Susceptibility and bioassay tests
the end of this Unit you should be able to:
measure the level of insecticide resistance in a vector
determine the residual efficacy of an insecticide deposit
on a sprayed surface or material at a specified time after the spraying One of the most important reasons to sample vector
populations is to determine their susceptibility to insecticides. When insecticides are used in malaria control, it is important that you monitor changes in the
susceptibility level of the target vectors from time to time. The residual efficacy of the insecticide used should also be
determined at intervals after its application or use. In this Unit, you will learn the skills required to carry out these
4.1 Susceptibility tests
Physiological resistance to insecticides
Susceptibility tests are carried out to determine the proportion of the
vector population that is physiologically resistant to a particular insecticide.
Physiological resistance to insecticides has been defined as the ‘ability
of a population of insects to tolerate doses of an insecticide which would prove lethal to the majority of
individuals in a normal population of the same species.
The efficacy of indoor residual spraying (IRS) and insecticide impregnated
nets (ITNs) depends, among other things, on the proportion of vectors resting on the sprayed surface and on the
susceptibility of the vectors to the insecticide used. It is therefore important to monitor the development and
extent of insecticide resistance in a given vector population.
Susceptibility test kit (including exposure/holding tubes, copper
and silver rings, insecticide-impregnated filter papers, oil-impregnated control papers, sucking tubes),
thermometer, wooden box with large holes, towels, cotton wool, paper cups with cover nets, rubber
bands, markers or wax pencils, mosquito cage.
Method of determining the susceptibility of adult mosquitoes
The standardized WHO method involves checking the mortality of several
female Anopheles of
a known species exposed in special tubes to filter papers impregnated with a lethal concentration (known as discriminating dose) of a given insecticide dissolved in mineral oil.
as many mosquitoes by means of an aspirator (Fig. 4.1a).
Transfer 15-25 fed mosquitoes to a special plastic holding tube lined with insecticide free paper (Fig. 4.1b).
Connect a plastic tube lined with filter paper impregnated with mineral oil (used as control) with the holding
tube and transfer 15-25 mosquitoes to the tube through a hole in the slide between the 2 tubes (Fig. 4.1c); also transfer
the same number of osquitoes to plastic tube with insecticide-impregnated filter paper. (The filter papers are held in place
by special rings). Use separate sucking tubes to transfer mosquitoes to the exposure and control tubes to avoid contamination.
Close the slide and allow the exposure and control tubes to stand upright for the determined period, which is usually
one hour (Fig. 4.1d).
After the exposure period transfer the mosquitoes back to the holding tube, which should stand upright for 24 hours,
with a piece of moist cotton wool on the gauze end and in a wooden box with large holes for ventilation and covered with a
damp towel (Fig. 4.1e); you should monitor temperature and humidity in the box.
Count dead mosquitoes killed by the contact with the insecticide and those killed in the control tube at the end
of this recovery period.
You will need four replicates of the experiment to calculate % mortality in the exposure and control tubes.
The rates of mortality are:
mortality where C = (number of dead mosquitoes)/(total number of mosquitoes)
in control tube
mortality where E = (number of dead mosquitoes)/(total number of mosquitoes) in tube with insecticide
Where, E is the (uncorrected) exposure mortality expressed in % and C is
the control mortality expressed in %.
For instance, if control mortality C is 10% and crude exposure mortality
is 40%, the corrected exposure mortality is [(40 – 10)/100
– 10)] * 100 =
33% . If control mortality is greater than 20%, the experiment should
4.2 Bioassay tests
The residual efficacy of an insecticide on a sprayed
surface is determined by bioassay
tests. This is done by checking mortality of the target mosquito vector exposed to the sprayed
surface at intervals of weeks or months after the spraying. This technique can be also used to evaluate the quality
of a residual spraying operation. It is also used to dermine residual efficacy of an insecticide on bed nets. This can
be used to decide when to re-treat bed nets and also to assess the quality of treatment.
Bioassay kit (including plastic cones, adhesive sponge tape, bent
sucking tube, normal sucking tubes), cardboard paper, small nails, hammer, cotton wool, paper cups with cover
nets, rubber bands, markers, mosquito cage, wooden box with large holes, towels The plastic
cone and the special (bent) sucking tube are shown in Fig. 4.2.
Residual efficacy of insecticide
on sprayed surfaces
Residual efficacy of insecticide
on bed nets
The bioassay procedure for insecticide-treated bed nets is similar
to the procedure used for sprayed walls, except that the cone is attached to the fabric with a rubber band,
and the mosquitoes are usually exposed only for three minutes.
Susceptibility tests: You will be provided with live, fed female
mosquitoes in the laboratory. In pairs, prepare holding tubes and introduce 15 fed females to the holding
tubes. When all groups have introduced the mosquitoes, half the groups will prepare exposure
tubes and the other half control tubes. After all tubes have been prepared, all groups will be asked to transfer
the mosquitoes to the exposure and control tubes and to label each tube with the group number indicating whether it is
an exposure or a control tube, and the time of the day. After one hour of exposure, you should transfer
mosquitoes back to the holding tubes for 24 hours of observation. After 24 hours, all results will be combined
to calculate mortality rates.
Bioassay tests: Again, as in Exercise 4.1, you will form pairs and
half the groups will install cones on treated bed nets and half on untreated bed nets. Each pair should transfer
10 fed mosquitoes to the cones, and after three minutes the mosquitoes should be taken out and placed in paper
cups for 24 hours of observation. Again, the results will be combined for calculation of mortality rates.
(Please visit the WHO website for full & detailed
document) to complete your referene.