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Insecticidal Bioassay

Pesticides, including residual grain protectants and fumigants are used extensively in grain industry. Nearly one thousand species of insects have been associated with stored products throughout the world, of which the majority belong to Coleoptera (60%) and Lepidoptera (8-9%). Resistance to one or more of these materials has occurred in most major pest species. This relentless development of resistance is a serious threat to the future use of these materials and consequently, there is an urgent need to develop economically safer and sounder pest control techniques.

Method: Impregnated Filter Paper Method

The test samples are loaded on filter paper which are then exposed to different species of insects. After incubation the number of survival are determined and percent growth inhibition is calculated.

Species used in the assay:

1) Tribolium castaneum (Red flour beetle)
2) Sitophilus oryzea (Rice weevil)
3) Rhyzopertha dominica (Lesser grain borer)
4) Trogoderma granarium (Khapra beetle)
5) Callosobruchus analis (Pulse beetle)

Flour, wheat and pulse are used as rearing media.

Nature of Compound Tested
Crude plant extracts

Material and Method

Requirements: Test insects (Tribolium castaneum, Sitophilus oryzea, Rhyzopertha dominica, Trogoderma granarium and Callosobruchus analis), volatile organic solvent (ethanol, methanol, acetone etc.),standard insecticide (Permethrin), petri plates (9 cm diameter), micropipette (1000 µl), growth chamber, test sample, filter paper, glass vials, brush.

Preparation of Test Sample

• Crude sample: 200 mg test sample + 3 ml volatile solvent

• Pure sample: 20 mg test sample + 3 ml volatile solvent

Rearing Technique:
The stored grain pests are reared in the laboratory under controlled conditions (temperature and humidity) in plastic bottles containing sterile breeding media. Insects of uniform age and size are used for the experiment.

Procedure

Day-1

• Cut the filter paper according to the size of petri plate (9 cm or 90 mm) and put them in the plate.

• Load the whole sample over the filter paper with the help of micropipette.

• Leave the plates for 24 hours to evaporate the solvent completely.

Day-2

• Next day (after the evaporation of solvent) put 10 insects of each specie in each plate (test and control) with the help of a clean brush. Take healthy and active insects of same size and age.

• Incubate the plates at 27 °C for 24 hours with 50% relative humidity in growth chamber.

Day-3

• Assess the survival of the insects (count the number of survivals of each specie)

• Calculate the Percentage Inhibition or Percentage Mortality [5] with the help of the following formula:}

Percentage Mortality = 100 - No. of insects alive in test X 100
No. of insects alive in control

Control

Run positive and negative control with test compounds.
Positive control: It contains standard insecticide (Permethrin) and test insects.
Negative control: It contains volatile solvent and the test insects.

Name of Insects	+ ve Control		- ve Control		Test sample
Name of Insects	No. of insects alive	% Mortality	No. of insects alive	% Mortality	No. of insect alive	% Mortality
Tribolium castaneum
Sitophilus oryzea
Rhyzopertha dominica
Trogoderma granarium
Callosobruchus analis

Key: Concentration are reported in µg/c