Toxicon 58 (2011) 415–425

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Zootoxic effects of reduviid Rhynocoris marginatus (Fab.) (Hemiptera:

Reduviidae) venomous saliva on Spodoptera litura (Fab.)
K. Sahayaraj*, S. Muthukumar
Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St. Xavier’s College (Autonomous), Palayamkottai 627 002,
Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Rhynocoris marginatus is a predominant and potential reduviid predator of many
Received 22 December 2010 economically important pests in India. The venomous saliva (VS) was collected by milking
Received in revised form 3 June 2011 method and diluted with HPLC grade water to prepare different concentrations (200, 400,
Accepted 6 June 2011
600, 800 and 1000 ppm). The VS from R. marginatus was found to be toxic and the LD50 of
Available online 18 July 2011
the VS in Spodoptera litura third instar were 768 and 929 ppm at 48 and 96 h for micro-
injection and oral toxicity studies, respectively. Level of hydrolase and detoxification
Keywords:
enzymes significantly decreased in a dose-dependent manner after treating the host with
Venomous saliva
Microinjection VS for 96 h. A decrease in carbohydrate (21%) and lipid (46%) contents and an increase in
Oral administration the protein content (50%) were prominent in the experimental category. The VS reduced
Mortality the relative growth rate, approximate digestibility, efficiency of conversion of ingested and
Macromolecules digested food of S. litura in the oral toxicity study. Salivary venom inhibits the haemocytes
Enzyme level from aggregation and affects spreading behavior of haemocytes separated from the fifth
Haemocytes aggregation stadium larvae of S. litura. The result showed that VS toxins caused mortality, changed the
nutritional indices, and altered the levels of macromolecule quantity and digestive
enzymes of S. litura. We concluded that the VS of R. marginatus is venomous to a prey
species, S. litura.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction hemipterans, reduviids constitute an important predator,

The venom of arthropods has attracted considerable
interest as a potential source of bioactive substances. Their
biological properties and proteinaceous nature render
them useful in biological pest management as suggested
early in the 1990s (Maeda et al., 1991; Mc Cutchen et al.,
1991; Stewart et al., 1991; Tomalski and Miller, 1991;
Hammock et al., 1993). The venom of poisonous predators
has novel peptides which have been isolated from snakes,
scorpions, marine cone snails, spiders and other animals
including predatory insects. In arthropods, enormous
information is available about the insecticidal activity for
spiders, scorpions and parasitoids. Among the predatory
* Corresponding author. Tel.: þ91 4624264376; fax: þ91 4622561765.
E-mail address: ksraj42@gmail.com (K. Sahayaraj).
distributed worldwide and have been utilized in the bio-
logical control of cotton, soybean, groundnut and coconut
pests. Venoms of reduviid predators are known to possess
long-term, non-lethal paralytic effects on their prey. The
immobilized or partially digested (Blum, 1978; Cohen,
1990; Sahayaraj, 2007) prey are then used to feed by the
reduviid predator. Such unique paralytic activity (Edwards,
1961; Haridass and Ananthakrishnan, 1981; Mc Mahan,
1983; Maran and Ambrose, 2000) was due to the pres-
ence of novel neurotoxin compounds in the venom of
reduviid predators (Corzo et al., 2001). To date, only few
neurotoxin compounds have been isolated and character-
ized from reduviid predators (Corzo et al., 2001).
Tobacco caterpillar, Spodoptera litura (Fabricius) is one of
the most destructive pests of about 120 species of plants
belonging to 44 families (Qin et al., 2004; Nandagobal and
Gunathilagaraj, 2008). The use of insecticides for the

0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2011.06.001
416 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
control of S. litura has limitations due to its resistance
against many insecticides (Kodandaram and Dhingra,
2007) including pyrethroids (Munir et al., 2009). The
reduviid predator, Rhynocoris marginatus (Fab.) is an ento-
mophagous insect distributed in many agroecosystems and
feeding on more than twenty economically important
insect pests in India (Sahayaraj, 2007). The potential of R.
marginatus as a biocontrol agent under laboratory
(Sahayaraj, 2000; Sahayaraj and Balasubramanian, 2009;
Sahayaraj et al., 2003, 2004) and field conditions
(Sahayaraj, 1999; Sahayaraj and Martin, 2003; Sahayaraj
and Ravi, 2007) has been reported earlier. Maran (2000)
studied the paralytic potential of R. marginatus salivary
gland extract against selected pests. In our previous study,
the antimicrobial activity of R. marginatus salivary venom
against selected human pathogens has been recorded
(Sahayaraj et al., 2006a). However, none of them has
studied the toxicological, physiological and immunological
activities of this reduviid venomous saliva on any pests.
The true venoms of arthropods possesses insecticidal
activity against many economically important pests
(Wudayagiri et al., 2001; Parkinson et al., 2002; Tedford et al.,
2004; Dani et al., 2005; Ergin et al., 2006; de Lima et al., 2007;
Nicholson, 2007; Chaim et al., 2011; Baeka et al., 2011). The
venoms saliva of hunter reduviids possesses insecticidal
activity against many crop pests (Edwards, 1961; Ambrose
and Maran, 2000; Maran, 2000; Corzo et al., 2001).
However, studies of the effects of venomous saliva on various
gut enzymes in the insect have been seriously neglected.
There is a vast literature regarding the stage and age variation
of digestive enzymes in whole gut preparations of all groups
of insects (see Terra et al.,1996a, b; Chapman,1998; Guo et al.,
2011). In most insects, food digestion largely occurs in the
alimentary canal, in which most of the enzymes are produced
and secreted, including protease, lipases, carboxylases,
amylase, invertases, and maltases. Insect gut also produces
a variety of detoxification enzymes which play important
roles in adapting to an environment altered by endo and
exogenic compounds (Zhu et al., 2011). There is, however,
relatively little known about the factors controlling the
release of digestive enzymes in insects. This knowledge is
a prerequisite for developing methods of control of pests
based on inactivation of digestive enzymes. The chief aim of
this research therefore was to determine what extrinsic
factors (incorporating venom into the diet) are most impor-
tant in the regulation of enzyme release and food
consumption indices. The second aim was to examine the
action of mixture of neurotoxic components in the venomous
saliva of R. marginatus adult’s on mortality, whole body total
carbohydrates, proteins and lipids and inhibition of haemo-
cytes aggregation and spreading of S. litura third instar larvae.
2. Materials and methods
2.1. Insect collection and rearing
Laboratory colonies of the host species, S. litura and
reduviid predator were established from individuals that
were collected from cotton fields of Tamil Nadu, India. R.
marginatus were reared on the larvae of the host, S. litura at
30  2  C, 70–80% RH and with a photoperiod of 11:13 h D:L.
Host colony was maintained on fresh cotton leaves up to
second instars, and then transferred in to the freshly prepared
artificial diet (Mani and Rao, 1998) for further rearing.
2.2. Venom collection and preparation
The venomous saliva (VS) was collected from the 10-day
old freshly emerged adult reduviid as described by
Sahayaraj et al. (2006b) and Sahayaraj and Kanna (2009).
The salivary venom collected from more than 50 reduviid
predators were pooled and then stored on ice until their
use in our toxicity experiments within 12 h VS was
collected from each predator only once. Concentrations of
the VS (200, 400, 600, 800 and 1000 ppm) (1 ppm ¼ 1 ml of
crude venom in 1000 ml phosphate buffer) were prepared
by diluting with HPLC grade water (Qualigens, India).
2.3. Determination of toxicity
The toxicity of R. marginatus VS was evaluated against
third instar larvae of S. litura using microinjection
(Escoubas et al., 1995) and oral toxicity (Fitches et al., 1997)
methods. In microinjection method, different concentra-
tions of the VS were tested for toxicity by injecting 1.0 ml of
VS into third stadium S. litura larvae of approximately
120 mg in weight. Control category larvae were injected
with HPLC grade water. Salivary venom and water-injected
larvae were placed individually in a plastic container
(5.5 cm h  3.8 cm d) and maintained in Biological Oxygen
Demand (BOD) incubator with artificial diet. Larval
mortality was observed at 24 h interval up to 96 h.
Behavioral changes if any, in the host insect was observed
and recorded up to 3 h post-injection. A soybean seed
based artificial diet (Mani and Rao, 1998) was used to assay
VS by oral delivery against newly hatched third stadium S.
litura larvae (starved for 6 h prior to exposure to diet). For
each treatment, thirty larvae were maintained in sterilized
plastic container containing moist filter paper to prevent
diet desiccation. For oral toxicity bioassay, 1 ml of VS of
different concentrations (200, 400, 600, 800 and
1000 ppm) was blended thoroughly with the 100 mg of
artificial diet separately and provided to the larvae. Diets
containing an equal amount of HPLC grade water were
controlled. Survival was monitored daily up to 96 h. Then,
all the live insects were used for estimating macromole-
cules and enzyme profile analysis.
2.4. Macromolecular studies
Live insects obtained from the previous study have been
used for the estimation of whole body total carbohydrates,
proteins and lipids. The total carbohydrate (Sadasivam and
Manickam, 1997), protein (Lowry et al., 1951) and lipids
(Bragdon,1951) were estimated using glucose, bovine serum
albumin (BSA) and cholesterol as standards, respectively.
2.5. Preparation and quantification of enzymes
Third instars of VS treated S. litura larvae were used to
quantify enzyme activities. Enzyme extracts were prepared
by the method of Applebaum (1964) and Applebaum et al.
 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
(1961). Individuals were anaesthetized with cotton soaked
in chloroform and the entire digestive tract was dissected
out in ice cold insect Ringer’s solution (NaCl2 – 6.5 g; KCl2 –
0.25 g; CaCl2 – 0.25 g; Na2Co3 – 0.25 g in 1000 ml distilled
water). The malpighian tubules, adhering tissue and gut
contents were removed carefully. Then the gut was split
into foregut, midgut and hindgut regions, weighed and
each region was homogenized for 3 min at 4  C in ice cold
citrate–phosphate buffer (pH 6.8) (0.1 M citric acid and
0.1 M sodium citrate) using a tissue homogenizer with
a Teflon pestle. Homogenized gut sections were suspended
in buffer and diluted into 4 ml. The homogenate was
centrifuged at 8000 rpm for 15 min and supernatant was
used as enzyme source.
The amylase and invertase (Bernfeld, 1955; Ishaaya and
Swriski, 1970), trehalose (Ishaaya and Swirski, 1976), acid
phosphatase (ACP) and alkaline phosphates (ALP) (Beaufay
et al., 1954), protease (Morihara and Tsuzuki, 1977), lipase
(Cherry and Crandall, 1932), lactate dehydrogenase (King,
1965), asparate (AAT) and alanine aminotransferases
(ALAT) (Bergmeyer and Bernt, 1965), Glutathione S-Trans-
ferase (Yu, 1982), and esterase (Van Asperen, 1962) were
quantified using standard procedures.
2.6. Esterase assay in gel electrophoresis
SDS (non-native) discontinuous polyacrylamide gel
electrophoresis (SDS PAGE) was performed using a vertical
electrophoresis kit (BioTech, India), and a 6.5% acrylamide
separating gel with Tris–glycine buffer system (Cho et al.,
1999). Prior to electrophoresis, samples (gut region
enzyme extract) were diluted with the sample buffer [40%
sucrose, 0.154% dithiothreitol, 0.0372% EDTA and 0.2%
Triton X-IOO in Tris-glycine buffer (pH 8.3)] at 1:1 ratio.
Thirty microliters of the sample was loaded on each well
routinely and electrophoresis was performed at a constant
voltage of 25 mV. Gel was calibrated using Geni broad
range molecular weight marker (Geni, Bangalore) and
stained with 0.2 M sodium phosphate buffer (pH 6.5)
containing 1% a-naphthyl acetate, 1% b-naphthyl acetate
and 0.13% fast blue RR salt [4-Benzoylamino-2,5-
dimethoxybenzenediazonium chloride hemi(zinc chlo-
ride) salt]. The gel was then processed with destaining
solution (10% acetic acid and 50% methanol).
2.7. Nutritional indices
In another study, VS mixed artificial diet (500 mg) was
provided to the preweighed third stadium larvae (1 larvae/
petriplate), the weight gain, food consumption and eges-
tion by host animals were recorded. Fresh weight of each
larva was measured after 24 h up to 96 h after the treat-
ments. All uneaten food and faeces were collected and
immediately placed in the hot air oven at 60  C for 12 h and
weighed using a monopan balance. Fresh food was accu-
rately weighed and supplied in 500 mg portions. To
measure dry weight of the food supplied and to avoid
inaccuracy due to desiccation, food was along maintained
in a Petridish, its dry weight measured the next day and
used as a reference to determine the amount food
consumed by the insects. Consumption rate (CR) was
417
determined from the amount of total food ingested per
feeding day. Relative growth rate (RGR) was calculated
from the CR per mean body mass after test larva (RGR ¼ mg
wt gained per mg initial larval wt per day). Efficiency of
conversion of ingested food [ECI ¼ (wt gained/food
ingested)  100], efficiency of conversion of digested food
[ECD ¼ (wt gained/food ingested  wt of faces)  100] and
approximate digestibility [AD ¼ (food ingested  wt of
faces)/wt of food ingested  100] were calculated as sug-
gested by Waldbauer (1968). Data from dead insects were
not considered during calculation.
2.8. Inhibition of haemocytes aggregation and spreading
In another study, fifth instar larvae of S. litura insect
were individually swabbed with 95% ethanol (v/v), dried
and a small cut was made in mid-proleg with a minute
sterile pin under sterilized conditions. Haemolymph was
collected into a sterile Eppendorf tube containing a few
crystals of anticoagulant (1-phenyl-2-thiourea – PTU). The
haemocytes sample was prepared for aggregation bioas-
says according to the method described by Richards and
Dani (2008), and Dani and Richards (2009), with a slight
modification i.e., the haemocyte concentration increased to
2  105 cells per well. Ten microliters of crude venom was
diluted to 100 fold with phosphate buffer (pH 7.2). Twenty
microlitres of the diluted venom was added into each well
of 12-well strip ELISA plate containing 100 ml of haemo-
cytes per well. Then Ampicillin (Hi-Media) and phenolox-
idase inhibitor phenylthiocarbamide (PTC) (Hi-Media)
were added to each well to make the final concentration of
100 mg/ml and 20 mM respectively. Heamocytes with and
without venom treatments were incubated at 20  C, 65%
relative humidity until observations were made. After
incubation for 20 h, the aggregation of heamocytes was
observed using a phase contrast microscopy at 40
magnification (Olympus CX41, Japan). The haemocytes
were then visualized by fixing and staining them in 0.1%
Commassie Brilliant Blue G250 in acetic acid and methanol
in 1:4 ratio. After staining, cells were destained with 1:4
ratio of acetic acid and methanol mixture. Then the
monolayer of haemocytes were covered with sterile phos-
phate buffered saline (PBS) (pH 7.4, 0.138 M NaCl, 0.0027 M
KCl, 0.0073 M Na2HPO4, 0.00147 M KH2PO4) and the
aggregation of haemocytes was observed in four randomly
chosen fields of view at 200 magnification. The degree of
aggregation (DA) was recorded as the difference in the
transmittance (T) calculated from A405 before and after
incubation [(T405)20  (T405)0] using an ELISA strip
reader (SR 601-Qualisystems) and calculated using the
following formula: DA ¼ [(T405)20  (T405)0].
Impact of VS on the haemocytes spreading was studied
as described by Zhang et al. (2005) and Yu et al. (2007) with
slight a modification. Haemocytes with or without venom
treatment were incubated at 27  C. After 30 min and
240 min of incubation, the spreading of haemocyte was
observed using phase contrast microscope (Olympus CX 41,
Japan). Spreading and non-spreading plasmatocytes (PC)
and granular cells (GC) (Gupta, 1979) were counted from
three randomly chosen fields of view at 40 magnification.
418 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
Approximately 80 cells were counted in each field of
view and a total of 240 cells were counted. The spreading
percentage and spreading inhibitory ratio of Plasmatocytes
and granular cells respectively were calculated as follows:
ðNo of spreading PC or GC observedÞ
% spreading ¼
ðTotal no of spreading and non  spreading PC or GC observedÞ
ð% spreading of PC or GC without venom treatment as the
control  %spreading of PC or GC with venom treatmentÞ
Spreading inhibitory ratio ¼
% spreading of PC or GC of the control
Five wells were evaluated for each venom concentration in
three replicates.
2.9. Statistical analysis
The LD50 value was calculated following Finney (1971).
Control animal data was compared with different concen-
trations of VS. All data were subjected to one way ANOVA and
post hoc Tukey’s test using the SPSS statistical software
(Version 11.5). The significance was expressed as df, F and P [df
– degrees of freedom, F – Fisher distribution and P ¼ 5% level].
3. Results
3.1. Determination of toxicity
Individual S. litura injected with minimum concentrations
(200 and 400 ppm) exhibited no initial response, after
90 min, wriggling and restless movement, rapid mastication
action of mandible began and fell on the lateral side and
became motionless. The onset of these symptoms seemed to
occur faster (30–40 min) with increasing concentrations.
None of the control injections of 1.0 ml HPLC grade water
resulted in fatality or symptoms of envenomation within
96 h, the maximum period of observation. Larvae injected
with VS died within 48 h and showed an LD50 value of
768 ppm. However, in oral toxicity bioassay method, at 96 h
observation, dose-dependent mortality was observed (40.0%,
50.0%, 60.0%, 90.0% and 90.0% for 200, 400, 600, 800 and
1000 ppm respectively) and the LD50 value was 929 ppm.
3.2. Food consumption indices
Relative growth rate (RGR), approximate digestibility
(AD), efficiency of conversion of ingested (ECI) and digested
food (ECD) of S. litura larvae fed with VS mixed artificial diet
(Table 1) were significantly lower (P < 0.05) than those of
an insect fed with non-VS diet. RGR, AD, ECI and ECD were
decreased in dose-dependent manner. However, RGR, AD,
ECI and ECD increased from 24 h to 96 h in control category.
Highest RGR (F ¼ 1.2; df ¼ 9,30; P < 0.05), AD (F ¼ 1.23;
df ¼ 9,30; P < 0.05), ECI (F ¼ 1.42; df ¼ 9,30; P < 0.05) and
ECD (F ¼ 2.2; df ¼ 9,30; P < 0.05) were observed at 96 h in
non-venomous saliva category. But it decreased from 24 h
to 96 h in all VS concentrations. Lowest RGR (F ¼ 5.2;
df ¼ 9,30; P < 0.05), AD (F ¼ 2; df ¼ 9,30; P < 0.05), ECI
(F ¼ 7.74; df ¼ 9,30; P < 0.05) and ECD (F ¼ 3.2; df ¼ 9,30;
P < 0.05) was observed at 96 h for 1000 ppm concentration.
 100
 100
3.3. Macromolecules of S. litura
Carbohydrate and lipid contents decreased with the
increase in the concentration of VS, whereas protein
content increased significantly (F ¼ 8.3; df ¼ 2,12; P < 0.05)
with the increasing concentration of R. marginatus VS. In VS
treated insects, both carbohydrate (F ¼ 21.0; df ¼ 1,13;
P < 0.05) and lipid (F ¼ 1.4; df ¼ 2,12; P < 0.05) contents
were lower than control and an opposite trend was recor-
ded for protein (Table 2).
3.4. Digestive enzyme profile S. litura
Amylase, invertase, acid phosphatase, alkaline phos-
phatase, alanine aminotransferase, asparate aminotrans-
ferase, protease, lactate dehydrogenase trehalase and lipase
activities in foregut, midgut and hindgut of control and VS
treated S. litura larvae are shown in Table 3. While
comparing the control with other categories, the enzyme
activity was decreased from low to high concentrations.
Among the three regions, all enzymes showed high activity
in the midgut. There was reduction in amylase activity of
1000 ppm concentration at 90% (F ¼ 1.1; df ¼ 2, 12;
P < 0.05), 87% (F ¼ 4.7; df ¼ 2, 12; P < 0.05) and 86% (F ¼ 4.7;
df ¼ 2, 12; P < 0.05) in mid, hind and foregut respectively
while compared to the control. Similarly, in the same
concentration, the lipase activity was reduced more in
midgut (82%) (F ¼ 2.6; df ¼ 2, 12; P < 0.05), than in foregut
(79%) (F ¼ 6.3; df ¼ 2, 12; P < 0.05) and in hindgut (64%)
(F ¼ 4.3; df ¼ 2, 12; P < 0.05). Furthermore, 23% (F ¼ 7.9;
df ¼ 2, 12; P < 0.05), 61% (F ¼ 4.0; df ¼ 2, 12; P < 0.05), 33%
(F ¼ 1.2; df ¼ 2, 12; P < 0.05), and 40% (F ¼ 6.3; df ¼ 2, 12;
P < 0.05) reduction were more in the foregut than midgut
and hindgut for invertase, acid phosphatase, AAT and tre-
halase respectively. Similarly for AP, ASAT, protease and LD,
53% (F ¼ 7.3; df ¼ 2, 12; P < 0.05), 65% (F ¼ 5.9; df ¼ 2, 12;
P < 0.05), 30% (F ¼ 1.7; df ¼ 2, 12; P < 0.05) and 25% (F ¼ 9.4;
df ¼ 2, 12; P < 0.05) reduction respectively were recorded in
hindgut rather than foregut and midgut.
3.5. Detoxification enzymes
Glutathione S-Transferase (GST) activity in the foregut,
midgut and hindgut of the 3rd instar stadium larvae
 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425 419

Table 1
Nutritional indices of third instar larvae of S. litura after the treatments (oral toxicity assay) of salivary venom of R. marginatus.

Exposed hours Nutritional indices Concentrations of the venom in ppm

Control 200 400 600 800 1000

24 RGR 20.77  0.35 15.27  0.88* 13.15  0.73* 11.75  1.23* 10.75  0.95* 09.03  1.38*
AD 63.55  1.50 52.10  1.17* 38.60  1.26* 26.36  0.92* 25.30  1.18* 24.40  1.20*
ECI 31.37  0.61 29.10  1.18 * 15.18  1.11* 13.17  1.25* 11.35  1.36* 10.82  1.15*
ECD 80.77  0.74 69.97  2.28* 67.85  2.15* 63.16  1.24 * 61.66  0.98* 56.76  1.57*
48 RGR 21.85  1.02 14.48  0.80* 12.31  0.92* 11.29  1.17* 9.35  0.78* 8.48  1.12*
AD 65.42  1.26 38.40  3.05* 35.70  2.10* 25.34  1.19* 24.79  1.34* 20.69  0.86*
ECI 38.39  2.44 22.35  1.65* 19.06  0.75* 16.30  1.18* 13.65  1.20* 11.45  0.76*
ECD 83.40  1.24 68.87  2.46* 59.59  2.53* 63.00  0.87 * 62.46  0.85* 56.14  1.00*
72 RGR 23.30  0.79 13.45  1.10* 11.56  0.81* 10.49  0.77* 8.60  0.77* 7.03  0.91*
AD 68.41  1.46 35.65  1.08* 30.69  0.86* 25.76  1.21* 24.60  1.25* 17.69  1.07*
ECI 36.65  0.97 18.20  0.85* 16.61  0.86* 13.09  0.67* 11.19  0.93* 09.54  1.58*
ECD 85.49  1.33 65.25  2.11* 57.59  1.42* 51.50  1.04* 51.00  0.82* 47.60  1.07*
96 RGR 25.09  1.22 13.08  0.70* 10.93  0.9* 9.79  0.98* 7.43  0.93* 7.01  0.75*
AD 73.77  1.40 27.34  1.35* 21.73  1.18* 23.84  0.91* 23.64  0.88* 14.49  1.58*
ECI 39.07  2.30 16.29  1.20* 13.59  1.21* 12.39  1.14* 12.09  1.39* 10.79  1.04*
ECD 85.77  1.23 64.95  1.69* 56.58  1.46* 46.68  1.28* 45.60  1.18* 43.05  1.90*

Mean  SE, *The mean difference significance at 5% level (P < 0.05); RGR, relative growth rate; AD, approximate digestibility; ECI, efficiency of conversion of
ingested; ECD, efficiency of conversion digested food.

exposed to different doses of VS for 4 days are shown in the very clear when compared to control. The intensity of all the
Table 4. GST activity was high and low in midgut (1.4 times) bands was highly pronounced at 1000 ppm concentration.
(F ¼ 1.7; df ¼ 3, 12; P < 0.05) (1371 13 mmoles/min/mg
protein) and hindgut (0.64 times) when compared to the
3.6. Inhibition of haemocyte aggregation and spreading
foregut of the untreated control category. The activity was
recorded by VS ingestion at midgut (1.033 times). Similar to
When S. litura haemocytes were plated out at a suitable
GST activity, effects of VS doses in diet on esterase activities
density (i.e. 1.5  105 haemocytes) in the well of a 12-well
of the third instar stadium larvae were also more significant
strip, they attached to the surface of the well within min,
in the midgut region (F ¼ 1.7; df ¼ 3,12; P < 0.05)
and the majority of plasmatocytes and granular cells
(7.420  0.004 mmoles/min/mg protein in 1000 ppm) than
attached themselves within 1 h (Fig. 2a and b). During this
foregut and hindgut of S. litura (Table 4). A significant
time, these haemocytes extended small pseudopods
increase (P < 0.005) in GST and esterase activities were
(Fig. 2c and d). After 240 min, the pseudopods were larger
found in the third instar stadium larvae of S. litura fed on
(Fig. 2e) and some migration towards the neighboring cells
diets with various doses of VS in comparison with that from
had occurred, resulting in the appearance of small groups
the untreated control animals. Generally the hindgut shows
of cells. During the same period, VS treatment caused
lower GST and esterase activities than the foregut and
surface blebbing, and disintegration of the plasma
midgut of S. litura.
membrane in granulocytes (Fig. 2e and f). After 20 h, it was
Esterase band patterns of S. litura were also observed
clear that individual haemocytes had moved across the
using non-denaturing polyacrylamide gel electrophoresis
surface of the well and had formed clearly distinguishable
(Fig. 1). Three bands corresponding to masses of 50 kDa,
haemocyte aggregation which composed of 10 or more
37 kDa and 31 kDa were noticed in gel to emphasize the
cells. By contrast, in reduviid predator venom treated
detoxification activity in gut of S. litura. The band at 50 kDa
category, inhibition of haemocyte aggregation was
was observed in all lanes including control, whereas 37 kDa
observed. The transmittance difference obtained from
and 31 kDa were appeared from 400 ppm and 600 ppm,
venom added wells doesn’t make much difference (0%),
after respectively (Table 5). When the concentration of VS
whereas in control category, it was 60%. Same results were
increased the esterase bands became thicker and appeared
recorded when the experiment was repeated.
Table 2 Plasmatocytes were easily identified by their marked
Macromolecular profile (mg/mg) of Spodoptera litura fed with Rhynocoris spreading behavior on glass. The growth of pseudopodia
marginatus salivary venom mixed artificial diet at five different doses could be observed within 30 min after VS was added with
(ppm).
haemocytes of the host. Depending upon the venom
Venom concentrations Protein Carbohydrate Lipid concentrations, it significantly inhibits the spreading of S.
(in ppm) litura plasmatocytes and granulocytes. However, at 400 and
Control 123.3  0.2 83.8  0.7 93.2  1.8 600 ppm concentrations VS did not cause any change in
200 95.8  0.2* 78.0  0.9* 80.9  0.6*
plasmatocytes. On 30 min of observation, the spreading
400 149.0  1.8NS 73.9  1.2* 79.4  1.8*
600 163.7  1.0* 68.4  0.7* 73.0  1.8* percentage of plasmatocytes was decreased in dose-
800 179.6  1.3NS 67.5  0.4* 52.9  0.4* dependent manner. The spreading percentage was 98% in
1000 185.3  3.1* 65.8  1.2* 50.5  0.5* control category and significantly decreased in 400 ppm
*The mean difference significant at 5% level (P < 0.05); NS
, not significant (91%) (F ¼ 25.43; df ¼ 4, 18; P ¼ 0.05), 500 ppm VS (59%)
at 5% level (P > 0.05). (F ¼ 3.1; df ¼ 4, 18; P ¼ 0.05). At 240 min observation,
420 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425

Table 3
Digestive enzyme activities in the foregut, midgut and hindgut of the third instar stadium larvae exposed to different does (in ppm) of salivary venom of
R. marginatus.

Enzymes Concentration (ppm) Parts of the digestive system

Foregut Midgut Hindgut

Amylase (mg maltose released/min/mg protein) Control 3.71  0.05a 6.14  0.02b 3.65  0.04c
200 1.44  0.04*a 1.42  0.04*a 0.48  0.02*c
400 0.88  0.02*a 0.97  0.02*b 0.73  0.02*c
600 0.73  0.03*a 0.82  0.02*b 0.63  0.45*c
800 0.62  0.03*a 0.74  0.03*b 0.57  0.02*c
1000 0.59  0.04*a 0.64  0.03*b 0.54  0.01*c
Invertase (mg glucose released/min/mg protein) Control 74  1a 95  2b 36  2c
200 65  2*a 93  1*b 34  2*c
400 64  1*a 87  1*b 34  2*c
600 63  1*a 82  1*b 32  2*c
800 61  1*a 79  2*b 31  1*c
1000 57  1*a 74  1*b 28  1*c
Acid phosphatase (mmol /mg/h1) Control 18.77  1.17a 25.55  1.15b 7.37  0.32c
200 16.51  0.71*a 23.62  1.68*b 6.46  0.37*c
400 15.85  0.83*a 21.24  0.88*b 4.23  0.12*c
600 11.98  0.83*a 17.16  1.05*b 4.36  0.27*c
800 10.60  0.76*a 17.74  0.37*b 3.33  0.18*c
1000 7.28  0.20*a 16.32  0.68*b 4.12  0.08*c
Alkaline phosphatase (mmol /mg/h1) Control 24.64  0.27a 32.33  0.68b 15.29  0.84c
200 23.05  0.82*a 32.48  0.47Ns b
14.57  0.70*c
400 21.18  0.78*a 29.31  0.45*b 11.47  0.51*c
600 20.72  1.61*a 25.13  2.61*b 10.83  1.48*c
800 17.40  2.28*a 21.36  1.38*b 8.22  0.41*c
1000 16.65  0.71*a 18.05  0.79*b 7.10  0.46*c
Protease (mg tyrosine/mg protein/min) Control 0.76  0.01a 0.94  0.01b 0.57  0.01c
200 0.73  0.02*a 0.89  0.02*b 0.53  0.01*c
400 0.69  0.02*a 0.85  0.01*b 0.49  0.01*c
600 0.66  0.01*a 0.80  0.01*b 0.48  0.01*c
800 0.63  0.01*a 0.75  0.01*b 0.46  0.01*c
1000 0.58  0.01*a 0.68  0.02*b 0.40  0.01*c
Trehalase (mg glucose released/min/mg protein) Control 4.27  0.08a 4.30  0.02a 3.30  0.04c
200 3.62  0.01*a 4.07  0.04*b 3.23  0.04*c
400 3.47  0.03*a 3.87  0.03*b 3.04  0.04*c
600 3.07  0.04*a 3.73  0.03*b 2.78  0.05*c
800 2.72  0.09*a 3.57  0.03*b 2.57  0.05*c
1000 2.55  0.03*a 3.34  0.02*b 2.36  0.03*c
Lipase (meq/min/g sample) Control 0.57  0.01a 0.77  0.01b 0.25  0.01c
200 0.45  0.02*a 0.64  0.03*b 0.30  0.02*c
400 0.33  0.05*a 0.51  0.01*b 0.27  0.04*c
600 0.22  0.02*a 0.41  0.02*b 0.16  0.01*c
800 0.23  0.06*a 0.30  0.01*b 0.15  0.03*c
1000 0.15  0.01*a 0.16  0.03*b 0.14  0.01*c

*For analysis between control to different concentrations. The mean difference significant at 5% level (P < 0.05); NS, not significant at 5% level (P < 0.05);
Means followed by the same letters between different column for same enzyme are not significant different (P < 0.05).

1000 ppm VS highly reduced the spreading of plasmato- 4. Discussion

cytes (57%) (F ¼ 5.0; df ¼ 3, 20; P ¼ 0.05). Similarly, gran-
ulocytes showed dose-dependent impact in spreading
activity. For instance, the spreading percentage of GC was
low (52%) (F ¼ 4.6; df ¼ 3, 14; P ¼ 0.05) and high (55%)
(F ¼ 11.8; df ¼ 3, 14; P ¼ 0.05) at 30 and 240 min of obser-
vation, respectively in 1000 ppm (Fig. 3). Generally, the
spreading inhibitory percentage (SIP) was higher at
240 min of incubation than at 30 min incubation, except at
400 ppm venom concentration (Fig. 3). Plasmatocytes
showed higher percentage of inhibition 39% (F ¼ 25.59;
df ¼ 3, 11; P < 0.05) and 40% (F ¼ 18.80; df ¼ 3, 14; P < 0.05)
in 30 and 240 min of incubation respectively, which is
significant when compared to the data in lower concen-
tration (Fig. 3). The granulocytes showed the highest inhi-
bition (69%) at 600 ppm, which is not significant when
compared to that in the lower concentration.
Results of this study show that the VS of the reduviid
predator is venomous to S. litura. Injection of venom caused
wriggling and restless movement, rapid mastication action
of mandible, falling off lateral and becoming motionless for
30 to 40 min and then resuming its routine activities.
Mortality of S. litura was observed at 48 and 96 h with LD50
768 and 929 ppm for microinjection and oral administra-
tion respectively. But, previously Corzo et al. (2001) tested
toxicity of three reduviids purified venom against S. litura.
They have not recorded any toxicity against the insects
showing that crude venom has more impact than purified
peptides like Ptul, Adl and Iobl.
In the nutritional indices experiments, R. marginatus
salivary venom significantly reduced the RGR, AD, ECI and
ECD of S. litura as reported by Morales et al. (2007). This
 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425 421

Table 4
Detoxification enzyme activities in the foregut, midgut and hindgut of the third instar stadium larvae exposed to different does (in ppm) of salivary venom of
R. marginatus.

Enzymes Concentration (ppm) Parts of the digestive system

Foregut Midgut Hindgut

Alanine aminotransferase (mmol/min/mg protein) Control 5.56  0.17a 6.44  0.19b 3.67  0.09c
200 5.36  0.15Ns a 6.51  0.12Ns b 3.60  0.11Ns c
400 4.62  0.20*a 5.54  0.23*b 3.46  0.15*c
600 4.27  0.15*a 5.55  0.15*b 2.66  0.13*c
800 4.53  0.18*a 5.40  0.06*b 2.62  0.26*c
1000 3.74  0.15*a 4.59  0.21*b 2.49  0.09*c
Asparate aminotransferase (mmol/min/mg protein) Control 5.58  0.26a 7.39  0.14b 3.35  0.16c
200 5.52  0.14Ns a 7.36  0.24Ns b 2.91  0.15*c
400 5.37  0.14*a 6.70  0.16*b 2.62  0.15*c
600 4.62  0.07*a 6.59  0.10*b 2.41  0.19*c
800 4.85  0.09*a 6.32  0.08*b 1.71  0.12*c
1000 4.35  0.15*a 6.21  0.13*b 1.18  0.04*c
Lactate dehydrogenase (mIU/mg protein/min) Control 10.32  0.37a 13.47  0.11b 10.09  0.05c
200 9.91  0.17*a 12.74  0.09*b 9.67  0.01*c
400 9.56  0.04*a 12.61  0.04*b 9.54  0.03*a,c
600 9.32  0.06*a 12.29  0.09*b 8.40  0.12*c
800 8.36  0.04*a 11.94  0.06*b 7.84  0.04*c
1000 8.04  0.05*a 11.46  0.05*b 7.74  0.04*c
Glutathione S-Transferase (nmoles/min/mg protein) Control 634  4a 857  3b 406  2c
200 652  4*a 874  1*b 519  1*c
400 976  16*a 1002  2*b 788  3*c
600 1036  7*a 1115  7*b 1034  6*a,c
800 1171  4*a 1251  7*b 1035  5*c
1000 1327  5*a 1371  13*b 1266  7*c
Esterase (nmoles/min/mg protein) Control 1.743  0.004a 2.606  0.012b 0.373  0.004c
200 2.200  0.007*a 3.366  0.004*b 0.381  0.004*c
400 2.911  0.006*a 4.216  0.010*b 0.727  0.004*c
600 4.721  0.004*a 5.314  0.006*b 2.212  0.004*c
800 5.376  0.004*a 6.015  0.002*b 3.131  0.005*c
1000 6.547  0.006*a 7.420  0.004*b 5.203  0.004*c

*For analysis between control to different concentrations. The mean difference significant at 5% level (P < 0.05); NS, not significant at 5% level (P < 0.05);
means followed by the same letters between different column for same enzyme are not significant different (P < 0.05).

indicates that the ingestion of VS blended diets exhibited
some chronic toxic impact on the host larvae. The
percentage reduction in RGR AD, ECI and ECD results from
a food conversion deficiency, which reduces growth
Fig. 1. R. marginatus venomous saliva impact on the esterase band patterns
of S. litura haemolymph using non-denaturing polyacrylamide gel electro-
phoresis stained fast glue RR salt.
perhaps through a diversion of energy from biomass
production into detoxification enzyme (Wheeler et al.,
2001) as observed in our study. Total body carbohydrate
and lipid contents decreased in the VS mixed diet fed S.
litura, whereas protein content increased significantly. The
increase of protein quantity might be due to higher
conversion of nitrogen to body matter and lower conver-
sion of carbohydrates may be attributed to lesser nitrogen
and greater carbohydrate utilization for maintenance.
Moreover, increase of protein content clearly indicated the
influence of VS on the protein synthesis of this insect.
R. marginatus VS reduced the digestive enzymes in
foregut, midgut and hindgut of S. litura in dose-depended
manner. Enzymes are catalysts that regulate all reactions
in all cells in the body (Penzlin, 2003). To date, several
different classes of arthropod venomous salivary compo-
nents have been shown to be insecticidal towards a range
of economically important insect pests when tested in
artificial diets (Fitches et al., 2004; Nghia et al., 2006;
Mukherjee et al., 2006). There is, however, relatively little
known about the factors controlling release of digestive
enzymes in insects (Lehane et al., 1996; Lwalaba et al.,
2009). There are relatively few studies addressing the
direct effect of food in the gut on the release of digestive
enzymes (prandial regulation), and very few studies on the
direct effect of specific nutrients on enzyme release.
However, there are almost no studies on the effect of VS on
422 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425

Table 5
Spodoptera litura whole digestive system esterase bands molecular weight (Dalton) profile after feeding with R. marginatus saliva in various concentrations
(in ppm).

Venom concentrations in ppm

Control 200 400 600 200 200

53,246 (0.523) 51,202 (0.541) 50,748 (0.545) 51,997 (0.534) 53,700 (0.519) 55,857 (0.500)
– – 38,373 (0.654) 37,919 (0.658) 37,465 (0.662) 41,325 (0.628)
– – – 31,561 (0.714) 31,561 (0.714) 36,216 (0.673)

Values in parentheses indicates the relative mobility (Rf) of the bands.

Fig. 2. Phase contrast light field microphotographs showing S. litura haemocytes maintained without salivary venom of R. marginatus and subsequently stained
with Commassie Brilliant Blue G250. Note the aggregated haemocytes (arrow head) monolayer (a) (40) without salivary venom of R. marginatus. Haemocytes
incubated in 0.02 ml/ml of salivary venom (b–f). Note the absence of haemocytes aggregation on the haemocytes monolayer (b) (4). Note the extended pseu-
dopods (single arrow) (c, d, e, f) and disintegration of plasma membrane (double arrow) (c, f) (100).
 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
Fig. 3. The spreading (SP) and spreading inhibitory percentage (SIP) of plasmatocytes and granulocytes with various concentrations of venom in vivo conditions
after 30 and 240 min of incubation at 27  C.
the release of digestive enzymes. Such studies can actually
be best carried out with short-term (4 days) feeding in the
third larval stage of S. litura with VS mixed artificial diet and
recorded digestive and detoxication enzymes. This is the
first report of the direct inhibition of enzyme release.
Reduction of digestive enzyme secretion implies either
a direct effect (entry into the cell) or indirect effect (docking
on receptors) of components present in the VS of R. mar-
ginatus, which then has an effect on the cell production
(synthesis) and/or release of the enzyme (Lwalaba et al.,
2010). This is not surprising, because there is already
considerable evidence for the effect of inhibitors on
increased or altered trypsin variants in the gut epithelial
cells of Spodoptera frugiperda in response to acute feeding
of SBTI and BTI-CMe (Lara et al., 2000; Brioschi et al., 2007).
We believed and concluded that the insecticidal and
enzymatic inhibition activities of the VS is due to a low
[MW ¼ 3798 Da (RmIT1) and 7500 Da (RmIT2)], and high
(30 kDa) molecular weight components recorded from the
venomous saliva of this predator using MALDI–TOFMS and
GC-MS respectively (unpublished data). Moreover, the VS
of R. marginatus is even more toxic when injected directly
into the insect haemocoel, which completely bypasses the
gut. We think it is more likely that most of the reductions in
enzyme activity we observe are secondary consequences of
a mixture of neurotoxic components in the venom
(consistent with the neuromuscular abnormalities we
observed after injection of VS).
Generally activities of asparate (AAT) and alanine
(ALAT) aminotransferase was low in S. litura larvae after
exposed to different doses of VS of the reduviid predator
for four days. It was indicated that specific types of
proteins are synthesized in the haemolymph from
423
precursors of amino acids by enzymatic transamination
reactions. Glutamic acid is formed by amino transfer from
aspartic acid by AAT or from alanine by ALAT. R. margin-
atus salivary toxins interfere with the transformation of
the amino acids and hence the AAT and ALAT level
decreased. Furthermore, normally, organisms primarily
spent more energy into the physiological responses with
high sensitivity and efficiency due to limited energy
reserves (Stone et al., 2002). Therefore, more energy might
be transferred to elevate the GST, esterase and LDH in
order to defend rapidly and stably the toxic response
caused by toxin, indicated in S. litura these enzymes were
acts as the prime detoxication enzymes rather than AAT
and ALAT. Aminotransferases are bioindicators of gluco-
neogenesis, the formation of carbohydrates from amino
acids. The AST and ALT serve as a strategic linkage between
the carbohydrates and protein metabolism that are known
to be altered during various physiological and pathological
conditions (Zibaee et al., 2008). There was no significant
increase of ALT and AST activities show the utilization of
protein for gluconeogenesis was very low. It was also
evidence in the level of whole body protein and carbo-
hydrate (Table 2). When ingested by larvae, toxin proteins
bind to specific receptors in the midgut region and toxin
binding in susceptible insects disrupts the insect metab-
olism, thereby causing overall toxic effects and ultimately
resulting in larval death.
There are no reports in the literature describing the host
haemocyte aggregation behavior of reduviid predator
venom. These results indicate that the haemocyte anti-
aggregation factor(s) present in R. marginatus venom
prevent aggregation occurring, rather than breaking up
aggregates after they have formed. This helps the predator
424 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
make perfect extra oral digestion in haemocoel without any
blockage. Similarly Pimpla hypochondriaca venom reduced
the spreading behavior of Lacanobia oleracea haemocytes
and abolishes the ability of these cells to migrate and form
aggregates in-vitro (Parkinson et al., 2002; Dani et al.,
2005). Presently, aggregation and spreading activity were
detected for crude venom that has previously been shown
to possess antimicrobial activity of this predator’s crude
venom in-vitro (Sahayaraj et al., 2006b).
5. Conclusion
The incorporation of R. marginatus salivary venom into the
artificial diet or directly injected into the body of S. litura
reduced the digestive physiology (food consumption and
hydrolase enzyme levels). Furthermore, R. marginatus VS
might have some toxic anti-aggregation factors which
interact with host haemolymph and haemocytes activities by
suppressing the immune response. Thus it alters the physi-
ological status of the prey for better extra oral digestion. The
salivary venom affects the prey’s haemocytes aggregation
and spreading behavior, nervous systems (wriggling move-
ments/ later motionless), nutritional indices, enzyme activi-
ties and increasing the induction of detoxification enzymes.
Further studies are in progress to identify the toxins as well as
their biologically active components in the salivary venom of
the reduviid predator R. marginatus.
Conflict of interest
None.
Acknowledgement
K. Sahayaraj gratefully acknowledges the Department of
Science and Technology (GS1) (Ref. No. SR/SO/AS – 33/
2006), New Delhi for the financial assistance. We thank
Rev. Fr. Dr. Alphonse Manickam, S.J., Principal, St. Xavier’s
College, Palayamkottai for the laboratory facilities and
encouragement. We are grateful to Dr. Usha Rani (India)
for her critical review of an early draft of the manuscript.
References
Ambrose, D.P., Maran, S.P.M., 2000. Polymorphic diversity in salivary and
haemolymph proteins and digestive physiology of assassin bug Rhy-
nocoris marginatus (Fab.) (Heteroptera: Reduviidae). J. Appl. Entomol
124, 315–317.
Applebaum, S.W., 1964. The action pattern and physiological role of
Tenebrio larval amylase. J. Insect Physiol. 10, 897–906.
Applebaum, S.W., Jankovic, M., Birk, Y.,1961. Studies on the mid gut amylase
activity of Tenebrio molitor L. larvae. J. Insect Physiol. 7, 100–108.
Baeka, J.H., Ji, Y., Shinb, J.S., Leec, S., Leec, S.H., 2011. Venom peptides from
solitary hunting wasps induce feeding disorder in lepidopteran
larvae. Peptides 32, 568–572.
Beaufay, H., Berihet, J., Duve, C.D., 1954. Les systeme hexose- phospha-
tasique. V. Influence de diverse agents sur I’ activite et la stabilite de
la glucose 6–phosphatase. Bull. Stereo Chem. Biol. 36, 1539–1550.
Bergmeyer, H.V.W., Bernt, E., 1965. In: Bergmeyer, H.U. (Ed.), Methods in
Enzymatic Analysis. Academic Press, New York, pp. 324–327.
Bernfeld, J.E., 1955. Amylases a and b. Methods. Enzymol. 1, 149–158.
Blum, S.M., 1978. Biochemical defenses of insects. In: Rockstein, M. (Ed.),
Biochemistry of Insects. Academic Press, New York, pp. 465–513.
Bragdon, T.H., 1951. Colorimetric determination of blood lipids. J. Bio-
chem. 190, 513.
Brioschi, D., Nadalini, L.D., Bengtson, M.H., Sogayar, M.C., Moura, D.S.,
Silva-Filho, M.C., 2007. General up regulation of Spodoptera frugiperda
trypsins and chymotrypsin allows its adaptation to soybean
proteinase inhibitor. Insect Biochem. Mol. Biol. 37, 1283–1290.
Chaim, O.M., Silva, D.T., Moreira, D.C., Wille, A.C.M., Ferrer, V.P.,
Matsubara, F.H., Mangili, O.C., da Silveira, R.B., Gremski, L.H.,
Gremski, W., Ribeiro, A.S., Veiga, S.S., 2011. Brown spider (Loxosceles
genus) venom toxins: tools for biological purposes. Toxins 3, 309–344.
Chapman, R.F., 1998. Alimentary canal, digestion and absorption. In:
Chapman, R.F. (Ed.), The Insects – Structure and Function. Cambridge
University Press, U.K., pp. 38–66.
Cherry, I.S., Crandall Jr., L.A., 1932. The specificity of pancreatic lipase in
appearance in the blood after pancreatic injury. Am. J. Physiol. 100,
266–273.
Cho, J.R., Kim, Y.J., Kim, J.J., Kim, H.S., Yoo, J.K., Lee, J.O., 1999. Electro-
phoretic pattern of larval esterases in field and laboratory-selected
strains of the tobacco cutworm, Spodoptera litura (Fabricius). J. Asia-
Pacific Entomol. 2 (1), 39–44.
Cohen, A.C., 1990. Feeding adaptations of some predaceous heteropterans.
Ann. Entomol. Soc. Am. 83, 1215–1223.
Corzo, G., Adachi-Akahane, S., Nagao, T., Kusui, Y., Nakajima, T., 2001.
Novel peptides from assassin bugs (Hemiptra: Reduviidae): isolation,
chemical and biological characterization. FEBS Lett. 499, 256–261.
Dani, M.P., Edwards, J.P., Richards, E.H., 2005. Hydrolase activity in the
venom of the pupal endoparasitic wasp, Pimpla hypochondriaca. J.
Comp. Physiol. B 141, 373–381.
Dani, M.P., Richards, E.H., 2009. Cloning and expression of the gene for an
insect haemocyte anti-aggregation protein (VPR3), from the venom of
the endoparasitic wasp, Pimpla hypochondriaca. Arch. Insect Biochem.
Physiol. 71 (4), 191–204.
Edwards, J.S., 1961. The action and composition of the saliva of an assassin
bug Platymeris rhadamanthus Gaerst. (Hemiptera: Reduviidae). J. Exp.
Biol. 8, 61–77.
Ergin, E., Uckan, F., Rivers, D.B., Sak, O., 2006. in vivo and in vitro activity of
venom from the endoparasitic wasp Pimpla turionellae (L.) (Hyme-
noptera: Ichneumonidae). Arch. Insect Biochem. Physiol. 61, 87–97.
Escoubas, P., Palma, M.F., Nakajima, T., 1995. A microinjection technique
using Drosophila melanogaster for bioassay-guided isolation of
neurotoxins in arthropod venoms. Toxicon 33, 1549–1555.
Finney, D.J., 1971. Probit Analysis. Cambridge University, London, 333 pp.
Fitches, E., Angharad, M.R., Gatehouse, J., Gatehouse, A., 1997. Effects of
snowdrop lectin (GNA) delivered via artificial diet and transgenic
plants on the development of tomato moth (Lacanobia oleracea) larvae
in laboratory and glasshouse trials. J. Insect Physiol. 43, 727–739.
Fitches, E., Edwards, M.G., Mee, C., Grishin, E., Gatehouse, A.M.R.,
Edwards, J.P., Gatehouse, J.A., 2004. Fusion proteins containing insect-
specific toxins as pest control agents: snowdrop lectin delivers fused
insecticidal spider venom toxin to insect haemolymph following oral
ingestion. J. Insect Physiol. 50, 61–71.
Guo, J.Y., Wu, G., Wan, F.H., 2011. Temporal allocation of metabolic
tolerance to transgenic Bt cotton in beet armyworm, Spodoptera exi-
gua (Hübner). Sci. China Life Sci. 54, 152–158.
Gupta, A.P., 1979. Identification key for hemocyte type in hanging drop
preparations. In: Gupta, A.P. (Ed.), Insect Hemocytes: Development,
Forms, Functions, and Techniques. Cambridge University Press, Lon-
don, pp. 527–529.
Hammock, B.D., Mc Cutchen, B.F., Beetham, J., Choudary, P.V., Fowler, E.,
Ichinose, R., Ward, V.K., Vickers, J.M., Bonning, B.C., Harshman, L.G.,
1993. Development of recombinant viral insecticides by expression of
an insect-specific toxin and insect-specific enzyme in nuclear poly-
hedrosis viruses. Arch. Insect Biochem. Physiol. 22 (3, 4), 315–344.
Haridass, E.T., Ananthakrishnan, T.N., 1981. Functional morphology of
salivary system in some Reduviidae (Insecta: Heteroptera). Proc. Indi.
Acad. Sci. (Anim. Sci.) 90, 145–160.
Ishaaya, I., Swriski, E., 1970. Invertase and amylase activity in the armored
scales Chrysomphalus aonidum and Aonidiella avantii. J. Insect Physiol.
16, 1599–1606.
Ishaaya, I., Swirski, E., 1976. Trehalase, invertase and amylase activities in
the black scale, Saissetia oleae, and their relation to host adaptability.
J. Insect Physiol. 22, 1025–1029.
King, J., 1965. The deydrogenases or oxidoreductase-N lactate dehydro-
genase. In: Practical Clinical Enzymology. Van D. nor strand Company,
London, pp. 83–93.
Kodandaram, M.H., Dhingra, S., 2007. Variation in the toxicity of organ-
ophosphate insecticides to field populations of Spodoptera litura. Ind.
J. Plant Prot. 35 (1), 53–56.
Lara, P., Ortego, F., Gonzylez-Hidalgo, E., Castnera, P., Carbonero, P., Diaz, I.,
2000. Adaptation of Spodoptera exigua to barley trypsin inhibitor BTI-
CMe expressed in transgenic tobacco. Transgenic Res. 9, 169–178.
 K. Sahayaraj, S. Muthukumar / Toxicon 58 (2011) 415–425
Lehane, M.J., Bühler, H.M., Crisanti, A., 1996. Mechanisms controlling the
synthesis and secretion of digestives enzymes in insects. In:
Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect Midgut.
Chapman & Hall, London, pp. 195–205.
de Lima, M.E., Figueiredo, S.G., Pimenta, A.M.C., Santos, D.M., Borges, M.H.,
Cordeiro, M.N., Richardson, M., Oliveira, L.C., Stankiewicz, M.,
Pelhate, M., 2007. Peptides of arachnid venoms with insecticidal
activity targeting sodium channels. Comp. Biochem. Physiol C 146,
264–279.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randell, R.J., 1951. Protein
measurement with Folin phenol reagent. J. Biochem. 193, 265–275.
Lwalaba, D., Hoffmann, K.H., Woodring, J., 2009. Control of the release of
digestive enzymes in the larvae of the fall armyworm, Spodoptera
frugiperda. Arch. Insect Biochem. Physiol. 72, 1–16.
Lwalaba, D., Weidlich, S., Hoffmann, K.H., Woodring, J., 2010. Exogenous
and endogenous protease inhibitors in the gut of the fall armyworm
larvae, Spodoptera frugiperda. Arch. Insect Biochem. Physiol. 74 (2),
114–126.
Maeda, S., Volrath, S.L., Hanzlik, T.N., Harper, S.A., Majima, K., Maddox, D.
W., Hammock, B.D., Fowler, E., 1991. Insecticidal effects of an insect-
specifc neurotoxin expressed by a recombinant baculovirus.
Virology 184 (2), 777–780.
Mani, C., Rao, P.J., 1998. Comparative biology of Spodoptera litura (Fab.) on
semisynthetic diet and natural food. Shaspha 5 (2), 141–144.
Maran, P.M., 2000. Chosen Reduviid Predators–Prey Interaction: Nutri-
tional and Pheromonal Chemical Ecology (Insecta: Heteroptera:
Reduviidae). Manonmaniam Sundaranar University, India.
Maran, S.P.M., Ambrose, D.P., 2000. Paralytic potential of Catamiarus
brevipennis (Serville), a potential biological control agent (Insecta:
Heteroptera: Reduviidae). In: Ignacimuth, S., Sen, A., Janarthanan, S.
(Eds.), Biotechnological Applications for Integrated Pest Management.
Oxford Publishing Co. Pvt. Ltd., New Delhi, pp. 125–131.
Mc Cutchen, B.F., Choudary, P.V., Crenshaw, R., Maddox, D., Kamita, S.G.,
Palekar, N., Volrath, S., Fowler, E., Hammock, B.D., Maeda, S., 1991.
Development of a recombinant baculovirus expressing an insect-
selective neurotoxin: potential for pest control. Biotechnology (NY)
9, 848–852.
Mc Mahan, E.A., 1983. Adaptations, feeding preferences and biometrics of
a termite – baiting assassin bug. (Hemiptera: Reduviidae). Ann.
Entomol. Soc. Am. 76, 483–486.
Morales, J., Medina, P., Vinuela, E., 2007. The influence of two endopara-
sitic wasps, Hyposoter didymator and Chelonus inanitus, on the growth
and food consumption of their host larva Spodoptera littoralis.
BioControl 52, 145–160.
Morihara, K., Tsuzuki, H., 1977. Production of protease and elastrase by
Pseudomonas aeuroginosa strains isolated from patients. Infect.
Immun. 15, 679–685.
Mukherjee, A.K., Sollod, B.L., Wikel, S.K., King, G.F., 2006. Orally active
acaricidal peptide toxins from spider venom. Toxicon 47, 182–187.
Munir, A., Ahmed, S.M., Ali, H.S., 2009. Efficacy of insecticide mixtures
against pyrethroid- and organophosphate-resistant populations of
Spodoptera litura (Lepidoptera: Noctuidae). Pest. Manage. Sci. 65 (3),
266–274.
Nandagobal, V., Gunathilagaraj, K., 2008. Groundnut Entomology. Satish
Serial Publishing House, New Delhi, pp. 570.
Nghia, P.T., Fitches, E., Gatehouse, J.A., 2006. A fusion protein containing
a lepidopteran-specific toxin from the South Indian red scorpion
(Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to
target insects. Omonrice 14, 28–43.
Nicholson, G.M., 2007. Insect-selective spider toxins targeting voltage-
gated sodium channels. Toxicon 49, 490–512.
Parkinson, N., Smith, I., Audsley, N., Edwards, J.P., 2002. Purification of
pimplin, a paralytic heterodimeric polypeptide from venom of the
parasitoid wasp Pimpla hypochondriaca, and cloning of the
cDNA encoding one of the subunits. Insect Biochem. Mol. Biol. 32,
1769–1773.
Penzlin, H., 2003. In: Lehrbuch der Tierphysiologie, seventh ed. Gustav
Fischer Verlag, Jena, Germany.
Qin, H., Ye, Z., Huang, S., Ding, J., Luo, R., 2004. The correlations of the
different host plants with preference level, life duration and survival
rate of Spodoptera litura Fabricius. Chin. J. Eco-Agri. 12, 40–42.
Richards, E.H., Dani, M.P., 2008. Biochemical isolation of an insect hae-
mocyte antiaggregation protein from the venom of the endoparasitic
wasp, Pimpla hypochondriaca and identification of its gene. J. Insect
Physiol. 54, 1041–1049.
Sadasivam, S., Manickam, A., 1997. In: Biochemical Methods, second ed.
New Age International Publication, India, pp. 8–9.
Sahayaraj, K., 1999. Field evaluation of Rhynocoris marginatus (Fab.) against
two groundnut defoliators. Inter. Arach. Newslett. 19, 41–42.
425
Sahayaraj, K., 2000. Evaluation of Biological control potential of Rhyno-
coris marginatus (Fab.) on four groundnut pests under laboratory
conditions. Inter. Arach. Newslett. 20, 72–74.
Sahayaraj, K., 2007. Pest Control Mechanism of Rediviids. Oxford Book
Company, Narayan Niwas, Jaipur, India, pp. 204.
Sahayaraj, K., Martin, P., 2003. Assessment of Rhynocoris marginatus (Fab.)
(Hemiptera: Reduviidae) as augmented control in groundnut pests. J.
Cent. Eur. Agric. 4 (2), 103–110.
Sahayaraj, K., Ravi, C., 2007. Evaluation of reduviid predators and plant
products against chosen groundnut pests. Arch. Phytopathol. Pfl. 40
(4), 281–290.
Sahayaraj, K., Balasubramanian, R., 2009. Biological control potential of
artificial diet and insect hosts reared Rhynocoris marginatus (Fab.) on
three pests. Arch. Phytopathol. Pfl. 42 (3), 238–247.
Sahayaraj, K., Kanna, A.V., 2009. Starvation impact on venom quantity of
reduviid predator, Catamiarus brevipennis Servile. Entomon 34 (2),
119–121.
Sahayaraj, K., Martin, P., Karthikraja, S., 2003. Suitable sex ratio for the
mass rearing of reduviid predator Rhynocoris marginatus (Fab.). J.
Appl. Zool. Res. 14 (1), 34–37.
Sahayaraj, K., Thangarani, S., Delma, J.C.R., 2004. Comparative prey suit-
ability of Helicoverpa armigera and Spodoptera litura larvae for Rhy-
nocoris marginatus (Fab.) (Heteroptera: Reduviidae). Belgium J.
Entomol. 6, 383–392.
Sahayaraj, K., Borgio, J.F., Kumar, S.M., Anandh, G.P., 2006a. Antimicrobial
activity of Rhynocoris marginatus (Fab) and Catamirus brevipennis
(Servile) (Hemiptera: Reduviidae) venom on selected human patho-
gens. J. Venom. Anim. Toxins incl. Trop. Dis. 12, 487–496.
Sahayaraj, K., Kumar, S.M., Anandh, G.P., 2006b. Evaluation of milking and
electrict shock methods for venom collection from hunter reduviids.
Entomon 31, 165–168.
Stewart, L.M., Hirst, M., Lopez, F.M., Merryweather, A.T., Cayley, P.J.,
Possee, R.D., 1991. Construction of an improved baculovirus insecti-
cide containing an insect specific toxin gene. Nature 352, 85–88.
Stone, D., Jepson, P., Laskowski, R., 2002. Trends in detoxification enzymes
and heavy metal accumulation in ground beetles (Coleoptera: Cara-
bidae) in habiting a gradient of pollution. Comp. Biochem. Physiol. C.
132, 105–112.
Tedford, H.W., Sollod, B.L., Maggio, F., King, G.F., 2004. Australian
funnel web spiders: master insecticide chemists. Toxicon 43,
601–618.
Terra, W.R., Ferreira, C., Jordao, B.P., 1996a. Digestive enzymes. In:
Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect Midgut.
Chapman & Hall, London, pp. 153–194.
Terra, W.R., Ferreira, C., Baker, J.E., 1996b. Compartmentalization of
digestion. In: Lehane, M.J., Billingsley, P.F. (Eds.), Biology of the Insect
Midgut. Chapman & Hall, London, pp. 206–235.
Tomalski, M.D., Miller, L.K., 1991. Insect paralysis by baculovirues medi-
ated expression of a mite neurotoxin gene. Nature 352, 82–85.
Van Asperen, K., 1962. A study of housefly esterases by means of
a sensitive colorimetric method. J. Insect Physiol. 8, 401–416.
Waldbauer, G.P., 1968. The consumption and utilization of food by insects.
Adv. Insect Physiol. 5, 229–288.
Wheeler, G.S., Slansky, F., Yu, S.J., 2001. Food consumption, utilization and
detoxification enzyme activity of larvae of three polyphagous noctuid
moth species when fed the botanical insecticide rotenen. Entomol.
Exp. Appl. 98, 225–239.
Wudayagiri, R., Inceoglu, B., Herrmann, R., Derbel, M., Choudary, P.V.,
Hammock, B.D., 2001. Isolation and characterization of a novel
lepidopteran-selective toxin from the venom of South Indian red
Scorpion, Mesobuthus tumulus. BMC Biochem. 2, 16.
Yu, H., Yang, H., Ma, D., Lv, Y., Liu, T., Zhang, K., Lai, R., Liu, J., 2007. Vespid
chemotactic peptide precursor from the wasp, Vespa magnifica
(Smith). Toxicon 50, 377–382.
Yu, S.J., 1982. Host plant induction of glutathione S-transferase in the fall
armyworm. Pest. Biochem. Physiol. 18, 101–106.
Zhang, Z., Ye, G.Y., Cai, J., Hu, C., 2005. Comparative venom toxicity
between Pteromalus puparum and Nasonia vitripennis (Hymenoptera:
Pteromalidae) toward the hemocytes of their natural hosts, non-
target insects and cultured insect cells. Toxicon 46, 337–349.
Zhu, Y.C., Guo, Z., Chen, M.S., Zhu, K.Y., Liu, X.F., Scheffler, B., 2011.
Major putative pesticide receptors, detoxification enzymes, and
transcriptional profile of the midgut of the tobacco budworm,
Heliothis virescens (Lepidoptera: Noctuidae). J. Invertebr. Pathol. 106,
296–307.
Zibaee, A., Sendi, J.J., Alinia, F., Etebari, K., 2008. A study on biochemical
differences among five different groups of rice striped stem borer
Chilo suppressalis walker (Lepidoptera: Pyralidae). Invertebr. Surv. J. 5,
20–29.