Insecticidal activity of venomous saliva from Rhynocoris fuscipes ( Reduviidae ) against Spodoptera litura and Helicoverpa armigera by microinjection and oral administration

Rhynocoris fuscipes is a potential predator of many economically important pests in India. In the present study, its venomous saliva (VS) was collected by milking and diluted with HPLC grade water to different concentrations (200, 400, 600, 800 and 1000 ppm). Microinjection of Rhynocoris fuscipes VS was more toxic than its oral administration in Helicoverpa armigera (cotton bollworm) and Spodoptera litura (tobacco cutworm). Thus, R. fuscipes VS was found to be toxic to third instar S. litura and H. armigera with respective LD50s of 846.35 and 861.60 ppm/larva at 96 hours after microinjection. The current results showed that VS of Rhynocoris fuscipes caused mortality of H. armigera and S. litura. Active peptides from VS may be isolated, identified and assessed for their impact in order to ascertain how they alter the physiology of these pests, information that could be applicable in pest management programs.


INTRODUCTION
Arthropod venom 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 previously suggested (1)(2)(3)(4)(5).The venom of poisonous predators has novel peptides that have been isolated from snakes, scorpions, marine cone snails, spider and other animals including predatory insects.In arthropods, copious information is available on spiders and parasitoids.Among the predatory hemipterans, reduviids constitute an important predator on account of presenting worldwide distribution and having been utilized in the biological 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 prey is then used as food by the reduviid predator (6)(7)(8).Such unique paralytic activity was due to the presence, in the venom of reduviid predators, of novel neurotoxic compounds, only a few of which have been isolated and characterized to date (2,(9)(10)(11)(12).
The tobacco caterpillar, Spodoptera litura (Fabricius) is one of the most destructive pests and consists of about 120 species of plants belonging to 44 families (13,14).The chemical control of S. litura presents limitations due to its resistance against many insecticides including pyrethroids (15,16).The cotton bollworm, Helicoverpa armigera (Hübner), one of the most important pests, affects crop production globally (17).Insecticidal resistance of this pest has also been reported in the literature (18,19).
The reduviid predator, Rhynocoris fuscipes Fab.(Hemiptera: Reduviidae) is an entomophagous insect distributed in many agroecosystems and feeds on more than 40 economically important insect pests in India (8).The potential of R. fuscipes as a biological control agent under laboratory and field conditions has been described previously (20)(21)(22)(23)(24). Maran (23) studied the paralytic potential of R. fuscipes salivary gland extract against selected pests.However, no one has studied the toxicological, physiological and immunological activities of this reduviid salivary venom on any pests.In the present work, we evaluated for the first time the biological activities of adult R. fuscipes salivary venom against H. armigera and S. litura third instar larvae.

Insect Collection and Rearing
Laboratory colonies of the host species, H. armigera and S. litura, and the reduviid predator were established from individuals that were collected from cotton fields in Tamil Nadu, India.R. fuscipes were reared on the larvae of the host, S. litura at 30.0 ± 1°C and 70-80% relative humidity with a photoperiod of 11/13 hours darkness/ light.The host colonies were maintained on fresh cotton leaves up to second instars, and then transferred to the freshly prepared artificial diet for further rearing.

Venom Collection and Preparation
The venomous saliva (VS) was collected from the ten-day-old freshly emerged adult reduviid as described previously (25,26).The salivary venom collected from more than 50 reduviid predators was pooled and then stored on ice until used in our toxicity experiments within 12 hours.VS was collected from each predator only once.Concentrations of the VS (200, 400, 600, 800 and 1000 ppm) were prepared by diluting with HPLCgrade water (Qualigens, India).

Determination of Toxicity
The toxicity of R. fuscipes VS was evaluated against third instar larvae of H. armigera and S. litura using microinjection and oral toxicity methods (27,28).In the microinjection method, different VS concentrations were tested for toxicity by injecting 1.0 µL of VS into each third stage S. litura larva 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 height x 3.8 cm diameter) and maintained in a BOD incubator on an artificial diet.Larval mortality was observed at 24 hours intervals up to 96 hours.Behavioral changes, if any, in the host insect were observed and recorded up to three hours post-injection.A soybean seed-based artificial diet was used to assay VS by oral delivery against newly hatched third stage S. litura larvae (starved for six hours prior to exposure to diet) (29).
For each treatment, six larvae were maintained in a sterilized plastic container containing moist filter paper to prevent diet desiccation.For the oral toxicity bioassay, 1 mL of VS of different concentrations (200, 400, 600, 800 and 1000 ppm) was blended thoroughly with 100 mg of artificial diet separately and provided to the larvae.Control diets contained an equal amount of HPLC-grade water.Survival was monitored daily up to 96 hours.A similar procedure was used for H. armigera third instar larvae.

Statistical Analysis
The LD 50 value was calculated by the method of Finney (29).Control animal data were compared with different VS concentrations.All data were subjected to one-way ANOVA and post hoc Tukey's test using the statistical software SPSS (Version 11.5).The significance level was set at 5 or 1%.

RESULTS
Individual H. armigera and S. litura injected with minimum concentrations (200 and 400 ppm) exhibited no initial response; but within 90 minutes the following sequence was observed: wriggling, restless movement, rapid mastication of the mandible, lateral fall and, finally, motionlessness.The onset of these symptoms seemed to occurring faster (30 to 40 minutes) with increasing concentrations.None of the control injections of 1.0 µL HPLC-grade water resulted in fatality or symptoms of envenomation within 96 hours, the maximum period of observation.

DISCUSSION
In the present study, the saliva from the reduviid predator was determined to be venomous Toxicity of three reduviids (Peirates turpi, Agriosphodrus dohrni and Isyndus obsurus) venoms were tested against S. litura larvae.The fact that they did not show any toxicity against the insects indicates that crude venom has more impact than such purified peptides as Ptul, Adl and Iobl (2).We concluded that the toxic nature of the VS is due to its protein content.However, isolation and identification VS peptides will be required in the near future.Previous reports of the toxic nature of Platymeris rhadamanthus, Peirates affinis and Haematorrhophus nigroviolaceous, Catamiarus brevipennis salivary venoms support our findings (9,10,12,25).Injection of such venoms caused wriggling and restless movement, rapid mastication action of the mandible, a lateral fall and motionlessness for 30 to 40 minutes prior to resuming its routine activities.The paralysis was due to medium and low molecular weight neurotoxin (proteins).Edwards (9) reported that the salivary proteins of P. rhadamanthus provoke rapid loss of nervous conduction followed by loss of muscle contraction and relaxation.The study indicates that the venomous saliva is more toxic when injected directly into the insect haemocoel, completely bypassing the gut, which might be: consistent with the neuromuscular abnormalities we observed after injection of venom, or secondary consequences of a mixture of neurotoxic components in the venomous saliva of the reduviid.It is suggested to isolate, purify and identify the insecticidal compounds from reduviid VS.