BIOASSAY AND PLANT SCREENING SECTION
INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES
HEJ RESEARCH INSTITUTE OF CHEMISTRY
UNIVERSITY OF KARACHI
INSECTICIDAL ACTIVITY
Introduction
Freedom from insect infestation and contamination has become an important
consideration in storage of grain and to maintain a high quality product [1]. 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%).
Pesticides, including residual grain protectants and fumigants are used extensively in
grain industry. 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 more sound pest control techniques.
Commonly used bioassay techniques for the determination of insecticidal activity are:
Insecticide impregnated dust on grain [2]
Direct spray on grain [2]
Impregnated filter paper test [3]
Toxicity on various surfaces [4]
Common Stored Grain Pests
1. Red flour beetle, Tribolium castaneum
Rearing temperature 30 °C
Relative humidity 50 – 70%
Rearing media Wheat flour
Life cycle 22-25 days
2. Rice weevil, Sitophilus oryzea
Rearing temperature 25 °C
Relative humidity 50 – 70%
Rearing media Wheat and rice
Life cycle 26-28 days
3. Lesser grain borer, Rhyzopertha dominica
Rearing temperature 30 °C
Relative humidity 50 – 70%
Rearing media Wheat and gram seeds
Life cycle 30 days
4. Khapra beetle, Trogoderma granarium
Rearing temperature 30 °C
Relative humidity 50 – 70%
Rearing media Wheat
Life cycle 30 days
5. Pulse beetle, Callosobruchus analis
Rearing temperature 25-35 °C
Relative humidity 50 – 70%
Rearing media Mung seeds
Life cycle 25-30 days
IMPREGNATED FILTER PAPER METHOD
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
a) Crude sample: 200 mg test sample + 3 ml volatile solvent
b) 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
1. Cut the filter paper according to the size of petri plate (9 cm or 90 mm) and put
them in the plate.
2. Load the whole sample over the filter paper with the help of micropipette.
3. Leave the plates for 24 hours to evaporate the solvent completely.
Day-2
1. 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.
2. Incubate the plates at 27 °C for 24 hours with 50% relative humidity in growth
chamber.
Day-3
1. Assess the survival of the insects (count the number of survivals of each specie)
2. 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. We use
that concentration of insecticide, which is effective against all test insects.
Negative control: It contains volatile solvent and the test insects.
Name of Insects + ve Control - ve Control Test sample
No. of % No. of % No. of %
insects Mortality insects Mortality insect Mortality
alive alive alive
Tribolium castaneum
Sitophilus oryzea
Rhyzopertha dominica
Trogoderma granarium
Callosobruchus analis
Key: Concentration of test sample ----------- µg/cm2 (crude)
Concentration of test sample ------------- µg/cm2 (pure)
Concentration of Std. Drug ----------- µg/cm2
References
1. Collins, P. J. (1998) Resistance to grain protectants and fumigants in insect pests
of stored products in Australia. Presented in Australian Postharvest Technical
conference (1998) pp. 55-57.
2. Champ, B. R. (1981) Methods for detecting Pesticide resistance in storage pests.
Proc. Aust. Dev. Asst. Course on preservation of stored cereals, pp. 691-696.
3. Tabassum, R., Naqvi, S. N. H., Azmi, M. A., Nurulain, S. M. and Khan, M. F.
(1997) Residual effect of a neem fraction, nimolicine and an insect growth
regulator, dimilin, against stored grain pest Callosobruchus analis. [Link]
Congr. Zool., vol. 17, pp. 165-170.
4. Atta-ur-Rahman, Choudhary, M. I. and William, J. T., (2001) Bioassay techniques
for drug development. Harward academic Publisher, pp. 67-68.