Biological Control 30 (2004) 181–192

www.elsevier.com/locate/ybcon

Evaluation of host responses to envenomation as a means to assess

ectoparasitic pteromalid waspÕs potential for controlling
manure-breeding flies
David B. Rivers*
Department of Biology, Loyola College in Maryland, Baltimore, MD, USA

Received 21 January 2003; accepted 13 January 2004

Abstract

For most of the pteromalid wasps currently used in biocontrol programs, key fundamental aspects of the waspÕs biology are
lacking, including information on the factors that make one fly more attractive than another as a host and which flies are best suited
for wasp development, and hence mass propagation for later release. This study examined the physiological relationships between
two species of filth flies (Musca domestica and Fannia canicularis) and eight species of ectoparasitic wasps (five solitary and three
gregarious species). Seven of eight wasp species preferred the larger pupae of house flies over the smaller F. canicularis, and all
solitary species induced death in fly hosts within 24 h after injection of venom. In contrast, the gregarious wasps induced a de-
velopmental arrest in parasitized hosts, the duration of which was host species dependent. This host arrest was accompanied by an
increase in host hemolymph lipids and correlated with clutch sizes of the two gregarious wasps, Nasonia longicornis and Musci-
difurax raptorellus. A similar relationship was not observed for any of the solitary species tested. Isolated crude venoms from all
wasps induced similar changes in the morphology of cultured insect cells and the venoms displayed overlapping lethality (LC50 s),
but the time required for venoms from gregarious species to trigger death was significantly longer than solitary wasp venoms. The
possible use of host responses to envenomation as parameters to screen the biological control potential of parasitoids is discussed.
Ó 2004 Elsevier Inc. All rights reserved.

Keywords: Filth fly; Musca domestica; Fannia canicularis; Ectoparasitoid; Spalangia endius; Spalangia cameroni; Muscidifuruax zaraptor;
Muscidifurax raptor; Muscidifurax raptorellus; Nasonia longicornis; Melittobia digitata; Pachycrepoideus vindemiae; Venom

1. Introduction (Walker) is also an important biocontrol agent (Kauf-

Biological control programs aimed at reducing fly
populations in poultry operations have achieved mixed
ratings (Axtell, 1986; Petersen, 1993). In general, release
of indigenous species of pteromalid wasps have yielded
encouraging results, particularly in poultry facilities lo-
cated in the southern US (Axtell and Arrends, 1990;
Petersen, 1993). Most of these efforts have been with
augmentative releases of Spalangia endius Walker and
Muscidifurax raptor Girault and Sanders (Legner, 1981;
Rutz and Axtell, 1979), two species of solitary, pupal
ectoparasitoids. In northern states, Nasonia vitripennis
*
Fax: 1-410-617-5682.
E-mail address: drivers@loyola.edu.
man et al., 2001), probably due to its higher cold tol-
erance than other pteromalids (Rivers et al., 2000).
Improvements in fly control using these biocontrol
agents have been empirical only; i.e., integrated pest
management programs using biological control have
relied almost entirely on augmenting the natural popu-
lations of indigenous wasp species (Axtell and Rutz,
1986), which should and does reduce fly densities to
some extent. Such use of pteromalid wasps does not
depend on a thorough understanding of the host or
parasitoid (DeBach and Rosen, 1991). To some extent,
this is actually good because there is only limited in-
formation available concerning the biology and host/
parasite associations of filth flies breeding in poultry
manure, and even less knowledge about the pteromalid
wasps being sold commercially to control the flies

1049-9644/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2004.01.004
182 D.B. Rivers / Biological Control 30 (2004) 181–192
(Petersen, 1993). Therefore, some fly control has been
achieved despite inadequate knowledge of the biological
control agents. This underscores the tremendous po-
tential of using filth fly parasitoids in biocontrol pro-
grams in poultry rearing facilities.
The current augmentative approach, however, does
not promote maximum effective usage of the biological
control agents. There are clear examples of how our
sparse knowledge of filth fly parasitoids has produced
unsatisfactory fly control. For example, attempts to
suppress fly pests in poultry houses using exotic para-
sites and predators has essentially failed miserably
(Axtell, 1986). Augmentation efforts with different spe-
cies and strains of indigenous pteromalids have also met
with similar results (Hokkanen and Pimentel, 1984;
Rutz and Axtell, 1979). Why have these efforts failed
while other attempts have worked? The answers to the
question get to the core of most problems associated
with biological control: research emphasis in this area
has been focused on trial and error releases (release the
natural enemy and then determine if pest populations
decline below economic injury levels) and on the dis-
covery of new exotic and indigenous biocontrol agents
to be used in trial and error releases. Few studies have
examined the relationships between the filth fly hosts
and the parasitic wasps being released to control them,
leaving unanswered several important questions. What
factors influence host selection behavior of the wasps?
Which features of the fly make one host more suitable
than another for oviposition or parasitoid offspring de-
velopment? Do ectoparasitic pteromalids alter the
physiology of their fly hosts like their endoparasitoid
counterparts? And if they do, are the changes host
specific and/or wasp specific? The trend to overlook
these research areas has continued despite recommen-
dations from the USDA and other sources that our
fundamental knowledge of biological control agents
must be improved if we are to increase the efficacy of
natural enemies in pest control programs (Baum, 1993;
Jervis and Copeland, 1996; King et al., 1988; Petersen,
1993).
Several studies (Rivers and Denlinger, 1994, 1995a,b;
Rivers et al., 1998) using N. vitripennis as a model sys-
tem with several species of sarcophagid (flesh flies),
calliphorid (blow flies), and muscoid flies have shown
that alterations in host lipid content of hemolymph and
fat body tissues correlates with (1) host selection by the
wasp, and (2) host suitability for the parasitoids in terms
of offspring production, duration of development, adult
body sizes, and sex ratio allocation. All manipulations
of fly hosts are induced by venom from female wasps,
and the resulting host changes can be used as indicators
of host selection in the laboratory and field for N. vit-
ripennis. This study examined whether the effects of ve-
nom from several species of ectoparasitic pteromalid
wasps on two important pest species (Musca domestica
L. and Fannia canicularis L.) of poultry mimic the action
of N. vitripennis venom. If so, then a rapid and novel
screening assay using muscoid fly pupae and cultured
insect cells can be developed to assess the potential of
ectoparasitic wasps in reducing populations of a given
fly species. The possibility of using cultured insect cells
and isolated wasp venoms in an in vitro assay is
presented.
2. Materials and methods
2.1. Insect collection
Adults and larvae of M. domestica and F. canicularis
were field collected using two methods from three
poultry rearing facilities in Lancaster, PA. Light traps
baited with either roosters or pork liver were placed
within poultry houses and checked daily for adult flies
and eggs. Baited Malaise traps were placed outside the
rearing facilities and also checked daily for flies. Species
identity was determined using the criteria of Greenberg
(1971).
Larvae and pupae of M. raptor, S. endius, and
Pachycrepoideus vindemiae (Rondani) were field col-
lected using sentinel bag trapping (with house fly pupae)
within caged-layer houses (Rutz, 1986). Species identity
of field-collected wasps was confirmed using the keys of
Rueda and Axtell (1985). Larvae and pupae of Musci-
difurax raptorellus Kogan and Legner, Spalangia came-
roni Perkins, and Muscidifurax zaraptor Kogan and
Legner were gifts from Dr. Chris Geden with the
USDA-ARS (Gainesville, FL). Nasonia longicornis
Darling was provided by Dr. Jack Werren, Department
of Biology at the University of Rochester (Rochester,
NY). Pupae of Melittobia digitata Dahms, a eulophid,
were obtained from Carolina Biological Supply Com-
pany (Raleigh, NC).
2.2. Insect rearing
M. domestica and F. canicularis were maintained as
laboratory colonies as previously described (Rivers and
Denlinger, 1995a). To generate fly pupae, adults and
larvae were maintained under long-day conditions (15 h
light: 9 h dark, 25 °C) throughout development. Wasp
strains were reared as laboratory colonies as previously
described (Rivers, 1996) using house fly and flesh fly
pupae as hosts. Parasitoids were held at 25 °C and 15 h
light: 9 h dark.
2.3. Host suitability
To determine if the reproductive success of ptero-
malid wasps is dependent on venom-induced physio-
logical changes in filth fly hosts, the host suitability of
 D.B. Rivers / Biological Control 30 (2004) 181–192
the two species of filth flies was determined for each
wasp species. These experiments were carried out as
described previously (Rivers and Denlinger, 1995b;
Rivers et al., 1998). Pupae (2 days after pupariation at
25 °C) of M. domestica and F. canicularis were exposed
singly to individual females (3–7 days after emergence
from host puparia) of M. raptor, M. raptorellus, M.
zaraptor, N. longicornis, S. endius, S. cameroni, M. dig-
itata, or P. vindemiae. Oviposition was restricted to the
posterior one-third of the fly puparia (puparia were
wrapped in aluminum foil; Rivers and Denlinger, 1994)
to facilitate egg counting.
In parallel experiments, flies were exposed to parasi-
toids as described above with the exception that wasp
oviposition was not restricted. Following egg laying,
adult wasps were discarded and parasitized hosts kept
separately in plastic 1 oz cups at 25 °C and 15 h light: 9 h
dark until adult wasp emergence. Clutch sizes, sex ra-
tios, duration of development, and adult body length
(measured in mm from head to the tip of the abdomen)
were determined for each wasp species reared on each
type of fly host (Rivers et al., 1998).
For each wasp and host species pairing, each exper-
iment was replicated three times, using 40 host pupae for
each replicate. Differences in fecundity, length of de-
velopment, sex ratio and adult body sizes were com-
pared by two-way ANOVA with wasp species and host
species as factors. Means were analyzed by Student–
Newman–KeulÕs multiple comparisons tests (Sokal and
Rohlf, 1969) using SuperANOVA statistical software
(Abicus, Fairfax, VA).
2.4. Arrestment of fly development
Fly hosts that enter developmental arrest following
venom injection by N. vitripennis yield more adult wasps
than those hosts that die rapidly after envenomation
(Rivers and Denlinger, 1995a). Therefore, to examine
this relationship in filth flies envenomated by other
pteromalid wasps, M. domestica, and F. canicularis
served as hosts for the eight species of ectoparasitic
parasitoids. Pupae of each fly were wrapped in alumi-
num foil to restrict oviposition, and exposed singly to
individual wasp females. After host exposure, adult
wasps were discarded, and the posterior cap of the fly
puparia opened to expose parasitoid eggs. The eggs were
counted and then removed. Each venom-injected fly was
kept separately in plastic 1 oz cups at 25 °C with a
photoperiod of 15:9 (light:dark) h. The number of fly
hosts that entered developmental arrest, deposited eye
pigment, formed body bristles, and died following en-
venomation were scored as described by Rivers and
Denlinger (1994).
For each wasp and host species pairing, each exper-
iment was replicated three times, using 40 host pupae for
each replicate. Differences in the occurrence of host
183
development arrest, eye pigment formation, and bristle
formation were compared by two-way ANOVA with
wasp species and host species as factors. Percentage data
were normalized by arcsine transformation (Sokal and
Rohlf, 1969) before means were analyzed by Student–
Newman–KeulÕs multiple comparisons tests using Su-
perANOVA statistical software (Abicus, Fairfax, VA).
The relationship between wasp fecundity and the dura-
tion of host development arrest was compared using
simple linear regression with the number of parasitoid
eggs as the dependent variable and duration of host
development arrest as the independent variable.
2.5. Host lipids
To determine if the reproductive success of ptero-
malid wasps is dependent on venom-induced changes in
host lipids, parasitic wasps were exposed to flies as de-
scribed above. The lipid content of hemolymph and fat
body from hosts with and without developing parasite
larvae was determined as described by Rivers and
Denlinger (1995a). Hemolymph was collected from filth
flies parasitized in the posterior body regions by col-
lecting blood from each fly species at 12 h intervals for
the first 3 days after parasitism. Samples from unpara-
sitized flies served as controls. Fat body was extracted in
PringleÕs saline (Pringle, 1938) as described previously
(Rivers and Denlinger, 1995a), centrifuged in glass test
tubes at 3000g at 25 °C for 3 min, the supernatants were
discarded, and the fat body pellets then washed twice in
PringleÕs saline. After the second wash, the pellets were
briefly (
5 min) dried to remove excess water, and then
weighed on an analytical balance to determine wet
weights. Isolated fat body were solubilized by adding
30% KOH and then placed in a heating block preset at
90 °C (Thompson, 1986). Hemolymph and fat body
lipids were extracted using a modified version of the
chloroform:methanol procedure of Bligh and Dyer
(1959), the lipids were converted to sulfonic acid deriv-
atives (Rivers and Denlinger, 1995a), and quantified
colorimetrically using vanillin reagent (Van Handel,
1985). Sesame oil (Sigma) was used to generate a stan-
dard curve.
Fifteen pupae were used at each time interval (six
time intervals) for each species of host and wasp. Dif-
ferences in fat body and hemolymph lipid content ex-
tracted from fly hosts were compared by two-way
ANOVA with wasp species and host species as factors.
Means were analyzed by Student–Newman–KeulÕs
multiple comparisons tests (Sokal and Rohlf, 1969) us-
ing SuperANOVA statistical software (Abicus, Fairfax,
VA). The relationship between wasp fecundity and host
hemolymph lipids was compared using simple linear
regression with the number of parasitoid eggs as the
dependent variable and host hemolymph lipid content as
the independent variable.
184 D.B. Rivers / Biological Control 30 (2004) 181–192
2.6. Isolation of crude venom
Venom from each wasp species was isolated as de-
scribed previously for N. vitripennis (Rivers et al., 1993).
Venom glands and reservoirs were dissected from female
wasps in phosphate buffered saline (PBS) [10 mM so-
dium phosphate, 15% (w/v) sucrose, 0.9% (w/v) NaCl,
and 1 mM EDTA, pH 8.0]. Glands and reservoirs were
concentrated by centrifugation (7000g for 10 min at
4 °C) and then homogenized in 100 ll PBS in an ice bath.
Homogenized material was centrifuged (12,000g for
30 min at 4 °C), the supernatant (crude venom) was then
passed through a syringe membrane filter (0.22 l, cel-
lulose acetate), and the crude venom preparation stored
frozen at )70 °C until used.
2.7. Venom assays
Venom assays were originally proposed to use cul-
tured cells from the house fly, M. domestica, but efforts
to establish a continuously growing cell line from lar-
vae or pupae were unsuccessful. As an alternative ap-
proach, venom assays used the well-characterized
cultured cells from the flesh fly, Sarcophaga peregrina
(NIH-SaPe4), and cabbage looper, Trichoplusia ni
(BTI-TN-5B1-4) by the methods of Rivers et al. (1999).
Cells were grown (approx. 2–3 days at 27 °C) to con-
fluent monolayers in 96-well microtiter plates contain-
ing (100 ll/well) SchneiderÕs insect medium with 3%
fetal bovine serum for fly cells or TC-100 with 10%
fetal bovine serum for caterpillar cells. Following the
addition of wasp venom, cell viability was assessed
using trypan blue dye exclusion staining (Rivers et al.,
1993). Cell responses (i.e., morphological changes) were
monitored continuously with a Sony CCD camera
mounted on a phase-contrast inverted microscope and
connected to a Macintosh G4 containing a Scion CG-7
frame grabber. Changes in cell shape and membrane
integrity (i.e., swelling and lysis) were determined from
captured images following the criteria of Trump and
Berezesky (1995).
A LC50 for venom from each wasp species was de-
termined by exposing cells to 5–6 concentrations of ve-
nom and incubating for 24 h at 27 °C before observing
mortality (Rivers et al., 1993). Cell viability was assessed
using trypan blue dye exclusion staining. LT50 s were
calculated by exposing cells to an LC99 dose of each
wasp venom and observing cell mortality at 10 time
intervals. Values in parentheses represent 95% confi-
dence intervals determined by probit analysis (Finney,
1971).
2.8. Fly pupae assay
Isolated crude venom from each pteromalid wasp was
injected into the body cavity of pupae from M. domes-
tica as described previously (Rivers et al., 1993). The
capacity to enter developmental arrest and induce lipid
increases in hemolymph and/or fat body was determined
for each wasp species as described above. The experi-
ments were repeated using pupae from F. canicularis.
The results from this objective were used to (1) deter-
mine if the physiological changes brought about by
isolated venom were identical to those induced by nat-
ural envenomation, (2) assess whether the physiological
changes generated in the two fly hosts were identical or
were there species-specific differences, and (3) correlate
the physiological responses of filth fly hosts with wasp
progeny production.
3. Results
3.1. Host suitability
As a precursor to determining if the reproductive
success of pteromalid wasps is dependent on venom-
induced physiological changes in the filth fly hosts, the
suitability of M. domestica and F. canicularis as hosts
for each wasp was assessed. Adult females of S. endius,
M. raptor, P. vindemiae, M. zaraptor, S. cameroni, and
M. raptorellus preferred the larger pupae of M. do-
mestica versus the smaller F. canicularis. Parasitism
rates (as defined by injection of venom and oviposition)
for all six species when exposed singly to individual fly
pupae exceeded 80% when exposed to house fly pupae,
but was never greater than 50% for any wasp species
using F. canicularis. In contrast, females of N. longi-
cornis showed no preference for either fly species and
parasitism rates were low (<42%) for both types of
hosts. M. digitata, the only non-pteromlaid wasp tes-
ted, rejected F. canicularis as a host: females displayed
drumming behavior on host puparia, but were never
observed to initiate drilling attempts on pupae. Fe-
males of M. digitata did parasitize house fly pupae
(<85% parasitism), but required a much longer period
of time (nearly 24 h) to initiate oviposition behavior
and complete egg laying by comparison to the other
wasp species, which typically oviposited within 2–3 h
after host exposure.
Host species did not affect fecundity (egg deposition)
for the five solitary wasp species (S. endius, S. cameroni,
M. zaraptor, M. raptor, and P. vindemiae): all of these
wasps deposited a single egg/host if oviposition was
completed. In contrast, far more parasitoids were pro-
duced on pupae of M. domestica than on F. canicularis
when successfully parasitized by the gregarious M.
raptorellus and N. longicornis (Table 1). For all of the
wasps, regardless of reproductive strategy (solitary ver-
sus gregarious), adult body sizes were significantly larger
and the duration of development was shorter when de-
veloping on house fly pupae than on F. canicularis
 D.B. Rivers / Biological Control 30 (2004) 181–192 185

Table 1
Impact of host species on oviposition, development, sex ratio, and adult body length of seven species of ectoparasitic wasps
Wasp species Parasitoid response (Mean  SEM)
(host)
Host response No. of adult Duration of Frequency Body length (mm)
to parasitism parasitoids/host development (days) of males
Male Female
S. cameroni
Md Dead 1.0  0.0a 26.9  1.1a 0.39  0.04a 2.56  0.07a 3.10  0.14a
Fc Dead 1.0  0.0a 29.0  1.5b 0.41  0.07a 2.38  0.17b 2.77  0.20b

S. endius
Md Dead 1.0  0.0a 24.6  0.9a 0.35  0.09a 2.61  0.21a 3.11  0.11a
Fc Dead 1.0  0.0a 28.5  1.6b 0.34  0.07a 2.50  0.05a 3.08  0.04a
M. raptor
Md Dead 1.0  0.0a 26.0  2.0a 0.41  0.10a 3.05  0.15c 3.81  0.22c
Fc Dead 1.0  0.0a 27.4  1.2ab 0.41  0.02a 2.87  0.16d 3.54  0.18d
M. zaraptor
Md Dead 1.0  0.0a 21.8  1.4c 0.37  0.08a 2.98  0.15c 3.62  0.11d
Fc Dead 1.0  0.0a 23.7  1.2a 0.40  0.11a 2.76  0.19d 3.50  0.13d

P. vindemiae
Md Dead 1.0  0.1a 31.4  2.1b 0.27  0.06b 1.05  0.08e 1.32  0.13e
Fc Dead 1.1  0.3a 35.1  1.1d 0.32  0.10b 0.95  0.02f 1.21  0.14f
N. longicornis
Md Arrested 13.1  0.7b 17.3  1.8e 0.22  0.10c 1.23  0.11g 1.56  0.07g
Fc Arrested 7.7  0.9c 19.0  0.5f 0.19  0.12c 1.15  0.14eg 1.50  0.12g
M. raptorellus
Md Arrested 4.9  0.5d 22.3  1.3cf 0.45  0.01a 1.30  0.17g 1.66  0.10g
Fc Arrested 2.7  0.2e 22.9  1.2cf 0.42  0.07a 1.17  0.09e 1.45  0.06h
For host species, Md ¼ Musca domestica and Fc ¼ Fannia canicularis. All hosts were true pupae that had been maintained at 25 °C, L15: D9 prior
to parasitoid exposure. Values in the same column followed by the same letter are not significantly different at P < 0:05.

(Table 1). Despite these differences, the proportion of

eggs that became male wasps appeared unaffected by
host species (Table 1). M. digitata was not used for
comparison in this study.

3.2. Arrestment of fly development

Before laying eggs, all wasp species injected venom

into pupae of F. canicularis and M. domestica. Larvae of
the seven pteromalid species (but not M. digitata) typi-
cally consume all or nearly all of the host tissues within
5–7 days at 25 °C (depending on species) following
oviposition, and thus eggs of each parasite were re-
moved from hosts prior to hatch to distinguish the ef-
fects of envenomation from the impact of larval feeding.
All five solitary wasps tested killed the host flies, re- Fig. 1. Developmental fate of M. domestica and F. canicularis fol-
gardless of species, within 24 h after venom injection and lowing envenomation by N. longicornis and M. raptorellus. The pro-
thus none induced developmental arrest (Table 1). In gression of pharate adult development in envenomated hosts was
contrast, M. raptorellus and N. longicornis, both gre- assessed by observing the deposition of eye pigment and formation of
garious wasps, induced a halt in fly development, which body bristles. Deposition of eye pigment always preceded bristle for-
mation, but bristles never developed on a host if eye pigment had not
lasted until host death (Fig. 1). The duration of the first been deposited. None of the fly hosts survived envenomation by
developmental arrest as well as the developmental fate either wasp, regardless of developmental fate.
of hosts following envenomation was dependent on host
species. For example, host arrest lasted the longest pe- pharate adult development (e.g., deposition of eye pig-
riod of time in envenomated pupae of M. domestica and ment and formation of body bristles) than those of F.
far more house fly pupae showed some progression in canicularis (Fig. 1).
186 D.B. Rivers / Biological Control 30 (2004) 181–192

Fig. 2. Relationship between the numbers of eggs deposited by N. longicornis (A,B) and M. raptorellus (C,D) on M. domestica (A,C) and F. ca-
nicularis (B,D), and the duration of developmental arrest induced in each host by venom injection. Regressions were significant (P < 0:001) for N.
longicornis and M. raptorellus on both fly hosts.

A positive relationship was observed between the
number of eggs deposited per host and the duration of
developmental arrest induced by N. longicornis and M.
raptorellus (Fig. 2). This relationship was most pro-
nounced for N. longicornis in that clutch sizes were nearly
twice as large as those of M. raptorellus when reared on
M. domestica (Fig. 2), and the duration of host arrest was
also significantly longer [X  SEM days was 22.7  1.5
versus 13.5  2.3 for N. longicornis and M. raptorellus,
respectively (F ¼ 11:6, df ¼ 1, 134, P < 0:01)] than in
house fly pupae parasitized by M. raptorellus.
3.3. Host lipids
Lipid levels in fat body and hemolymph from un-
parasitized pupae of F. canicularis and M. domestica
remained relatively constant throughout the experi-
mental period (Figs. 3A and B). Envenomation of ei-
ther fly host by any of the five solitary wasp species did
not elevate the total lipids extracted from host fat body
or hemolymph (data not shown). In fact, in enveno-
mated hosts, hemolymph lipid titers declined to 1/3 the
levels extracted from healthy flies within 48 h, regard-
less of host species. Fat body lipid content also de-
clined, although the rate of decrease was slower than
that observed for hemolymph lipids. In contrast, lipid
levels in fly hosts envenomated by the two gregarious
wasps, N. longicornis and M. raptorellus, displayed
significant increases in hemolymph lipids within 24 h
after venom-injection (Fig. 3A), and this was followed
by an accumulation of lipid in fat body 4 days after
parasite attack (Fig. 3B). Similar trends in host lipids
 D.B. Rivers / Biological Control 30 (2004) 181–192 187

Fig. 3. Changes in the lipid composition of hemolymph and fat body in pupae of M. domestica and F. canciularis in response to envenomation by N.
longicornis and M. raptorellus. (A,B) are the mean  SEM hemolymph lipid levels (mg/ml) on each day following envenomation. (C,D) are the
mean  SEM fat body lipid levels (mg/g) on each day following envenomation.

were observed when parasite larvae were allowed to
feed on the hosts, although the total amount of lipid
extracted from hemolymph and fat body was less (data
not shown).
A positive relationship was observed between the
numbers of eggs deposited by N. longicornis and M.
raptorellus and the hemolymph lipid content of the
two fly hosts on the 2nd day following venom injec-
tion (Figs. 4A and B). This lipid titer corresponds
with the peak elevation in host hemolymph lipids for
both M. domestica and F. canicularis induced by the
two wasps (Fig. 3). Such a relationship was not ob-
served for the solitary wasp species because they killed
the hosts rapidly and did not stimulate elevations in
host lipids.
3.4. Toxicity of wasp venoms in vitro
Isolated crude venom from the five solitary and three
gregarious wasp species was assayed for cytotoxicity in
vitro using two cell lines, BTI-TN-5B1-4 and SaPe4
cells. Preliminary observations revealed that the two cell
lines were nearly identical in susceptibility toward the
wasp venoms, therefore only BTI-TN-5B1-4 cells were
used for all subsequent assays. These cells have been
used extensively to examine the mode of action of N.
vitripennis venom and their morphology permitted
tracking chronological changes in cell morphology in-
duced by venom much easier than the SaPe4 cells.
A comparison of the cytotoxic activities of the eight
wasp venoms revealed that all were nearly equal in
188 D.B. Rivers / Biological Control 30 (2004) 181–192

Fig. 4. Relationship between the numbers of eggs deposited by N. longicornis (A,B) and M. raptorellus (C,D) on M. domestica (A,C) and F. ca-
nicularis (B,D), and the hemolymph lipid content of each host on the 2nd day (peak in lipid elevation, Fig. 3) after envenomation. Regressions were
significant (P < 0:001) for N. longicornis and M. raptorellus on both fly hosts.

toxicity toward BTI-TN-5B1-4 cells (Table 2). The cal-
culated LC50 values; i.e., the concentration of crude
venom necessary to evoke mortality in 50% of the cell
population, were not significantly different from each
other and the 95% confidence intervals and slopes
overlapped (Table 2). The venoms did differ, however, in
the time required to induce death. LT50 s were signifi-
cantly lower for the solitary wasp venoms than those
from the gregarious wasps (Table 2).
Though the timing of cell responses was different, all
of the venoms induced similar morphological changes in
BTI-TN-5B1-4 cells when incubated with an LC99 dose
of wasp venom. Untreated and saline-treated cells were
fibroblast-like in appearance, with numerous cytoplas-
mic extensions positioned in a multipolar arrangement.
Cells in the logarithmic phase of growth form adherent
monolayers, and begin to retract the cytoplasmic ex-
tensions if multiple points of cell–cell contact occur. For
all venom assays in this study, venom was added to cells
in the logarithmic phase of growth.
When an LC99 dose of venom from any wasp was
added to the adherent cells, the cells responded by re-
tracting cytoplasmic extensions, followed by blebbing of
the plasma membrane, swelling of the plasma and nu-
clear membranes, and eventual lysis. Crude venom from
each species did not induce lysis at the same rate, as
evident by the different LT50 values (Table 2).
3.5. Toxicity of wasp venoms in vivo
When isolated crude venom from each wasp was in-
jected into pupae of the house fly using an LC99 dose
 D.B. Rivers / Biological Control 30 (2004) 181–192
Table 2
Susceptibilitya of BTI-TN-5B1-4 cells to venoms from eight species of ectoparasitic wasps
Wasp species LC50 (VREb /ll)
Muscidifurax zaraptor 0.0006 (0.0003, 0.0021)
Muscidifurax raptor 0.0002 (0.0000, 0.0017)
Spalangia endius 0.0011 (0.0004, 0.0023)
Spalangia cameroni 0.0014 (0.0009, 0.0033)
Pachycrepoideus vindemiae 0.0009 (0.00002, 0.0036)
Muscidifurax raptorellus 0.0015 (0.0007, 0.0032)
Nasonia longicornis 0.0009 (0.0001, 0.0022)
Melittobia digitata 0.0016 (0.0006, 0.0035)
a
An LC50 for venom from each wasp was determined by exposing cells to 5–6 concentrations of venom and incubating for 24 h at 27 °C before
observing mortality. Cell viability was assessed using trypan blue dye exclusion staining. LT50 s were calculated by exposing cells to an LC99 dose of
venom and observing cell mortality at 10 time intervals. Values in parentheses represent 95% confidence intervals determined by probit analysis.
b
VRE ¼ venom reservoir equivalent.
determined from the in vitro assays, the flies showed
signs of necrosis within 1 h at the site of injection and all
were dead by 24 h post-injection. Dissections of the
pupae at 24 h post-injection revealed that fly hemol-
ymph had become darkened from melanization. Addi-
tionally, fragments of tissue apparently sloughed off
from muscle and fat body were observed throughout the
hemocoel. Attempts to isolate and quantify hemolymph
and fat body lipids were unsuccessful as melanization
and rapid tissue decay likely contributed to low or un-
detectable lipid titers using the colorimetric assay.
When the F. canicularis was used as a host for in-
jections, similar observations were made: necrosis was
apparent at the wound site within 15 min and all fly
pupae were dead and necrotic by 24 h. Similarly, fat
body and hemolymph lipid levels were not quantifiable
at 24 h post-injection from the heavily decayed hosts.
4. Discussion
Selection of a host for parasitism by any of the eight
ectoparasitic wasps (seven pteromalids and one eulop-
hid) was dependent on host species. The impact of host
species on these parasitoids was evident in the larger
clutch sizes (gregarious species), higher production of
offspring (gregarious species), more rapid development
time, and larger adult body sizes of each wasp species
reared on M. domestica than on F. canicularis. M. dig-
itata did not attempt to drill on puparia of F. canicularis,
suggesting that the wasp used cues derived from the fly
puparium for host recognition. This cue recognition
would be surprising if found to be true because dipter-
ans are not considered natural hosts for M. digitata
(Assem et al., 1982). As suggested previously for N.
vitripennis (Rivers and Denlinger, 1995a), though ovi-
position is known to be influenced by chemical or
physical parameters of the fly puparium (Edwards, 1954;
Wylie, 1970), oviposition restraint on pupae of F. ca-
nicularis likely reflects a nutritional deficiency that was
detected by M. raptorellus and N. longicornis during
189
LT50 (min)
9.5 (0.5, 32.7)
6.3 (1.2, 34.7)
11.6 (5.7, 40.7)
13.0 (0.7, 29.1)
6.5 (2.9, 28.0)
68.9 (41.5, 128.9)
83.8 (25.1, 99.3)
144.6 (54.1, 345.2)
venom injection, an event that always precedes egg
laying by ectoparasitic pteromalids (Dawei and Dingxi,
1987; Rivers, 1996). The longer development times and
reduced adult body sizes of all seven wasps parasitizing
pupae of F. canicularis are features of a nutritionally
unsuitable host (Herbert and Clouthier, 1990; Nechols
and Kikuchi, 1985; Vinson and Barbosa, 1987). These
observations are also consistent with the view that host
acceptance by a parasitoid does not imply host suit-
ability for parasitoid development (Mackauer, 1973;
Vinson and Iwantsch, 1980).
Host suitability may not be limited by nutritional
inadequacies of the host, but rather by the inability of
the parasitoid to acquire or utilize specific nutrients
(Beckage and Riddiford, 1978; Vinson and Iwantsch,
1980). The ability to induce a developmental arrest in
the host appears to be critical for maximizing the yield
of parasitoids (Rivers and Denlinger, 1995b; Rivers
et al., 1998). N. vitripennis has been shown to use a re-
productive strategy in which females manipulate fly
hosts through venom injection to maximize progeny
production (Rivers and Denlinger, 1995a,b). For ex-
ample, N. vitripennis is known to retard the development
and alter lipid titers of pupae and pharate adults of the
flesh fly, Sarcophaga bullata Parker, as well as several
other fly species from the families Sarcophagidae and
Calliphoridae (Rivers and Denlinger, 1994, 1995a,b). In
this study, the same types of host responses was ob-
served in M. domestica and F. canicularis parasitized by
the gregarious wasps N. longicornis and M. raptorellus,
although in both cases the duration of the develop-
mental suppression was much shorter than that induced
by N. vitripennis (Rivers and Denlinger, 1994; Rivers
et al., 1998). These differences in the length of host de-
velopmental arrest induced by the three ectoparasitoids
are not related to the time needed by each wasp to
complete development, as would be expected for endo-
parasitic species (Beckage, 1985). This is evident by
observing that the duration of wasp development is far
longer than the time the flies remain alive following
parasitism: shortly (5–7 days at 25 °C) after egg hatch,
190 D.B. Rivers / Biological Control 30 (2004) 181–192
larvae from each species of wasp consume all or most of
the fly host. In contrast, at 25 °C, N. longicornis pro-
gresses from egg to adult emergence in 17–19 days,
depending on host species, while M. raptorellus require
22–23 days to complete development. Thus, although
envenomation can elicit a prolonged halt in host devel-
opment, the presence of feeding parasitoid larvae leads
to a rapid deterioration of the host.
Differences in the length of developmental suppres-
sion between the three gregarious parasitoids are largely
if not entirely attributed to host preference/range. N.
vitripennis prefers pupae and pharate adults of sar-
cophagids over calliphorids and muscoid flies (Rivers
and Denlinger, 1995a), rejects muscoid pupae in choice
tests with sarcophagids (Rivers, 1996, unpublished), and
the venom of this wasp kills house fly pupae rapidly
without inducing a developmental delay (Rivers and
Denlinger, 1995a). As this study demonstrated, both N.
longicornis and M. raptorellus readily parasitized and
manipulated both species of muscoid flies using venom
without triggering rapid decay of host tissues and a
quick death. The implications at least within the genus
Nasonia are that though the host ranges of the three
described species in the genus overlap (Darling and
Werren, 1990), there are clearly greater differences be-
tween the species than geographical distribution and
morphological characters. N. longicornis may have far
greater potential to reduce mucoid fly populations in
poultry facilities than N. vitripennis has demonstrated
(Axtell, 1986; Petersen, 1993), particularly in terms of
progeny production on pupae of M. domestica.
The host responses to envenomation by the two
gregarious parasitoids were vastly different from those
evoked by the five solitary species tested. Injection of
venom into fly pupae by females of S. endius, S. came-
roni, P. vindemiae, M. raptor, and M. zaraptor resulted
in necrosis development in tissues at the site of enven-
omation within 15–60 min, depending on host species,
and fly death by 24 h post-envenomation. Induction of
host developmental arrest was never observed in any
host parasitized by a solitary wasp, which is consistent
with previous findings with M. zaraptor when reared on
muscoid flies (Rivers et al., 1998). These observations
lend support to the prediction of Rivers et al. (1998) that
the developmental events following host arrestment are
linked to biochemical changes that are essential to
progeny development of gregarious wasps (Rivers and
Denlinger, 1995a; Rivers et al., 1998), but are not im-
portant to solitary parasitoids like those used in this
study. There are several lines of evidence that suggest a
linkage between an ectoparasitic pteromalid waspÕs re-
productive strategy and host responses to envenoma-
tion:
(i) Host developmental arrest was only induced by the
venoms from the two gregarious species, N. longicor-
nis and M. raptorellus.
(ii) There was a positive correlation between the num-
bers of eggs deposited per host and the duration
of developmental arrest induced by N. longicornis
and M. raptorellus, but not for the five solitary
wasps.
(iii) An arrest in host development was accompanied by
elevations in hemolymph lipid titers that also posi-
tively correlated with clutch sizes of the two gregar-
ious parasitoids.
The results are also in agreement with earlier predic-
tions for N. vitripennis and other gregarious parasitoids
that the flyÕs response to envenomation reflects its suit-
ability as a host (Rivers and Denlinger, 1995b). For ex-
ample, pupae of M. domestica were more likely to enter a
developmental arrest, remained in arrest longer, and
more lipid was extracted from hemolymph and fat body
of house flies than from F. canicularis. Correspondingly,
larger clutch sizes were deposited on M. domestica by the
two gregarious wasps than on F. canicularis.
Envenomation of either host by M. raptorellus or N.
longicornis resulted in elevated hemolymph lipid con-
tent in both fly species within 1–2 days following ve-
nom injection. Such increases did not occur in either fly
when parasitized by any of the solitary wasps examined
in this study. The fact that a positive relationship was
observed between the peak in host hemolymph titer
and clutch sizes for N. longicornis and M. raptorellus,
but not for the five solitary species argues that the
gregarious parasitoids alter host lipid metabolism
through venom injection to yield a host that has more
nutrients available per individual wasp larva (Rivers et
al., 1998). This is especially critical for gregarious
pteromalids that uses pupal hosts, because (1) larvae
compete with each other and host tissues for available
nutrients, and (2) the pupal host cannot feed to re-
plenish nutrients and thus is a fixed resource at the
time of oviposition. Gregarious pteromalid species that
parasitize fly pupae appear to be unique idiobiont
parasitoids in that they have the ability to increase the
quality of a host through venom injection from an
otherwise finite condition at the initial stage of para-
sitism (Jervis and Copeland, 1996).
A comparison of the cytotoxic activities of the five
solitary and three gregarious wasp venoms revealed that
all were nearly equal in toxicity toward BTI-TN-5B1-4
cells and that each venom-induced similar morphologi-
cal changes in the cultured cells. The venom-induced
changes in cell morphology (e.g., swelling of the plasma
and nuclear membranes and lysis) are consistent with a
colloid-osmotic mechanism of cell death (Rivers et al.,
1999), a mode of action shared by all eight wasps tested.
Such findings argue, at least for the seven pteromalid
species, that these venoms evolved under similar selec-
tion pressures, particularly because their modes of
actions are believed to reflect a receptor-mediated,
G-protein dependent pathway (Rivers et al., 2002).
 D.B. Rivers / Biological Control 30 (2004) 181–192
Where the venoms appear to have diverged is in the
timing of the induced changes: venoms from the two
gregarious pteromalids (and even the gregarious eu-
lophid) required a much longer period of time to
trigger cell swelling and ultimately, cell death. Rivers
et al. (2002) recently demonstrated that during the
period of time between the onset of cell swelling and
death, venom from N. vitripennis activates phospholi-
pase A2 (PLA2 ). The significance of this finding is that
PLA2 has been found to stimulate fatty acid synthesis
in fat body from cockroaches (Ali and Steele, 1997).
Thus, if PLA2 evokes the same regulation of fat syn-
thesis in other insects, the delay in cellular responses
observed in vitro with gregarious wasp venoms are in
agreement with the differences in host responses trig-
gered by the solitary and the gregarious wasps during
natural parasitism.
The primary goal of this study was to determine if the
responses of filth fly hosts to envenomation by ecto-
parasitic wasps correlated with cytotoxic activities of
isolated crude venoms, so that a rapid screening assay
could be developed for evaluating parasitoids potential
in fly control. It appears that such a relationship does
exist, and distinctions can be made based on the re-
productive strategy used by the wasp species to be
evaluated. For example, since successful parasitism by
gregarious pteromalids is dependent on the ability to
induce developmental arrest and redirect fly lipid me-
tabolism, host suitability of other fly species can be de-
termined within 3–4 days using the physiological
parameters assessed in this study. Results from the in
vitro and in vivo assays also indicate that current
practices of using multiple parasitoids in augmentative
or inoculative release programs likely decrease the rates
of successful parasitism by gregarious species, or at least
lower the production of offspring. In a scenario in which
solitary parasitoids are released first (parasitize a host
first), this study has shown that the venom will quickly
kill the host and tissues will deteriorate rapidly. Sub-
sequent multi-parasitism by a gregarious species either
will not occur (Wylie, 1970), or the dead or dying host
will not enter developmental delay and hemolymph lipid
levels cannot increase. If oviposition occurs at all, the
result should be low clutch sizes, increased time to
complete development, stunted adult body sizes, all of
which will perpetuate a further reduction in female fit-
ness in the subsequent generation (Godfray, 1994).
Gregarious parasitoids should be used for fly control
either alone, which is not really feasible; in releases with
other species of compatible gregarious species (Kauf-
man et al., 2001), meaning their venoms evoke similar
host responses; or in inoculative or augmentative re-
leases in which the gregarious species are allowed to
establish several days to weeks prior to sequential re-
lease of solitary species.
191
Acknowledgments
This research was supported by a grant from the US
Egg and Poultry Association (#457).
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