptional and translational inhibitors (Seligman et al., 1977). Larvae treated with these inhibitors cannot undergo pupariation as retraction of
anterior segments, longitudinal contraction, and
sclerotization and tanning are blocked. Subsequent
injection of fly larvae with ARF reverses inhibition
of retraction but PTF treatment does not rescue tanning (Seligman et al., 1977). Similarly, in this
study, injection of 20E into venom-injected prered spiracle larvae reversed the inhibition of retraction as well as tanning. Consistent with this
scenario is the observation that a high percentage
of late-red spiracle larvae, a stage of development
in which ARF and PTF may have already been released prior to treatment, injected with venom
retracted anterior segments, contracted longitudinally, and tanned. Development of envenomated pupae or pharate adults of S. bullata,
however, does not resume following 20E treatment
(Rivers and Denlinger, 1994b), arguing that venom
is not inhibiting ecdysteroid release.
Rather than triggering an ecdysteroid deficiency,
it is more likely that venom alters the ability of
target tissues in the CNS or elsewhere to bind hormone or suppresses tissue responsiveness by inhibiting transcription and/or translation. Supporting
the latter scenario are the findings that the tanning response can be initiated by cAMP in fly larvae treated with transcriptional inhibitors but not
in flies injected with translational blockers (Seligman et al., 1977), and that crude venom has been
Rivers et al.
shown to elevate cAMP levels 4–5-fold in cultured
cells within 1 h after exposure (Rivers, unpublished
data). Collectively, these observations suggest that
translational activity may have been reduced or
abolished in certain tissues within the CNS of
venom-injected larvae. This possibility is certainly
not surprising since the venom has been shown to
alter expression of genes encoding for specific heat
shock proteins in envenomated pharate adults of
S. crassipalpis (Rinehart et al., 2002).
The altered behaviors of extrication and posteclosion can alternatively be explained by venom
disturbing motor programs of the CNS, as argued
for pupariation. For example, extrication is accomplished by cycles of stereotypic behaviors composed of anteriorly directed peristaltic muscular
contractions and relaxations, resulting in abdominal and thoracic flexions and deformations (Reid
et al., 1987a). Crude venom’s effect on shortening
the duration of extrication behavior in an eclosing
adult implies that the venom affects the motor neurons of the CNS that command the muscular system engaged in extrication, since the periodicity
and coordination of patterned muscular movements are under the control of the CNS (Reid et
al., 1987b).
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The study is a part of a research project of the
AS CR No. Z4 055 905, which was supported by a
grant of the Czech Granting Agency no. 522/01/0501
(J.Z.). Partial support was also provided by USDANRI grant no. 98-35302-6659 (D.L.D.), USDANRICGP Seed grant 2001-1005 (D.B.R.), and by a
Loyola College Faculty Development Grant (D.B.R.).
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vitripennis, disruption, hymenopterapteromalidae, walker, nasonia, parker, sarcophaga, wasps, venok, bullata, dipterasarcophagidae, eclosion, pupariation, flesh, behavior, ectoparasitic, fly
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