Different methods for monitoring insecticide resistance in mosquito vectors and their limitations.
Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world’s population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa. Important global efforts have been made to eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this extraordinary success is mainly due the use of long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide. Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions. To help establish suitable resistance management strategies, it is vital to better understand the distribution of resistance, its mechanisms and impact on effectiveness of control interventions and malaria transmission. In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors as well as its impact on malaria transmission, and discuss the molecular mechanisms and future perspectives.
- insecticide resistance
- bed nets
- metabolic resistance
- cytochrome P450
- knockdown resistance
In 1993, Steven Spielberg produced ‘Jurassic Park’, one of the most internationally acclaimed movies at that time. This science-fiction story is based on the cloning of dinosaurs using its DNA from mosquitoes that had been preserved in amber. Although the idea is brilliant, the technical limitations to get entire genome of dinosaurs from ancient DNA make it impossible . However, the movie is right on one fact that mosquitoes existed at the same time as dinosaurs probably biting them as other animals before evolving to become human biters . But only few mosquitoes have specialised in biting humans (anthropophily), although those that succeeded have caused devastating consequences to mankind. From all diseases that mosquitoes can transmit, malaria has been and still is the one with the greatest health and socioeconomic impact, from the ancient Egypt to present time . For example, malaria has been suggested as one of the causes of the death of the great Tutankhamun , one of the Egypt’s famous pharaoh. Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world’s population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa . Recent global efforts have been made to control and eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this success is mainly due the use of insecticide-treated nets (ITNs), long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide . Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions . Insecticide resistance is spreading globally. Currently, of 73 countries with ongoing malaria transmission that provided data, 60 countries reported resistance to at least one class of insecticides, while 50 reported resistance to two or more insecticide classes . In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors, as well as its impact on the epidemiology, and discuss the molecular mechanisms and future perspectives.
2. Insecticide resistance in malaria vectors
The term insecticide resistance is defined as the ability of an insect to withstand the effects of an insecticide by becoming resistant to its toxic effects by means of natural selection and mutations . Repeated exposure to insecticides selects individuals possessing biochemical machineries that can detoxify the insecticides more rapidly or are less sensitive to it . These individual survivors could then pass the resistance mechanism to the successive generations resulting in pest populations that are more resistant.
2.1. Development of the insecticide resistance
Resistance has been observed in more than 500 insect species worldwide , including malaria mosquitoes. Mosquitoes are typical R-strategists (animals that reproduce fast and produce a large number of offspring), and can adapt fast to environmental changes. As a consequence of this and the widespread use of insecticides in agriculture and public health, resistance has arisen relatively rapidly in malaria vectors. Insecticide-resistant phenotypes are favoured where mosquitoes are exposed to sub-lethal doses of the insecticide. Under these conditions, resistant individuals have a better chance to survive and reproduce; this means selection pressure towards resistant populations. Such conditions can result from vector control through insecticide decay (on treated walls or nets) or bad spraying technique. Insecticide resistance was first reported in malaria vectors in the 1950s , and resistance to dichlorodiphenyltrichloroethane (DDT) and pyrethroids is now widespread . Resistance is predicted to impair malaria control efforts but evidences from field studies remain limited and potentially conflicting . To date, malaria vectors have developed resistance to the main chemical classes used in public health, i.e., pyrethroids (PYs), organochlorines (OCs), carbamates (CAs) and organophosphates (OPs). Although public health use of insecticide has an impact on the development of resistance in mosquitoes, one key source of resistance in malaria vectors remains the massive use of insecticides for control of agricultural pests . Other chemicals and factors aside from insecticides may create a selective environment, which favours build-up of resistant populations .
2.2. Monitoring of insecticide resistance
Surveillance to monitor the emergence and spread of resistance is an essential step in insecticide resistance management (IRM) providing baseline data for programme planning and choice of insecticide [15, 16]. Effective resistance monitoring can improve the efficacy of vector control and may also delay or prevent the onset and spread of resistance. Insecticide resistance is commonly assessed by exposing mosquitoes to a diagnostic dose using standard protocols published by WHO . However, if resistance alleles are partially or fully recessive, like
2.3. Worldwide pattern of insecticide resistance
The worldwide distribution of the dominant malaria vectors is represented in Figure 1.
2.3.1. Sub-Saharan Africa
Malaria morbidity in sub-Saharan Africa represents 90% of the total cases reported worldwide [5, 19]. Many vectors play an important role in malaria transmission across Africa, notably the four major malaria vector species, i.e.,
2.3.2. Southeast Asia and Western Pacific Region
After Africa, Southeast Asia is the area with a higher incidence of malaria, with 7% of the cases reported . A good number of vectors (belonging to complexes or groups of species that are difficult to distinguish) are involved in transmission, presenting an extraordinary biodiversity, heterogeneity in distribution, linked with a high variety in host feeding and ecological habitat preferences, as well as high differences in vector competence [35, 36, 37]. Currently in Southeast Asia, PY resistance has been detected in
2.3.3. Eastern Mediterranean
Malarial morbidity in this region accounts for only 2% of reported cases world over . Afghanistan, Pakistan, Sudan, South Sudan and Yemen account for the majority of the malaria cases. In Afghanistan, a 2016 study done in five different locations reported that
2.3.4. Latin America
Malaria cases have declined considerably in this region in the past two decades, with many of the countries going into pre-elimination phase . However, with 562,000 cases reported during 2015–2016, malaria is still a high burden, especially in countries in the Amazonia region such as Brazil, Colombia, Peru and most recently Venezuela , that showed an alarming increase over 76% of the reported cases (from 136,402 to 240,613) between 2015 and 2016, displaying an unprecedented 365% increase in malaria cases between 2000 and 2015 . This country now encompasses Brazil as the larger contributor to the malaria burden in the Americas.
In conclusion, resistance to insecticide is steadily spreading worldwide in most vectors as shown by the comparison of resistance profile between 1985 and 2000 (Figure 3A) and 1985 to 2017 (Figure 3B) from IR mapper (http://www.irmapper.com/). This represents a serious challenge to malaria control, which relies heavily on insecticide-based tools.
3. Insecticide resistance mechanisms
A proportion of insect populations can tolerate doses of insecticides which have been proved lethal to the majority of the individuals in a normal population of the same species through various mechanisms such as: (i) insecticide can be broken down or detoxified much faster in the resistant mosquitoes than in the susceptible ones, hence quickly eliminated from their body (metabolic resistance); (ii) the target of the insecticide can be genetically altered to prevent the insecticide from binding thereby reducing the insecticide effect (target-site resistance); or (iii) resistant mosquitoes may absorb the toxin slower than susceptible insects (penetration resistance). An illustration of these mechanisms is represented in Figure 4.
3.1. Methods used to study resistance mechanisms
Insecticide resistance monitoring is essential to understand the actual threat and how resistance is spreading among malaria vectors . Once resistance has reached very high levels (fixed in the population), most insecticide resistance management strategies, which are based to restore susceptibility, would not work. Thus, regular monitoring is crucial. Three detection methods (Table 1) can be used to monitor insecticide resistance, each method providing different information. Bioassays are the most popular way to monitor resistance where mosquitoes are exposed fixed doses of insecticides for a fixed time and the percentage mortality is recorded 24 h post-exposure . Even though they are simple to perform, bioassays have several disadvantages such as requiring a large number of mosquitoes, affected by variations in humidity, temperature and time of the day . Some authors argue that bioassays should be supplemented with DNA markers or even partially replaced by these DNA markers . It should be noted that DNA markers are usually specific to certain mechanisms hence the need to perform them is to avoid unknown mechanisms going undetected. Until now, no assay has been developed that is suitable to monitor cuticular or behavioural resistance.
|Susceptibility bioassay tests||Biochemical assays||Molecular assays|
|Vectors are exposed to fixed insecticide concentrations, and the level of vector mortality is subsequently recorded. The results are expressed as the percentage of vectors knocked down, alive or dead. Susceptibility testing requires samples of at least 100 live mosquitoes per testing site. These susceptibility tests are generally used for routine monitoring, as they can be applied in the field. They provide standardised data that are relatively easily interpreted. Either WHO paper bioassays or CDC bottle bioassays can be used. The results obtained with the two methods are not comparable. In order to observe longitudinal or temporal patterns in resistance, countries and academic institutions in all regions must therefore use the same method consistently over time.||Biochemical assays detect the presence of a particular resistance mechanism or an increase in enzyme activity. They require fresh mosquitoes, but much fewer than for bioassays. Unlike bioassays, biochemical assays can identify some specific resistance mechanisms and indicate an increase in metabolic enzyme activity. Biochemical assays are normally used in conjunction with synergist and molecular assays.||Molecular tests are used on the actual gene, allowing detailed and direct analysis of resistance genes. Testing can be done with straightforward polymerase chain reaction techniques (30) with DNA or in more elaborate microarray tests with RNA. More advanced molecular methods can provide complex genetic information including whether the mutation is unique or has spread. These are the most accurate tests for measuring resistance frequency in vector populations. Molecular tests must, however, be correlated with susceptibility testing.|
|Susceptibility tests identify the existence of resistance once it is at a detectable level but do not establish the resistance mechanism involved. They may also not identify resistance if the frequency is too low. Several countries have reported shortages in the supply of testing materials and have switched between the WHO and CDC tests, making results difficult to compare. In some cases, they have limited their testing.||The method is more difficult to use in the field as it requires sophisticated equipment, and interpretation of the results requires strong technical skills. Further, the correlation between chemical reactions in these tests and increased ability to metabolise insecticides is not yet well defined.||The method requires sophisticated equipment and entomological capacity. It can be used to detect target site resistance and a few identified metabolic mechanisms. Therefore, susceptibility tests should be used to complement molecular results, as the absence of identifiable genotypic resistance does not necessarily mean that resistance does not exist.|
3.2. Target-site resistance
One of the mechanisms mosquito becomes resistant is by altering the target site of the insecticide thereby preventing it from binding effectively hence the insecticide has little or no effect on the insect. Most insecticide targets are found within the nervous system and mutations in these target sites (mainly receptors) lead to reduced sensitivity. For example, PYs and DDT act on the voltage-gated sodium channels (VGSCs) and mutation in the amino sequence of this gene results in reduced sensitivity of the channels preventing PYs and DDT from binding . Insects with this mutation can withstand prolong exposure to insecticide without being knocked down, hence the name “knockdown resistance” (
3.3. Metabolic resistance
Metabolic resistance is the most common and challenging of all insecticide resistance mechanisms. Mosquitoes have enzyme systems that protect them from xenobiotic compounds and some of these enzyme systems can break down insecticide before it can reach its site of action. In metabolic resistance, enzymes that detoxify the insecticide can be overexpressed or alter the affinity of the enzyme for the insecticide through amino acid substitutions . Overexpression of insecticide resistance genes is the most frequent mechanism in resistant mosquitoes. This increased expression of insecticide resistance genes can be due to
3.3.1. Cytochrome P450 monooxygenases
Of the six families of P450s, genes belonging to the CYP4, CYP6 and CYP9 have been observed in resistant mosquitoes with increased transcriptional level , with the majority of those implicated in resistance belonging to the CYP6 family. For a P450 to be involved in resistance, it does not only have to be overexpressed but also must be able to metabolise/sequester the insecticide to which the insect is resistant and also be better metaboliser than those for the susceptible strain . In
3.3.2. Glutathione S-transferases
The GSTs are involved in the phase two of the detoxification of xenobiotic compounds where they conjugate the substrate with glutathione enhancing solubility thus facilitating the excretion. In insect, six classes of GSTs, i.e., delta, sigma, epsilon, omega, theta and zeta have been identified . Insects resistant to major classes of insecticide show elevated levels of GSTs activities. For example, GSTs confer resistance to DDT in mosquitoes including
CAs and OPs are the main insecticides that are metabolised or sequestered by esterase-mediated insecticide resistance. Esterase levels in the resistant mosquitoes can either be elevated like in
3.4. Cuticular or reduced penetration resistance
Cuticular resistance occurs when mosquitoes reduce the absorption of insecticide into their bodies by altering the structure or composition of the cuticle. A wide range of insecticides are threatened by this mechanism as for their lethal effect to occur, most insecticides must cross the cuticle in order to reach their site of action. Cuticular resistance enhances the resistance conferred by other mechanisms. This mechanism has not been extensively studied as compared to the other mechanisms because there are very few examples. Recently, Yahouédo et al.  studied the role of the cuticular resistance in PY-resistant strain of
4. Impact of current insecticide resistance in parasite transmission: a global warning based on reported level of resistance?
4.1. Fitness cost of resistant lab and field
The use of insecticide selects small proportion of individuals possessing resistance genes allowing them to resist and survive the effects of the insecticide, transferring the genetic modifications conferring resistance to the progeny. This should most likely increase the proportion of resistant individuals within the population. However, mutations or genes conferring resistance are usually associated with a fitness cost and may disrupt normal physiological functions [106, 107]. For example, resistant vectors may have lower mating success [108, 109], lower fecundity and fertility, higher developmental time and lower longevity. Resistant individuals may be also more susceptible to natural predators  or more prone to mortality during overwintering. Most insecticide resistance management strategies rely on the fact that fitness cost may impact the spread and persistence of resistance alleles in the vector populations .
4.2. Impact of resistance on life traits: longevity, fecundity and mating male competitiveness
Resistance caused by overproduction of metabolic enzymes generally shows lower fitness cost than target site resistance, most probably because the primary function of the enzyme is not disrupted . But to date, little is known about the effective impact of metabolic resistance on the life traits of the vector due to the absence of DNA-based molecular marker. Nevertheless, many studies demonstrated that resistant strains of arthropods often present lower fitness compared to their susceptible counterparts . For example, it was shown that resistance strains may be associated with relatively slower larval development, reduced survival rates among larvae and adults, reduced fecundity in females and reduced fertility [106, 113, 114]. It was shown that target-site resistance due to
4.3. Epidemiological consequences of the insecticide resistance on malaria incidence
4.3.1. Past and current evidences
There are large number of confounding factors threatening the assessment of epidemiological consequences of the insecticide resistance on malaria incidence and data interpretation . For this reason, only few studies have assessed the epidemiological impact of insecticide resistance. Impact of PY resistance on control failure was reported from the borders of Mozambique and South Africa. In 1996, the malaria control programme in KwaZulu-Natal (South Africa) switched from using DDT to deltamethrin for indoor spraying . After four years of deltamethrin spraying, reported malaria cases increased approximately fourfold.
5. Behavioural resistance to insecticides used in public health
As we have mentioned previously, the extraordinary success of malaria reduction in Africa is largely due the use of insecticides applied indoors through LLINs and IRS . This malaria control approach takes advantage of the strong human preference, as well as the indoor feeding and resting behaviour of African malaria-transmitting mosquitoes . As we have shown in this chapter, progress has been made in understanding the genetic basis of the ability of mosquitoes to survive insecticide entering the body. However, little is known about the causes of increasingly reported changes in blood-feeding behaviour developed by certain species of malaria-transmitting mosquitoes to avoid exposure to insecticides . This phenomenon is known as behavioural resistance and it is defined as any modification in insect behaviour that helps to circumvent the lethal effects of insecticides. Thus, through intraspecific behavioural shifts in biting time, location and host preference, malaria-transmitting mosquitoes avoid exposure to insecticides, feeding on humans when most people are not protected , jeopardising the current control strategy in Africa primarily based on indoor application of insecticides [130, 131, 132]. Recent studies conducted in West and East Africa have shown that indoor application of insecticides may induce intraspecific behavioural shifts towards early biting, exophagic biting and exophilic resting behaviour in malaria-transmitting mosquitoes [130, 131, 133]. Similarly, current studies conducted in Central Africa showed a comparable shift towards exophilic resting behaviour . Mathematical modelling and field evidences have proved that these shifts in blood-feeding behaviour could threaten and impact on the current control programmes [132, 135]. The mechanisms driving these shifts have not yet been elucidated, although some studies have shown that both genetic and environmental factors play a key role [135, 136].
6. Conclusion and perspectives
Insecticide resistance is undoubtedly a major challenge to the control of malaria vectors worldwide as it limits the tools available to achieve the goal of controlling and eliminating this debilitating disease. It is therefore of the utmost importance that novel insecticides and new control tools be designed to help manage and mitigate the impact of resistance. Through the work of various partners such as Innovative Vector Control Consortium (IVCC), UNITAID and several manufacturers, the challenge of producing new insecticides and tools is beginning to be met. This is exemplified by the recent prequalification by the WHO of the new insecticide Sumishield (clothianidin, a neonicotinoid) in October 2017. This new insecticide together with the organophosphate Actellic (pirimiphos-methyl) could now allow countries to effectively design and implement suitable resistance management strategies for IRS interventions according to WHO’s Global Plan for Insecticide Resistance Management (GPIRM). With other new insecticides expected to enter the market in the near future, resistance management strategies such as rotation of insecticides could become more realistic to implement. However, even with new insecticides available, the community should avoid being complacent as the mosquitoes will surely develop resistance with time if consideration is not given to how to use such new insecticides including between public health and agriculture sectors. Detection of resistance markers notably for metabolic resistance is also urgently needed to not only track the spread of resistance but to better assess its impact on control interventions or mosquito fitness and malaria transmission. The recent detection of markers such as L119F-GSTe2 in
This work was supported by a Wellcome Trust Senior Fellowship in Biomedical Sciences (WT101893MA) to CSW.
Conflict of interest
No conflict of interest.
|CDC||Centers for Disease Control and Prevention|
|GPIRM||Global Plan for Insecticide Resistance Management|
|IRM||insecticide resistance management|
|IRS||indoor residual spraying|
|IVCC||Innovative Vector Control Consortium|
|LLIN||long-lasting insecticidal net|
|VGSC||voltage-gated sodium channel|
|WHO||World Health Organization|