Zootoxic effects of reduviid Rhynocoris marginatus (Fab.) (Hemiptera: Reduviidae) venomous saliva on Spodoptera litura (Fab

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Zootoxic effects of reduviid Rhynocoris marginatus (Fab.) (Hemiptera:Reduviidae) venomous saliva on Spodoptera litura (Fab.)

K. Sahayaraj*, S. MuthukumarCrop 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

Article history:Received 22 December 2010Received in revised form 3 June 2011Accepted 6 June 2011Available online 18 July 2011

Keywords:Venomous salivaMicroinjectionOral administrationMortalityMacromoleculesEnzyme levelHaemocytes aggregation

a b s t r a c t

Rhynocoris marginatus is a predominant and potential reduviid predator of manyeconomically important pests in India. The venomous saliva (VS) was collected by milkingmethod and diluted with HPLC grade water to prepare different concentrations (200, 400,600, 800 and 1000 ppm). The VS from R. marginatus was found to be toxic and the LD50 ofthe 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 detoxificationenzymes significantly decreased in a dose-dependent manner after treating the host withVS for 96 h. A decrease in carbohydrate (21%) and lipid (46%) contents and an increase inthe protein content (50%) were prominent in the experimental category. The VS reducedthe relative growth rate, approximate digestibility, efficiency of conversion of ingested anddigested food of S. litura in the oral toxicity study. Salivary venom inhibits the haemocytesfrom aggregation and affects spreading behavior of haemocytes separated from the fifthstadium larvae of S. litura. The result showed that VS toxins caused mortality, changed thenutritional indices, and altered the levels of macromolecule quantity and digestiveenzymes of S. litura. We concluded that the VS of R. marginatus is venomous to a preyspecies, S. litura.

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1. Introduction

The venom of arthropods has attracted considerableinterest as a potential source of bioactive substances. Theirbiological properties and proteinaceous nature renderthem useful in biological pest management as suggestedearly 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 predatorshas novel peptides which have been isolated from snakes,scorpions, marine cone snails, spiders and other animalsincluding predatory insects. In arthropods, enormousinformation is available about the insecticidal activity forspiders, scorpions and parasitoids. Among the predatory

hemipterans, reduviids constitute an important predator,distributed worldwide and have been utilized in the bio-logical control of cotton, soybean, groundnut and coconutpests. Venoms of reduviid predators are known to possesslong-term, non-lethal paralytic effects on their prey. Theimmobilized or partially digested (Blum, 1978; Cohen,1990; Sahayaraj, 2007) prey are then used to feed by thereduviid 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 ofreduviid predators (Corzo et al., 2001). To date, only fewneurotoxin compounds have been isolated and character-ized from reduviid predators (Corzo et al., 2001).

Tobacco caterpillar, Spodoptera litura (Fabricius) is one ofthe most destructive pests of about 120 species of plantsbelonging to 44 families (Qin et al., 2004; Nandagobal andGunathilagaraj, 2008). The use of insecticides for the

* Corresponding author. Tel.: þ91 4624264376; fax: þ91 4622561765.E-mail address: [email protected] (K. Sahayaraj).

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control of S. litura has limitations due to its resistanceagainst many insecticides (Kodandaram and Dhingra,2007) including pyrethroids (Munir et al., 2009). Thereduviid predator, Rhynocoris marginatus (Fab.) is an ento-mophagous insect distributed in many agroecosystems andfeeding on more than twenty economically importantinsect 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; Sahayarajand Ravi, 2007) has been reported earlier. Maran (2000)studied the paralytic potential of R. marginatus salivarygland extract against selected pests. In our previous study,the antimicrobial activity of R. marginatus salivary venomagainst selected human pathogens has been recorded(Sahayaraj et al., 2006a). However, none of them hasstudied the toxicological, physiological and immunologicalactivities of this reduviid venomous saliva on any pests.

The true venoms of arthropods possesses insecticidalactivity against many economically important pests(Wudayagiri et al., 2001; Parkinson et al., 2002; Tedford et al.,2004;Dani et al., 2005; Erginet al., 2006;de Lima et al., 2007;Nicholson, 2007; Chaim et al., 2011; Baeka et al., 2011). Thevenoms saliva of hunter reduviids possesses insecticidalactivity against many crop pests (Edwards, 1961; Ambroseand Maran, 2000; Maran, 2000; Corzo et al., 2001).However, studies of the effects of venomous saliva onvariousgut enzymes in the insect have been seriously neglected.There is avast literature regarding the stage and age variationof digestive enzymes inwhole gut preparations of all groupsof insects (see Terra et al.,1996a, b;Chapman,1998;Guoet al.,2011). In most insects, food digestion largely occurs in thealimentarycanal, inwhichmostof the enzymesareproducedand secreted, including protease, lipases, carboxylases,amylase, invertases, and maltases. Insect gut also producesa variety of detoxification enzymes which play importantroles in adapting to an environment altered by endo andexogenic compounds (Zhu et al., 2011). There is, however,relatively little known about the factors controlling therelease of digestive enzymes in insects. This knowledge isa prerequisite for developing methods of control of pestsbased on inactivation of digestive enzymes. The chief aim ofthis research therefore was to determine what extrinsicfactors (incorporating venom into the diet) are most impor-tant in the regulation of enzyme release and foodconsumption indices. The second aim was to examine theactionofmixtureof neurotoxic components in thevenomoussaliva of R. marginatus adult’s on mortality, whole body totalcarbohydrates, proteins and lipids and inhibition of haemo-cytes aggregation and spreadingof S. litura third instar larvae.

2. Materials and methods

2.1. Insect collection and rearing

Laboratory colonies of the host species, S. litura andreduviid predator were established from individuals thatwere collected from cotton fields of Tamil Nadu, India. R.marginatus were reared on the larvae of the host, S. litura at30� 2 �C, 70–80% RH andwith a photoperiod of 11:13 h D:L.

Host colony was maintained on fresh cotton leaves up tosecondinstars, and thentransferred in tothe freshlypreparedartificial diet (Mani and Rao, 1998) for further rearing.

2.2. Venom collection and preparation

The venomous saliva (VS) was collected from the 10-dayold freshly emerged adult reduviid as described bySahayaraj et al. (2006b) and Sahayaraj and Kanna (2009).The salivary venom collected from more than 50 reduviidpredators were pooled and then stored on ice until theiruse in our toxicity experiments within 12 h VS wascollected from each predator only once. Concentrations ofthe VS (200, 400, 600, 800 and 1000 ppm) (1 ppm¼ 1 ml ofcrude venom in 1000 ml phosphate buffer) were preparedby diluting with HPLC grade water (Qualigens, India).

2.3. Determination of toxicity

The toxicity of R. marginatus VS was evaluated againstthird 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 ofVS into third stadium S. litura larvae of approximately120 mg in weight. Control category larvae were injectedwith HPLC grade water. Salivary venom and water-injectedlarvae were placed individually in a plastic container(5.5 cm h� 3.8 cmd) and maintained in Biological OxygenDemand (BOD) incubator with artificial diet. Larvalmortality was observed at 24 h interval up to 96 h.Behavioral changes if any, in the host insect was observedand recorded up to 3 h post-injection. A soybean seedbased artificial diet (Mani and Rao, 1998) was used to assayVS by oral delivery against newly hatched third stadium S.litura larvae (starved for 6 h prior to exposure to diet). Foreach treatment, thirty larvae were maintained in sterilizedplastic container containing moist filter paper to preventdiet desiccation. For oral toxicity bioassay, 1 ml of VS ofdifferent concentrations (200, 400, 600, 800 and1000 ppm) was blended thoroughly with the 100 mg ofartificial diet separately and provided to the larvae. Dietscontaining an equal amount of HPLC grade water werecontrolled. 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 beenused for the estimation of whole body total carbohydrates,proteins and lipids. The total carbohydrate (Sadasivam andManickam, 1997), protein (Lowry et al., 1951) and lipids(Bragdon,1951)were estimatedusing glucose, bovine serumalbumin (BSA) and cholesterol as standards, respectively.

2.5. Preparation and quantification of enzymes

Third instars of VS treated S. litura larvae were used toquantify enzyme activities. Enzyme extracts were preparedby the method of Applebaum (1964) and Applebaum et al.

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(1961). Individuals were anaesthetized with cotton soakedin chloroform and the entire digestive tract was dissectedout 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 distilledwater). The malpighian tubules, adhering tissue and gutcontents were removed carefully. Then the gut was splitinto foregut, midgut and hindgut regions, weighed andeach region was homogenized for 3 min at 4 �C in ice coldcitrate–phosphate buffer (pH 6.8) (0.1 M citric acid and0.1 M sodium citrate) using a tissue homogenizer witha Teflon pestle. Homogenized gut sections were suspendedin buffer and diluted into 4 ml. The homogenate wascentrifuged at 8000 rpm for 15 min and supernatant wasused as enzyme source.

The amylase and invertase (Bernfeld, 1955; Ishaaya andSwriski, 1970), trehalose (Ishaaya and Swirski, 1976), acidphosphatase (ACP) and alkaline phosphates (ALP) (Beaufayet 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) werequantified using standard procedures.

2.6. Esterase assay in gel electrophoresis

SDS (non-native) discontinuous polyacrylamide gelelectrophoresis (SDS PAGE) was performed using a verticalelectrophoresis kit (BioTech, India), and a 6.5% acrylamideseparating gel with Tris–glycine buffer system (Cho et al.,1999). Prior to electrophoresis, samples (gut regionenzyme 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 wellroutinely and electrophoresis was performed at a constantvoltage of 25 mV. Gel was calibrated using Geni broadrange molecular weight marker (Geni, Bangalore) andstained with 0.2 M sodium phosphate buffer (pH 6.5)containing 1% a-naphthyl acetate, 1% b-naphthyl acetateand 0.13% fast blue RR salt [4-Benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chlo-ride) salt]. The gel was then processed with destainingsolution (10% acetic acid and 50% methanol).

2.7. Nutritional indices

In another study, VS mixed artificial diet (500 mg) wasprovided 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 eachlarva was measured after 24 h up to 96 h after the treat-ments. All uneaten food and faeces were collected andimmediately placed in the hot air oven at 60 �C for 12 h andweighed using a monopan balance. Fresh food was accu-rately weighed and supplied in 500 mg portions. Tomeasure dry weight of the food supplied and to avoidinaccuracy due to desiccation, food was along maintainedin a Petridish, its dry weight measured the next day andused as a reference to determine the amount foodconsumed by the insects. Consumption rate (CR) was

determined from the amount of total food ingested perfeeding day. Relative growth rate (RGR) was calculatedfrom the CR per mean body mass after test larva (RGR¼mgwt gained per mg initial larval wt per day). Efficiency ofconversion of ingested food [ECI¼ (wt gained/foodingested)� 100], efficiency of conversion of digested food[ECD¼ (wt gained/food ingested�wt of faces)� 100] andapproximate digestibility [AD¼ (food ingested�wt offaces)/wt of food ingested� 100] were calculated as sug-gested by Waldbauer (1968). Data from dead insects werenot considered during calculation.

2.8. Inhibition of haemocytes aggregation and spreading

In another study, fifth instar larvae of S. litura insectwere individually swabbed with 95% ethanol (v/v), driedand a small cut was made in mid-proleg with a minutesterile pin under sterilized conditions. Haemolymph wascollected into a sterile Eppendorf tube containing a fewcrystals of anticoagulant (1-phenyl-2-thiourea – PTU). Thehaemocytes sample was prepared for aggregation bioas-says according to the method described by Richards andDani (2008), and Dani and Richards (2009), with a slightmodification i.e., the haemocyte concentration increased to2�105 cells per well. Ten microliters of crude venom wasdiluted to 100 fold with phosphate buffer (pH 7.2). Twentymicrolitres of the diluted venom was added into each wellof 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 of100 mg/ml and 20 mM respectively. Heamocytes with andwithout venom treatments were incubated at 20 �C, 65%relative humidity until observations were made. Afterincubation for 20 h, the aggregation of heamocytes wasobserved using a phase contrast microscopy at 40�magnification (Olympus CX41, Japan). The haemocyteswere then visualized by fixing and staining them in 0.1%Commassie Brilliant Blue G250 in acetic acid and methanolin 1:4 ratio. After staining, cells were destained with 1:4ratio of acetic acid and methanol mixture. Then themonolayer of haemocytes were covered with sterile phos-phate buffered saline (PBS) (pH 7.4, 0.138 M NaCl, 0.0027 MKCl, 0.0073 M Na2HPO4, 0.00147 M KH2PO4) and theaggregation of haemocytes was observed in four randomlychosen fields of view at 200� magnification. The degree ofaggregation (DA) was recorded as the difference in thetransmittance (T) calculated from A405 before and afterincubation [(T405)20� (T405)0] using an ELISA stripreader (SR 601-Qualisystems) and calculated using thefollowing formula: DA¼ [(T405)20� (T405)0].

Impact of VS on the haemocytes spreading was studiedas described by Zhang et al. (2005) and Yu et al. (2007)withslight a modification. Haemocytes with or without venomtreatment were incubated at 27 �C. After 30 min and240 min of incubation, the spreading of haemocyte wasobserved using phase contrast microscope (Olympus CX 41,Japan). Spreading and non-spreading plasmatocytes (PC)and granular cells (GC) (Gupta, 1979) were counted fromthree randomly chosen fields of view at 40�magnification.

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Approximately 80 cells were counted in each field ofview and a total of 240 cells were counted. The spreadingpercentage and spreading inhibitory ratio of Plasmatocytesand granular cells respectively were calculated as follows:

Five wells were evaluated for each venom concentration inthree 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 datawere subjected to onewayANOVAandpost hoc Tukey’s test using the SPSS statistical software(Version11.5). The significancewasexpressedasdf, FandP [df–degrees of freedom, F– Fisher distribution and P¼ 5% level].

3. Results

3.1. Determination of toxicity

Individual S. litura injectedwithminimumconcentrations(200 and 400 ppm) exhibited no initial response, after90 min, wriggling and restless movement, rapid masticationaction of mandible began and fell on the lateral side andbecamemotionless. The onset of these symptoms seemed tooccur faster (30–40 min) with increasing concentrations.None of the control injections of 1.0 ml HPLC grade waterresulted in fatality or symptoms of envenomation within96 h, the maximum period of observation. Larvae injectedwith VS died within 48 h and showed an LD50 value of768 ppm. However, in oral toxicity bioassay method, at 96 hobservation, dose-dependentmortalitywasobserved (40.0%,50.0%, 60.0%, 90.0% and 90.0% for 200, 400, 600, 800 and1000 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 digestedfood (ECD) of S. litura larvae fedwith VSmixed artificial diet(Table 1) were significantly lower (P< 0.05) than those ofan insect fed with non-VS diet. RGR, AD, ECI and ECD weredecreased in dose-dependent manner. However, RGR, AD,ECI and ECD increased from24 h to 96 h in control category.Highest RGR (F¼ 1.2; df¼ 9,30; P< 0.05), AD (F¼ 1.23;df¼ 9,30; P< 0.05), ECI (F¼ 1.42; df¼ 9,30; P< 0.05) andECD (F¼ 2.2; df¼ 9,30; P< 0.05) were observed at 96 h innon-venomous saliva category. But it decreased from 24 h

to 96 h in all VS concentrations. Lowest RGR (F¼ 5.2;df¼ 9,30; P< 0.05), AD (F¼ 2; df¼ 9,30; P< 0.05), ECI(F¼ 7.74; df¼ 9,30; P< 0.05) and ECD (F¼ 3.2; df¼ 9,30;P< 0.05) was observed at 96 h for 1000 ppm concentration.

3.3. Macromolecules of S. litura

Carbohydrate and lipid contents decreased with theincrease in the concentration of VS, whereas proteincontent increased significantly (F¼ 8.3; df¼ 2,12; P< 0.05)with the increasing concentration of R. marginatus VS. In VStreated insects, both carbohydrate (F¼ 21.0; df¼ 1,13;P< 0.05) and lipid (F¼ 1.4; df¼ 2,12; P< 0.05) contentswere 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 lipaseactivities in foregut, midgut and hindgut of control and VStreated S. litura larvae are shown in Table 3. Whilecomparing the control with other categories, the enzymeactivity was decreased from low to high concentrations.Among the three regions, all enzymes showed high activityin the midgut. There was reduction in amylase activity of1000 ppm concentration at 90% (F¼ 1.1; df¼ 2, 12;P< 0.05), 87% (F¼ 4.7; df¼ 2,12; P< 0.05) and 86% (F¼ 4.7;df¼ 2, 12; P< 0.05) in mid, hind and foregut respectivelywhile compared to the control. Similarly, in the sameconcentration, the lipase activity was reduced more inmidgut (82%) (F¼ 2.6; df¼ 2, 12; P< 0.05), than in foregut(79%) (F¼ 6.3; df¼ 2, 12; P< 0.05) and in hindgut (64%)(F¼ 4.3; df¼ 2, 12; P< 0.05). Furthermore, 23% (F¼ 7.9;df¼ 2, 12; P< 0.05), 61% (F¼ 4.0; df¼ 2, 12; P< 0.05), 33%(F¼ 1.2; df¼ 2, 12; P< 0.05), and 40% (F¼ 6.3; df¼ 2, 12;P< 0.05) reduction were more in the foregut than midgutand hindgut for invertase, acid phosphatase, AAT and tre-halase respectively. Similarly for AP, ASAT, protease and LD,53% (F¼ 7.3; df¼ 2, 12; P< 0.05), 65% (F¼ 5.9; df¼ 2, 12;P< 0.05), 30% (F¼ 1.7; df¼ 2,12; P< 0.05) and 25% (F¼ 9.4;df¼ 2,12; P< 0.05) reduction respectivelywere recorded inhindgut 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

% spreading ¼ ðNo of spreading PC or GC observedÞðTotal no of spreading and non� spreading PC or GC observedÞ � 100

Spreading inhibitory ratio ¼ð% spreading of PC or GC without venom treatment as thecontrol� %spreading of PC or GC with venom treatmentÞ

% spreading of PC or GC of the control� 100

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exposed to different doses of VS for 4 days are shown in theTable 4. GST activity was high and low in midgut (1.4 times)(F¼ 1.7; df¼ 3, 12; P< 0.05) (1371�13 mmoles/min/mgprotein) and hindgut (0.64 times) when compared to theforegut of the untreated control category. The activity wasrecorded by VS ingestion at midgut (1.033 times). Similar toGST activity, effects of VS doses in diet on esterase activitiesof the third instar stadium larvaewere alsomore significantin the midgut region (F¼ 1.7; df¼ 3,12; P< 0.05)(7.420� 0.004 mmoles/min/mg protein in 1000 ppm) thanforegut and hindgut of S. litura (Table 4). A significantincrease (P< 0.005) in GST and esterase activities werefound in the third instar stadium larvae of S. litura fed ondiets with various doses of VS in comparisonwith that fromthe untreated control animals. Generally the hindgut showslower GST and esterase activities than the foregut andmidgut of S. litura.

Esterase band patterns of S. litura were also observedusing non-denaturing polyacrylamide gel electrophoresis(Fig. 1). Three bands corresponding to masses of 50 kDa,37 kDa and 31 kDa were noticed in gel to emphasize thedetoxification activity in gut of S. litura. The band at 50 kDawas observed in all lanes including control, whereas 37 kDaand 31 kDa were appeared from 400 ppm and 600 ppm,after respectively (Table 5). When the concentration of VSincreased the esterase bands became thicker and appeared

very clearwhen compared to control. The intensity of all thebands was highly pronounced at 1000 ppm concentration.

3.6. Inhibition of haemocyte aggregation and spreading

When S. litura haemocytes were plated out at a suitabledensity (i.e. 1.5�105 haemocytes) in the well of a 12-wellstrip, they attached to the surface of the well within min,and the majority of plasmatocytes and granular cellsattached themselves within 1 h (Fig. 2a and b). During thistime, these haemocytes extended small pseudopods(Fig. 2c and d). After 240 min, the pseudopods were larger(Fig. 2e) and some migration towards the neighboring cellshad occurred, resulting in the appearance of small groupsof cells. During the same period, VS treatment causedsurface blebbing, and disintegration of the plasmamembrane in granulocytes (Fig. 2e and f). After 20 h, it wasclear that individual haemocytes had moved across thesurface of the well and had formed clearly distinguishablehaemocyte aggregation which composed of 10 or morecells. By contrast, in reduviid predator venom treatedcategory, inhibition of haemocyte aggregation wasobserved. The transmittance difference obtained fromvenom added wells doesn’t make much difference (0%),whereas in control category, it was 60%. Same results wererecorded when the experiment was repeated.

Plasmatocytes were easily identified by their markedspreading behavior on glass. The growth of pseudopodiacould be observed within 30 min after VS was added withhaemocytes of the host. Depending upon the venomconcentrations, it significantly inhibits the spreading of S.litura plasmatocytes and granulocytes. However, at 400 and600 ppm concentrations VS did not cause any change inplasmatocytes. On 30 min of observation, the spreadingpercentage of plasmatocytes was decreased in dose-dependent manner. The spreading percentage was 98% incontrol category and significantly decreased in 400 ppm(91%) (F¼ 25.43; df¼ 4, 18; P¼ 0.05), 500 ppm VS (59%)(F¼ 3.1; df¼ 4, 18; P¼ 0.05). At 240 min observation,

Table 2Macromolecular profile (mg/mg) of Spodoptera litura fed with Rhynocorismarginatus salivary venom mixed artificial diet at five different doses(ppm).

Venom concentrations(in ppm)

Protein Carbohydrate Lipid

Control 123.3� 0.2 83.8� 0.7 93.2� 1.8200 95.8� 0.2* 78.0� 0.9* 80.9� 0.6*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*800 179.6� 1.3NS 67.5� 0.4* 52.9� 0.4*1000 185.3� 3.1* 65.8� 1.2* 50.5� 0.5*

*The mean difference significant at 5% level (P< 0.05); NS, not significantat 5% level (P> 0.05).

Table 1Nutritional 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 ofingested; ECD, efficiency of conversion digested food.

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1000 ppm VS highly reduced the spreading of plasmato-cytes (57%) (F¼ 5.0; df¼ 3, 20; P¼ 0.05). Similarly, gran-ulocytes showed dose-dependent impact in spreadingactivity. For instance, the spreading percentage of GC waslow (52%) (F¼ 4.6; df¼ 3, 14; P¼ 0.05) and high (55%)(F¼ 11.8; df¼ 3, 14; P¼ 0.05) at 30 and 240 min of obser-vation, respectively in 1000 ppm (Fig. 3). Generally, thespreading inhibitory percentage (SIP) was higher at240 min of incubation than at 30 min incubation, except at400 ppm venom concentration (Fig. 3). Plasmatocytesshowed higher percentage of inhibition 39% (F¼ 25.59;df¼ 3, 11; P< 0.05) and 40% (F¼ 18.80; df¼ 3, 14; P< 0.05)in 30 and 240 min of incubation respectively, which issignificant 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 whencompared to that in the lower concentration.

4. Discussion

Results of this study show that the VS of the reduviidpredator is venomous to S. litura. Injection of venom causedwriggling and restless movement, rapid mastication actionof mandible, falling off lateral and becoming motionless for30 to 40 min and then resuming its routine activities.Mortality of S. liturawas observed at 48 and 96 h with LD50768 and 929 ppm for microinjection and oral administra-tion respectively. But, previously Corzo et al. (2001) testedtoxicity of three reduviids purified venom against S. litura.They have not recorded any toxicity against the insectsshowing that crude venom has more impact than purifiedpeptides like Ptul, Adl and Iobl.

In the nutritional indices experiments, R. marginatussalivary venom significantly reduced the RGR, AD, ECI andECD of S. litura as reported by Morales et al. (2007). This

Table 3Digestive enzyme activities in the foregut, midgut and hindgut of the third instar stadium larvae exposed to different does (in ppm) of salivary venom ofR. 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/h�1) 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/h�1) 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).

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indicates that the ingestion of VS blended diets exhibitedsome chronic toxic impact on the host larvae. Thepercentage reduction in RGR AD, ECI and ECD results froma food conversion deficiency, which reduces growth

perhaps through a diversion of energy from biomassproduction into detoxification enzyme (Wheeler et al.,2001) as observed in our study. Total body carbohydrateand lipid contents decreased in the VS mixed diet fed S.litura, whereas protein content increased significantly. Theincrease of protein quantity might be due to higherconversion of nitrogen to body matter and lower conver-sion of carbohydrates may be attributed to lesser nitrogenand greater carbohydrate utilization for maintenance.Moreover, increase of protein content clearly indicated theinfluence of VS on the protein synthesis of this insect.

R. marginatus VS reduced the digestive enzymes inforegut, midgut and hindgut of S. litura in dose-dependedmanner. Enzymes are catalysts that regulate all reactionsin all cells in the body (Penzlin, 2003). To date, severaldifferent classes of arthropod venomous salivary compo-nents have been shown to be insecticidal towards a rangeof economically important insect pests when tested inartificial diets (Fitches et al., 2004; Nghia et al., 2006;Mukherjee et al., 2006). There is, however, relatively littleknown about the factors controlling release of digestiveenzymes in insects (Lehane et al., 1996; Lwalaba et al.,2009). There are relatively few studies addressing thedirect effect of food in the gut on the release of digestiveenzymes (prandial regulation), and very few studies on thedirect effect of specific nutrients on enzyme release.However, there are almost no studies on the effect of VS on

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

Fig. 1. R. marginatus venomous saliva impact on the esterase band patternsof S. litura haemolymph using non-denaturing polyacrylamide gel electro-phoresis stained fast glue RR salt.

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Table 5Spodoptera liturawhole 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 stainedwith Commassie Brilliant Blue G250. Note the aggregated haemocytes (arrow head) monolayer (a) (40�) without salivary venom of R. marginatus. Haemocytesincubated 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�).

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the release of digestive enzymes. Such studies can actuallybe best carried out with short-term (4 days) feeding in thethird larval stage of S. liturawith VSmixed artificial diet andrecorded digestive and detoxication enzymes. This is thefirst report of the direct inhibition of enzyme release.Reduction of digestive enzyme secretion implies eithera direct effect (entry into the cell) or indirect effect (dockingon 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 alreadyconsiderable evidence for the effect of inhibitors onincreased or altered trypsin variants in the gut epithelialcells of Spodoptera frugiperda in response to acute feedingof SBTI and BTI-CMe (Lara et al., 2000; Brioschi et al., 2007).We believed and concluded that the insecticidal andenzymatic inhibition activities of the VS is due to a low[MW¼ 3798 Da (RmIT1) and 7500 Da (RmIT2)], and high(30 kDa) molecular weight components recorded from thevenomous saliva of this predator using MALDI–TOFMS andGC-MS respectively (unpublished data). Moreover, the VSof R. marginatus is even more toxic when injected directlyinto the insect haemocoel, which completely bypasses thegut. We think it is more likely that most of the reductions inenzyme activity we observe are secondary consequences ofa mixture of neurotoxic components in the venom(consistent with the neuromuscular abnormalities weobserved after injection of VS).

Generally activities of asparate (AAT) and alanine(ALAT) aminotransferase was low in S. litura larvae afterexposed to different doses of VS of the reduviid predatorfor four days. It was indicated that specific types ofproteins are synthesized in the haemolymph from

precursors of amino acids by enzymatic transaminationreactions. Glutamic acid is formed by amino transfer fromaspartic acid by AAT or from alanine by ALAT. R. margin-atus salivary toxins interfere with the transformation ofthe amino acids and hence the AAT and ALAT leveldecreased. Furthermore, normally, organisms primarilyspent more energy into the physiological responses withhigh sensitivity and efficiency due to limited energyreserves (Stone et al., 2002). Therefore, more energy mightbe transferred to elevate the GST, esterase and LDH inorder to defend rapidly and stably the toxic responsecaused by toxin, indicated in S. litura these enzymes wereacts as the prime detoxication enzymes rather than AATand ALAT. Aminotransferases are bioindicators of gluco-neogenesis, the formation of carbohydrates from aminoacids. The AST and ALT serve as a strategic linkage betweenthe carbohydrates and protein metabolism that are knownto be altered during various physiological and pathologicalconditions (Zibaee et al., 2008). There was no significantincrease of ALT and AST activities show the utilization ofprotein for gluconeogenesis was very low. It was alsoevidence in the level of whole body protein and carbo-hydrate (Table 2). When ingested by larvae, toxin proteinsbind to specific receptors in the midgut region and toxinbinding in susceptible insects disrupts the insect metab-olism, thereby causing overall toxic effects and ultimatelyresulting in larval death.

There are no reports in the literature describing the hosthaemocyte aggregation behavior of reduviid predatorvenom. These results indicate that the haemocyte anti-aggregation factor(s) present in R. marginatus venomprevent aggregation occurring, rather than breaking upaggregates after they have formed. This helps the predator

Fig. 3. The spreading (SP) and spreading inhibitory percentage (SIP) of plasmatocytes and granulocytes with various concentrations of venom in vivo conditionsafter 30 and 240 min of incubation at 27 �C.

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make perfect extra oral digestion in haemocoel without anyblockage. Similarly Pimpla hypochondriaca venom reducedthe spreading behavior of Lacanobia oleracea haemocytesand abolishes the ability of these cells to migrate and formaggregates in-vitro (Parkinson et al., 2002; Dani et al.,2005). Presently, aggregation and spreading activity weredetected for crude venom that has previously been shownto possess antimicrobial activity of this predator’s crudevenom in-vitro (Sahayaraj et al., 2006b).

5. Conclusion

The incorporationofR.marginatus salivaryvenominto theartificial diet or directly injected into the body of S. liturareduced the digestive physiology (food consumption andhydrolase enzyme levels). Furthermore, R. marginatus VSmight have some toxic anti-aggregation factors whichinteractwithhosthaemolymphandhaemocytes activities bysuppressing the immune response. Thus it alters the physi-ological status of the prey for better extra oral digestion. Thesalivary venom affects the prey’s haemocytes aggregationand spreading behavior, nervous systems (wriggling move-ments/ later motionless), nutritional indices, enzyme activi-ties and increasing the induction of detoxification enzymes.Further studies are inprogress to identify the toxinsaswell astheir biologically active components in the salivary venomofthe reduviid predator R. marginatus.

Conflict of interest

None.

Acknowledgement

K. Sahayaraj gratefully acknowledges the Department ofScience and Technology (GS1) (Ref. No. SR/SO/AS – 33/2006), New Delhi for the financial assistance. We thankRev. Fr. Dr. Alphonse Manickam, S.J., Principal, St. Xavier’sCollege, Palayamkottai for the laboratory facilities andencouragement. We are grateful to Dr. Usha Rani (India)for her critical review of an early draft of the manuscript.

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