1. Insecticide Resistance and Approaches to Resistance Management
PMI AIRS Entomology Training
Dereje Dengela
PMI AIRS Project
July 7, 2015

2. INSECTICIDES
Remain the most important element of malaria vector control tools (IRS, LLINS and larvicides)
Four main classes are recommended
Organochlorines, organophosphates, carbamates and pyrethroids
12 insecticides for IRS
1 Orgnochlorine, 3 OPs, 2 carbamates and 6 pyrethroids

3. Insecticides Recommended for IRS

4. LLINs

5. INSECTCIDE RESISTANCE
Many vector species of public health importance including mosquitoes have developed resistance to one or more insecticides
What is insecticide resistance?
WHO’s definition
“Is the ability of certain individuals to tolerate doses of toxicants which would prove lethal to the majority of individuals in a normal population of the same species”
Phenotypic resistance is the phenomenon most commonly referred to in the public health. Molecular genotyping of resistance is the identification of the underlying genes that confer the inherited resistance.. Identification of the underlying genes provides evidence of the underlying evolutionary process. It provides understanding of both the degree of resistance expressed in individual insects with the resistance gene and the freq. of such insects in the population.

6. INSECTCIDE RESISTANCE
Insecticide resistance Action Committee (IRAC) defines resistance as
“The selection of heritable characteristic in an insect population that results in the repeated failure of an insecticide product to provide the intended level of control when used as recommended”
based on operational performance of the insecticide
Resistance leading to control failure is the phenomenon most commonly referred to in agriculture. National malaria control programs shouldn’t ,however, wait for control failure to occur before implementing strategies to manage insecticide resistance. There is no acceptable level of control failure in public health, and waiting could result in delaying action until it is too late.

7. INSECTCIDE RESISTANCE
Resistance is a natural phenomenon. Resistance genes appear through random mutations in which individuals are born "resistant”
Individuals who are substantially less susceptible may be present, generally at low frequencies
The emergence of insecticide resistance in a vector population is an evolutionary phenomenon.

8. INSECTCIDE RESISTANCE
It is assumed that wild –type genes more fit than resistant genes
Fitness cost: Resistant insects- lower productivity, longevity and robustness
In the presence of the insecticide, it is the less susceptible individuals that survive and reproduce
Therefore, the pre-existing mechanisms are selected driving the evolution of resistant
Populations, not species, become resistant (why?)
Resistance is a concept which applies to populations, which are to a degree isolated from the reminder of species concerned. It is also comparative term that relates resistance population to a more susceptible normal population.

10. Resistance Mechanisms(RM)
RM are grouped in to four categories :
Metabolic, target site, reduced penetration and behavioral
Metabolic resistance:
most common resistance mechanism that occurs in insects.
it is based on enzyme systems.
It occurs when increased or modified activity of an enzyme system prevent an insecticide from reaching its intended site of action.
Nearly all of the strains of culex quinquefasciatus which resists a broad range of OP insecticide have been found to posses multiple copies of a gene for esterases, enabling them to overproduce this type of enzyme. In contrast strains of malathion resistant Anopheles have been found with non-elevated levels of an altered form of esterase the specifically metabolizes the OP malathion at a much faster rate than that in susceptible individuals. Metabolic resistance mechanisms have been identified in vector populations for all major classes of insecticides.

11. Resistance Mechanisms(RM)
The three main enzyme systems are: esterases, mono-oxygenases and glutathione S-transferases
(GST).
2. Target-site resistance:
Second most common resistance mechanism.
Insecticides generally act at specific site within the insect( the nervous system)
occurs when the site of action of an insecticide is modified in a resistant strains, such that the insecticide no longer binds effectively
Result: the insect is unaffected or less affected by the insecticide. Kdr=DDT&PY, Ace-1= OP and carbamates.
Reduced susceptibility to Pys conferred by kdr mutations has been confirmed in An. gambiae in West, Central and East Africa.

13. Resistance Mechanisms(RM)
3. Reduced penetration:
Reduces uptake of insecticide due to the modification of the insect cuticle that prevent or slow absorption of insecticides.
Only one study has suggested correlation between cuticle thickness and pyrethroid resistance in An. funestus.
if cuticular resistance emerges, it might have significant impact when combined with other resistance mechanisms.

14. Resistance Mechanisms(RM)
4. Behavioral Resistance
Any modification in insects behavior that helps to avoid the lethal effects of insecticides.
Changes in vectors resting and feeding behavior to minimize contacts with insecticides.
No enough data whether behavioral avoidance is is genetic or adaptive trait.
Eg An. farauti. In Solomon Islands and New Guinea

15. Insecticide Resistance
Cross-resistance: One mechanism confers resistance to more than one insecticide
Example: kdr confers resistance to DDT and pyrethroids
Multiple resistance: occurs when several different resistance mechanisms are present simultaneously in resistant insects.
selection from different insecticide applications
Cross resistance often occurs between insecticide class that have the same mode of action for killing vectors. For example, if a resistance gene creates a change in target site in a vector, it is likely to affect any other insecticides that attack the same target sites, thus conferring the cross resistance. Similarly, an alteration to an enzyme that affects susceptibility to one insecticide may result in cross-resistance to another. Eg, In metabolic resistance, cross- resistance between PY and carbamates associated with mutations in cytochrome P450 enzymes detected. Target site and metabolic resistance can both occur in the same vector population and some times within the same individual. The two types of resistance appear to have different capacities to reduce the effectiveness of insecticide-based vector control interventions, with metabolic resistance being the stronger and more worrying mechanism.

16. Factors that influence resistance development
1)Frequency of application: many applications over a large geographic area quicken the development of resistance
- overuse and misuse
2) Persistence of residues: long persistence of the insecticides for IRS and LLINS have a strong selection effect leading to quicker resistance

17. Factors that influence resistance development
3) Rates of reproduction: short life cycle and high rate of reproduction speeds resistance development
Mosquitoes have short life cycle and high fecundity
4) Population isolation: open population allows migration of susceptible individuals which will have diluting effect

18. Distribution of Insecticide Resistance PMI countries
Wide spread resistance to PY and DDT.
High frequency of resistance reported from West and central Africa.
Resistance to all four classes of insecticide reported from East Africa eg. Ethiopia.
High frequency of metabolic resistance reported from Malawi, SA, Zimbabwe , Zambia and Mozambique

20. Impact of insecticide resistance
May lead to reduced effectiveness
-lower vector mortality, increased blood feeding and limited increase in malaria transmission
Failure of vector control
- When an intervention has virtually no effect on transmission or fails to prevent an uncontrolled resurgence in malaria cases.
eg. South Africa in 2000 With An. funesuts ( metabolic resistance)
Most experts agree that if nothing is done to reduce selection pressure, insecticide resistance will ultimately have an operational impact that will lead to wide spread control failure. In West Africa high frequency of kdr resistance was not accompanied by in obvious attributable increase in the number of malaria cases. But several reports indicate that resistance could have an operational impact and lead to control failure.
The operational impact of resistance is difficult to measure( The impact of insecticide resistance on vector control operations should ideally be measured from an epidemiological perspective. The link to epidemiology of malaria is, however, difficult to establish: (1) Many confounding factors in both the implementation of vector control and factors unrelated to vector control could contribute to an increase in number of malaria cases (2) It is not possible to design a fully rigorous experimental trail to measure the impact of resistance as one can’t randomize for resistance ( allocate resistance randomly to some places and with hold it from others) (3) detection of an increase in the number of malaria cases requires effective diagnosis and epidemiological monitoring an reporting, which are sometimes in adequate.

21. Monitoring Insecticide Resistance
Regular resistance monitoring is essential :
to understand the evolution and threat of IR among malaria vectors
to detect resistance individuals when they are at low frequency in the population so that IRM can effectively be introduced.
to enable timely and rational decision making when selecting insecticides for VC.
Resistance monitoring should include all classes if possible.

22. IR Monitoring Methods
Three methods can be used to test and monitor resistance development.
These are
1. WHO tube test and or CDC Bottle bioassay/s.
2. Biochemical test methods
3. Molecular test methods.
Each method provides a different type and depth of information. They are complementary and the choice depends on the information needed , cost, and availability of facilities to perform the activity.

23. WHO/ CDC susceptibility test methods
easy to use in the field and interpret the data.
standard method for routine monitoring.
Identify presence of resistance once it is at the detectable level.
may not detect resistance if the frequency is low.
Require at least 100 mosquitoes per test
Can’t help to identify underlying mechanism of resistance but CDC bottle bioassay with synergists can be use to give us indication of metabolic resistance.

24. Advantages of Molecular and Biochemical test methods over bioassays
Can detect resistance at very low frequency within population
Can indicate the presence of heterozygous with recessive genes that are not detected through bioassays
Help to identify the underlying mechanism of resistance.
able to identify genotypes( heterozygous or homozygous resistance)
use to monitor trends in resistance gene frequency over time.
No need to test large number of mosquitoes.

25. Molecular test methods
Greatly complement Bioassays
useful to monitor trends in resistance gene frequency over time.
used to determine the frequency of kdr/ace-1 alleles.
Microarray : can be used to screen metabolic resistance from field caught mosquitoes.
Highly sensitive and specific as compared to biochemical assays.

26. Biochemical test methods
These methods rely on enzymatic action, which may or my not reflect metabolism of insecticidal compounds.
Typically P450 activity is measured using o- deethylation of 7-ethoxycoumarin
GST activity is measured using chlorodinitrobenezene(CDNB) or DCNB.
Non specific esterase activity using 1- nephthylacetate.

27. Strategies for Managing Resistance
Insecticide based in conjunction with non insecticide VC method
1. Conserve susceptible genes in the population by reducing selection pressure
Use non-persistent insecticides
Replacement of residual insecticides by non-residual insecticides
Problem?
Selected application of insecticides
Local (targeting hot spots) rather than area wide application
Partial application to selected resting sites
Seasonal application
use synergists to inhibit detoxification enzymes
Suppress metabolic resistance
e. g Piperonyl butoxide

28. Strategies for Managing Resistance
2. Rotations of insecticides
apply insecticides having different mode of action at different times
kill those insects that are developing resistance to one compound by using a different one

29. Strategies for Managing Resistance
3. Use mixture of insecticides :
-a single formulation containing two or more insecticides for IRS mixed in the sprayer
- LLINs treated with different insecticides
- Combined use of IRS and LLINs with different insecticides having different mode of action

30. Strategies for Managing Resistance
4. Mosaic application
Application of different insecticides with different modes of action targeting same population of a vector
e.g three different insecticides in three houses at a village

31. Thank you!