Distribution, Mechanisms, Impact and Management of Insecticide Resistance in Malaria Vectors: A Pragmatic Review

4.1. Metabolic resistance

Metabolic resistance is the most common resistance mechanism that occurs in insects. This mechanism is based on the enzyme systems which all insects possess to help them to detoxify naturally occurring xenobiotics/insecticides. It is commonly accepted that insect detoxification systems derived from the plant-insect evolutionary arm race and several insect detoxification enzymes have been associated to the detoxification of plant toxins and all types of chemicals, including insecticides [51]. Over-expression of enzymes capable of detoxifying insecticides or amino acid substitutions within these enzymes, which alter the affinity of the enzyme for the insecticide, can result in high levels of insecticide resistance (see [52] for review). Increased expression of the genes encoding the major xenobiotic metabolizing enzymes is the most common cause of insecticide resistance in mosquitoes. Over expression of detoxyfing enzymes can occur as the result of gene amplification (e.g. duplication) or due to changes in either trans-acting regulator elements or in the promoter region of the gene [5, 53, 54]. The consequence is a significant increase of enzyme production in resistant insects that enables them to metabolize or degrade insecticides before they are able to exert a toxic effect. Three categories of enzymes, namely esterases, P450s and glutathione-S-transferases are known to confer resistance to insecticides in insect pest such as malaria vectors. These large enzyme families contain multiple enzymes with broad overlapping substrate specificities, and one member of the family might be capable of metabolizing limited number of insecticides. Similarly, the level of resistance conferred can vary from low to very high and may differ from compound to compound. Metabolic resistance mechanisms have been identified in mosquito populations for all major classes of insecticides currently used for vector control, including organochlorine, organophosphates, carbamates, and pyrethroids.

Esterases. One of the most common metabolic resistance mechanisms is that of elevated levels, or activity, of esterase enzymes which hydrolyze ester bonds or sequester insecticides. A striking example comes from studies on Culex quinquefasciatusthatresist to a broad range of OP insecticides. In this species, multiple copies of EST-genes was already found hence enabling it to overproduce this type of enzyme [55]. In contrast to the situation in Culex,a number of Anophelesspecies (ieAn. culicifacies, An. stephensiand An. arabiensis) have a non-elevated esterase mechanism that confers resistance specifically to malathion through increased rates of metabolism. Malathion resistance in Anophelesspp was associated with an altered form of esterase that specifically metabolizes the molecule at a much faster rate than that in susceptible counterparts [56, 57]. Although Carboxylesterase (CCEs) have been mostly associated to organophosphate resistance in mosquitoes, their role in pyrethroid resistance is probable. Indeed, the ability of esterases to metabolize pyrethroids has been suggested in mosquitoes [58, 59] even if no specific mosquito CCE has yet been validated as a pyrethroid metabolizer [50]. Clearly much more information is needed on the esterase-mediated resistance in malaria vectors.

P450s.Cytochrome P450-dependent monooxygenases are an important and diverse family of enzymes involved in the metabolism of numerous endogenous and exogenous compounds. P450 belong to six families and increased transcription of genes belonging to the CYP4, CYP6, and CYP9 has been observed in various insecticide-resistant species from different taxa [60]. There is increasing number of reports demonstrating elevated P450 monooxygenase activities in insecticide-resistant mosquitoes, frequently in conjunction with altered activities of other enzymes. In most cases where a link between insecticide resistance and elevated P450 activity has been shown, the CYP gene belongs to the CYP6 family. Since the publication of the An. gambiaegenome [61], P450s were extensively studied in the primary malaria vector in Africa. A total of 111 P450 enzymes were identified [62] and, as in other insects, only a small number of these enzymes are capable of detoxifying insecticides. However, higher activity of enzymes and/or expression of detoxification genes in insecticide resistant colonies do not necessarily correlate with insecticide resistance. For example, some authors have shown elevated transcript levels of an adult-specific CYP6 P450 gene, CYP6Z1, in pyrethroid-resistant strain of An. gambiae[63, 64] and An. funestus[65]. Further validation studies conducted in An. gambiaeshowed that cyp6z1 was however not capable to metabolize pyrethroids but was capable to metabolize DDT [66]. Another study showed that CYP6z2 displays broad substrate specificity, which may be associated with xenobiotics metabolism and detoxification [67]. Despite, CYP6Z2 being able to bind to permethrin and cypermethrin, this genedoes not metabolise any of these insecticides. Microarray-based approaches have lately identified three new “candidate” P450 genes that were found to be repeatedly over-produced in pyrethroid resistant populations of An. gambiae: CYP6M2, CYP6P3 and CYP6Z2 [68-70]. All of these genes encode for enzymes that are able to bind to type I and type II pyrethroids but only CYP6P3 and CYP6M2 showed to metabolize the insecticides [10, 71]. More recently, some authors demonstrated that CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in An. gambiae, hence demonstrating the first evidence for a metabolic cross-resistance in malaria vectors [9]. Interestingly the putative ortholog of An. gambiaeCYP6P3, CYP6P9, as being the prime candidate for conferring pyrethroid resistance, have been identified in An. funestus[72, 73] but only the CYP6P9 showed to metabolize types I and II pyrethroids [74]. Recent works showed that over-production of CYP6P9 in An. funestusresult from gene duplication [72]. In An. minimusmosquito, CYP6AA3 and CYP6P7 were up-regulated in pyrethroid-resistance population of Thailand [75] and seem to possess activities toward pyrethroid degradation [76, 77].

Glutathione S-transferases (GSTs). Glutathione transferases (GSTs) are multifunctional enzymes involved in the detoxification of many endogenous and xenobiotic compounds. Conjugation of Glutathione (GSH) to such organic molecules enhances solubility, thus facilitating their eventual elimination [78]. Elevated GST activity has been implicated in resistance to at least four classes of insecticides in insects. Higher enzyme activity is usually due to an increase in the amount of one or more GST enzymes, either as a result of gene amplification or more commonly through increases in transcriptional rate, rather than qualitative changes in individual enzymes [52]. At least six classes of insect GSTs have been identified in An. gambiae[79], found in several large clusters on all three chromosomes. The Delta and Epsilon classes found exclusively in insects are the largest classes of insect GSTs. Members of both classes have been implicated in resistance to all the major classes of insecticide. The primary role of GSTs in mosquito insecticide resistance is in the metabolism of DDT to DDE (non toxic products), although they also have a secondary role in organophosphate resistance [80]. GST-based DDT resistance is common in a number of anopheline species including An. gambiae[81-83], reflecting the heavy use of this insecticide for malaria control over several decades. Molecular biology and in vitroexpression studies showed that aggst3-2was over expressed in resistant strain of An. gambiaeand that recombinant aggst3-2was very efficient at metabolizing DDT [84]. Most studies of GSTs suggested that regulation occurs at the transcriptional level. Several regulatory elements have been identified in the promoter regions of GSTs that may mediate their induction but the significance of these findings is unclear. Genetic mapping of the major genes controlling GST-based DDT resistance in An. gambiaeprovided however evidence for a trans-acting regulator [84], although in this species, mutations in promoter elements of the Epsilon GST cluster are also associated with resistance [81]. It has been suggested that GSTs may play a role in pyrethroid resistance by detoxifying lipid peroxidation products induced by pyrethroids and/or by protecting from insecticide exposure induced oxidative stress [85]. Furthermore, GST might confer secondary role in pyrethroid resistance by sequestering the insecticide hence reducing the total in vivoconcentration of insecticide [86].

Despite the great advance obtained recently in the identification of the role of detoxifying enzymes in insecticide resistance, force is to note that the function of >90% of metabolic genes is still unknown. Although only a limited number of resistance mechanisms have been implicated to date, the diversity within enzyme families involved in metabolic resistance is likely to contribute substantially to resistance to many insecticide classes. Further functional genomics and post-genomic technology are needed to reveal the contributions of hitherto unsuspected enzymes in insecticide metabolism and/or sequestration and to identify the causal mutations associated with metabolic resistance in mosquitoes. The contribution that these enzymes make towards various insecticide resistance phenotypes in malaria vectors is yet to be elucidated.

4.2. Target-site resistance

The second most common resistance mechanism encountered in insects is target-site resistance. Insecticides generally act at a specific site within the insect, typically within the nervous system (e.g. OP, carbamate, DDT and pyrethroid insecticides). The site of action can be modified in resistant strains of insects such that the insecticide no longer binds effectively. Reduce sensitivity of the target receptors to insecticide results from non-silent point mutations in the gene encoding the protein. For example, the target site for OP and carbamate insecticides is acetylcholinesterase (AChE) in the nerve cell synapses. Several mutations in the gene encoding for an acetylcholinesterase have been found in insects” [87] which result in reduced sensitivity to inhibition of the enzyme by these insecticides [88, 89]. In malaria vectors, the G119S mutation (i.e. glycine to serine substitution at position 119) responsible for carbamate and OP resistance has been reported in An. gambiaeand An. albimanus,essentially at the heterozygous state [90]. Recent sequence analysis of some resistant mosquitoes collected in Benin revealed the presence of a duplication of the ace-1gene in both A*n*. A. gambiaeM and S forms [91]. In addition, mutations at a single codon (position 302) in the Rdl (resistance to dieldrin) gene encoding one receptor subunit, from an alanine residue to a serine (or more rarely to a glycine), have been documented in dieldrin-resistant insect species [92] including the malaria vectors An. stephensi[93], An. gambiae s.l. [94] and An. funestus[31]. Similarly, mutations in the amino acid sequence in the voltage-gated sodium channels of nerve cell membranes leads to a reduction in the sensitivity of the channels to the binding of DDT and pyrethroid insecticides [95]. Alterations in the target site that cause resistance to insecticides are often referred to as knockdown resistance (kdr) in reference to the ability of insects with these alleles to withstand prolonged exposure to insecticides without being ‘knocked-down” [96]. One of the most common amino acid replacements associated with pyrethroid resistance in malaria vectors is a substitution of the leucine residue found at codon 1014 with either phenylalanine (1014F) [97] or serine (1014S) [98] in the Voltage-Gated Sodium Channel (VGSC). Interestingly, residue 1014 does not appear to interact directly with the insecticide but is predicted to alter channel activation kinetics [99]. Note that a de novomutation (N1575Y) recently emerged within domains III-IV of voltage gate sodium channel in pyrethroid resistant populations of An. gambiaeand seems to occurs only in a single long-range haplotype, also bearing 1014F allele [100]. It has been suggested that the N1575Y mutation may compensates for deleterious fitness effects of 1014F and/or confers additional resistance to pyrethroid insecticides.

4.3. Reduced penetration

Modifications in the insect cuticle or digestive tract linings that prevent or slow the absorption or penetration of insecticides can be found is some resistant insects. This resistance mechanism is not specific and can affect a broad range of insecticides. Reduced uptake of insecticide, often referred to as cuticular resistance, is frequently described as a minor resistance mechanism. Certainly for pests where the major route of insecticide delivery is via ingestion, this is likely to be the case. However, for malaria control, where insecticides are typically delivered on bed nets or on wall surfaces, uptake of insecticides is primarily through the appendages. An increase in the thickness of the tarsal cuticle, or a reduction in its permeability to lipophilic insecticides, could have a major impact on the bioavailability of an insecticide in vivo. Examples of reduced-penetration mechanisms are however limited; cuticular resistance was reported for the domestic Fly Musca domestica[101] and the lymphatic filariasis vector Culex quinquefasciatus[102]. In Anopheles, microarrays studies have recently identified two genes, cplcg3 and cplcg4, encoding cuticular proteins that were upregulated in pyrethroid resistant strains from four populations and two different species (i.e. An. gambiaeand An. stephensi) ([69, 103, 104]. Recently, measures of mean cuticle thickness in a laboratory strain of An. funestususing scanning electron microscopy (SEM) showed that the mean cuticle thickness was significantly greater in pyrethroid tolerant mosquitoes than their susceptible counterparts [105]. Clearly much more work is required in order to identify the significance of cuticular resistance in phenotypic resistance.

4.4. Behavioural resistance

Insecticide resistance in mosquitoes is not always based on biochemical mechanisms such as metabolic detoxification or target site mutations, but may also be conferred by behavioural changes in response to prolonged exposure to an insecticide. Behavioural resistance does not have the same “importance” as physiological resistance but may be considered to be a contributing factor, leading to the avoidance of lethal doses of an insecticide [106, 107]. For example, the first study on the irritant effect of DDT residual deposits was conducted using Anopheles quadrimaculatuswhere females were found to be irritated shortly after making contact with the treated surfaces resulting in a rapid escape response from a treated house prior to taking a blood meal [108]. This type of response can be further divided into direct contact excitation (sometimes referred to as ‘irritancy’) and non-contact spatial repellency that is used when insects move away from the insecticide-treated area before making direct contact [106, 109]. Examples of behavioral resistance or avoidance are few. Change in vector composition (i.e. switch from An. minimusto An. harrisoni) has been observed following implementation of ITNs in a village form central Vietnam [110]. With regard to An. funestus, recent findings showed a shift from indoor to outdoor biting preferences in Tanzania in relation to increasing coverage of pyrethroid-impregnated net [111]. Significant changes in the host-seeking behavior of the An. funestuspopulation was confirmed in Benin (West Africa) where scaling up of LLINs at community level induced a change from night biting to early-morning biting behaviour [112]. It is unclear however whether adaptation of malaria vectors species to insecticidal based vector control interventions such as LLIN may result from a phenotypic plasticity or from selected behavioral traits (see Durnez & Coosemans for details).

5.1. Bioassays

Guidelines for test procedures and interpretation of results are available from the WHOPES

World Health Organization Pesticide Evaluation Scheme

These diagnostic dose assays are simple to perform and provide standardized data sets that, assuming the guidelines are followed, can be readily compared to identify temporal and/or geographical variations in the resistant status of malaria vector populations. However, it is important to recognize some of the limitations of these susceptibility tests. As only a single concentration of insecticide is used, the results do not provide any information about the level of resistance in a population. For example if 50 % of population A and 20 % of population B were killed after exposure to the diagnostic dose of permethrin, it cannot be concluded that population B is more resistant than population A. The results only indicate that both populations are resistant (according to WHO definitions if there is < 80 % mortality, the population is defined as resistant) and that, subject to tests of significance, there is a higher frequency of resistant individuals in population B than in A. Dose response assays would be needed to compare the levels of resistance in two populations (e.g. by measuring the Resistant Ratios and their 95% confidence intervals). For pyrethroids, median knock down time (MKDT) is also a useful quantifiable variable [117]. Similarly, the results of these tests cannot be used to compare the levels of resistance to two different insecticides. If 50 % mortality was observed after exposure to the diagnostic dose of permethrin (0.75 %) whereas mortality was 70% after exposure to the diagnostic dose of deltamethrin (0.05%), it is not correct to state that the population is more resistant to permethrin than deltamethrin. Again, all that can be stated is that the population is resistant to both insecticides.

Partly due to the limitations of the diagnostic dose assays described above and partly due to the difficulties that are sometimes incurred in obtaining a regular supply of the insecticide impregnated papers from WHO, an alternative bioassay methodology has been developed [118] and is being adopted by some monitoring programmes. This method, known as the CDC bottle bioassay, uses glass bottles coated with a known concentration of insecticide. As these test kits are assembled in the users own laboratory, the concentration of insecticide can be readily adjusted enabling dose response curves to be developed to compare two or more strains. A caveat to this is that the flexibility, and the potential variation in the insecticide grade used in the tests, impairs comparison of results between two separate studies.

Both WHO diagnostic doses and CDC bottle bioassays can be modified to incorporate synergists. Synergists such as piperonyl butoxide, that block the activity of two major detoxification enzyme families, can be used to explore the role of different resistance mechanisms. If resistance is due to increased metabolism, exposure to an appropriate synergist prior to insecticide bioassays should increase the level of mortality observed.

5.2. Biochemical tests

Biochemical tests to detect alterations in activities of enzyme families associated with insecticide resistance have been available for over two decades and are sometimes used in combination with insecticide bioassays [119]. These assays employ model substrates to record the overall activity of glutathione transferases, carboxylesterases or cytochrome P450s in individual insects. Biochemical assays are also available to detect target site resistance to organophosphate and carbamate insecticides caused by insensitive acetylcholinesterase (AChE). The enzymatic reaction produces a colour change that is generally visible to the naked eye and hence these assays do not require access to expensive equipment (spectrophotometer is appropriate). However, it is important that the mosquitoes are kept on ice from the point of collection to the performance of the assay and this can often pose logistical challenges. Furthermore, there are sensitivity and specificity issues that limit the utility of some of these assays. For example, with over 100 different cytochrome P450 enzymes in malaria vectors, an assay that measures the total level or activity of this enzyme family may not have the sensitivity to detect over production of the single or small number of P450 enzymes that are thought to be involved in pyrethroid metabolism. This may explain the lack of significant correlation observed in many studies between cytochrome P450 activity and bioassay mortality results [120, 121]. In addition not all members of the enzyme family will have the same affinity for the model substrates used in these assays (e.g. CDNB (1-chloro 2-4, dinitrobenzene) is the substrate typically used to assess glutathione transferase activity but the Epsilon class of GSTs which are responsible for DDT resistance have relatively low activity with this substrate). In order to incorporate data from resistance monitoring into evidence based decisions on appropriate insecticide based interventions for malaria control, it is clearly essential that the data is both reliable and accessible. Although guidelines for conducting the various assays exist, there is little consensus on the number of sites and frequency with which resistance monitoring should occur [122]. It is clear that resistance is a dynamic trait, and wide fluctuations in resistance levels throughout the malaria transmission season have been reported [116, 123, 124]. Resistance can also be very focal, particularly when vector composition differs between sites [125], hence a minimum number of sampling sites should be established, taking into account patterns of vector distribution and insecticide usage. The WHO/AFRO African Network for Vector Resistance was established in 2000 and amongst its objectives was the important goal of improving the dissemination of resistance data. Accordingly a database was established to store the results of resistance monitoring activities by the African Network for Vector Resistance (ANVR) members but until recently, this database was not readily accessible by outside users. The recent establishment of new data base (see section 6), as an online centralized resource for collating data on insecticide resistance in disease vectors and the integration of this with the ANVR database, will hopefully ensure that both published and unpublished data on resistance in malaria vectors are more readily available to all interested parties.

5.3. Molecular tests

A multitude of molecular assays have been developed to detect kdralleles in malaria mosquitoes, several of which were recently compared in a study by Bass et al (see [126]). These are routinely used by research laboratories monitoring for insecticide resistance and are gradually being incorporated into some national malaria control resistance monitoring programmes. Unfortunately, despite the recent identification of the key enzymes responsible for metabolic resistance to pyrethroids in An gambiaeand An funestus, there are currently no simple DNA based assays to detect these resistance mechanisms. Detection of these genes is presently dependent on RNA based approaches using relatively sophisticated equipment (e.g. RT-qPCR). Assays to detect the genetic mutation(s) responsible for the resistance phenotype in individual insects can provide an early warning of the emergence of resistance which may not have been detectable by bioassays that can only record the population response. The presence of a single individual with an allele known to confer resistance should be cause for concern as experience dictates that resistance can spread very rapidly in a population unless the selection pressure is eased and/or the genetic cost associated with the resistant allele is high. Conversely, a negative result from a molecular assay should not lead to complacency. As discussed above, molecular assays are presently only available for target site resistance and the failure to detect kdrclearly cannot be interpreted as an absence of resistance in a population. Hence molecular assays should be seen as a complement rather than a substitute for bioassays.

6.1. Africa

Although the occurrence of insecticide resistance in malaria vectors in Africa is not a “new“ event (see section 2.), the speed at which pyrethroid-resistance recently evolved in field populations is worrying as it may jeopardize the current malaria vector control initiatives carried out in the continent. As shown in figure 2, pyrethroid resistance in Anophelessp. is widespread but not uniformly distributed among the different countries. In the 49 African countries that have been investigated (see [6] for details), 15 did not report any data on resistance in the last 10 years i.e. Algeria, Botswana, Democratic Republic of Congo, Djibouti, Sierra Leone, Lesotho, Liberia, Libya, Maurice, Mauritania, Namibia, Rwanda, Somalia, Swaziland, Tunisia. If a lot of data has been generated in West Africa (as far as An. gambiae s.l.is concerned), a lack of information is globally observed in Central, Eastern and Austral Africa. It is obvious that the frequent conflicts that has occurred in the last decades in some African countries has rendered difficult the conduct of routine monitoring surveys by NMCP, International Organisation (WHO/ANVR) and/or research institutions.

Globally, pyrethroid resistance is high in An. gambiae s.l.in West Africa including Benin [127], Burkina Faso [128], Guinea Konakry ([129], Ghana [130], Mali [131], Niger [132], Nigeria [133] and Cote d’Ivoire [134]). In this region, pyrethroid resistance is predominant in An. gambiaes.s., compared to An. arabiensis. Surprisingly, susceptibility to pyrethroids (permethrin and/or deltamethrin) was reported in An. gambiaes.l. in Guinea Bissau [135] despite the presence of the L1014F mutation. In Central Africa, pyrethroid resistance/tolerance is widespread in An. gambiae s.lin Cameroon [136-138], Chad [116, 139], Gabon [140, 141], Equatorial Guinea [8] and Sudan [142, 143]. In Chad, North Cameroon and Sudan, pyrethroid resistance is present essentially in An. arabiensis,which is consistent with the higher prevalence of this mosquito species in more arid areaswith higher mean annual temperatures [144]. In East and Austral Africa, An. gambiaeand An. arabiensispopulations are mostly susceptible to pyrethroids in Tanzania [145, 146], Mozambique [147] and Madagascar [148], but highly resistant in eastern Uganda [149, 150], Ethiopia [151], Kenya [152, 153], Zambia [154], South Africa [155] and the Gwave Region of Zimbabwe [120]. Regarding An. funestus, most of the literature reporting pyrethroid resistance comes from South Africa [39, 156] and Mozambique [157-159], most probably because An. funestusis the main malaria vector in these countries. The data available in other African countries is very limited in partly due to the difficulty to colonize An. funestusspecies in laboratory. Pyrethroid resistance/tolerance was detected in Malawi [160, 161] and suspected in Obusi and Kassena-Nankana Regions from Ghana [121, 162] and Benin [163] whereas full susceptibility to permethrin and deltamethrin was found in Burkina Faso [164] and Tanzania [145]. There is a lack of information on secondary vectors e.g. An. mouchetiandAn. niliwhich can play important role in malaria transmission in specific settings (e.g. Cameroon, Congo, Côte d’Ivoire). Regarding other vectors species, fullsusceptibility to pyrethroids has been reported in An. labranchiaein Morocco [165] and inAn. pharoensisin Egypt [166] and Ethiopia [167].

In Africa, the L1014F mutation is widespread (figure 2) and predominant in the molecular S form compared to the M form, except in Benin [168], Guinea Equatorial [8] and Niger [132]. Some authors suggested that the kdralleles may have arisen from at least four independent mutation events in the An. gambiaeS-form [169]. Regarding the M form, it is not clear whether the kdrmutation resulted from an introgression from the S form only [170, 171] and/or from independent mutation events, has recently suggested for Bioko Island [172]. The second mutation, a leucine–serine substitution at the same codon (L1014S), was identified first in a colony of An. gambiae s.l.from Kenya [98]. This substitution has been lately reported in Burundi [173], Cameroon [136, 138], Gabon [174], Equatorial Guinea [175], Uganda [176], Republic of Congo [177] and Angola [140], mainly in co-occurrence with the 1014F kdrallele. Although some authors have reported that the 1014S allele may confer lower level of pyrethroid resistance than the 1014F allele [178], its spread from eastern to central Africa and more recently to West Africa [124, 179] suggest a survival advantage of mosquitoes sharing this mutation in presence of pyrethroids. So far, the L1014S substitution has always been detected in the S molecular form [180] but recent findings showed the occurrence of the 1014S allele in the M form in Equatorial Guinea [181] and Cameroon [182]. In these two countries, the 1014S allele was present at very low frequencies, alone or associated with the L1014F allele. It is currently impossible to know whether the kdralleles have arisen first in Cameroon or Equatorial Guinea. The higher frequency of the 1014S allelein the S form compared with the M form could either be attributed to an introgression from the S taxon or to a de novomutation. Regarding the sister taxa An. arabiensis, both of these mutations were reported in Western [124], Central [183] and Eastern Africa [184]. Interestingly, a new kdrmutation (N1575Y) occurring within domains III-IV of voltage gate sodium channel was found in both S and M molecular forms of An. gambiaeand occurs upon a 1014F haplotypic background only [100]. Additive resistance of 1575Ywas demonstrated for permethrin and DDT in both molecular forms of An. gambiae. The prevalence of the 1575Y mutation has increased in West Africa in the last years hence indicating that the 1014F-1575Y haplotype is under strong selection pressure (Djégbé pers. com). It is possible that besides the 1014F/1014S kdrmutation, other mutations in the para-type sodium channel gene might be needed for mosquitoes to survive after exposure to a discriminating concentration of an insecticide. Further investigation is needed to better address the distribution and the role of the N1575Y mutation in pyrethroid resistance as well as to assess the fitness benefits conferred by this allele on the L1014F mutation in malaria vectors.

Beyond the spread of kdralleles, metabolic-based resistance due to detoxifying enzymes namely oxidase, the GST (epsilon) and CCE families have expanded in African malaria vectors. InAn. gambiae s.l.metabolic resistance involving increased levels of P450 has been reported at least in Kenya [185], Cameroon [186], Benin [69], Nigeria [69], Ghana [70], Mozambique [147], South Africa [187] and Zimbabwe [120]. Up to now, only genes encoding CYP6P3 and CYP6M2 P450 enzymes have been clearly involved in cellular mechanisms known to metabolize deltamethrin and permethrin [10, 71].These genes were found over-expressed in pyrethroid-resistant An. gambiaepopulations from Benin, Nigeria and Ghana [69, 70], mainly in co-association with the kdrL1014F allele. In An. funestus, pyrethroid resistance involving increased activity of P450 monooxygenase and/or GST was demonstrated in South Africa [157], Mozambique [188] and Malawi [161].

To conclude, the immense challenge in Africa will be not to manage and control kdr-resistant mosquitoes only but to deal with the development of “multiple resistant” populations that could resist to different class of insecticides used in public health. One other issue is the occurrence and development of carbamate resistance in some countries (eg Benin, Nigeria) where this chemical class is in use for IRS through the PMI programme [47, 48]. The spread of carbamate resistance in malaria vectors in Africa is worrying for insecticide resistance management and alternative insecticides, and innovative strategies are urgently needed to better reduce the vectorial capacity of mosquitoes and hence effectively reduce the burden of malaria in the region. Resistance management strategy for malaria control is discussed in section 8.

6.2. South-East Asia and India

The South East Asia Region (SEAR) that account for 13% of the total malaria cases worldwide (2^nd position after Africa) [1] is not spare of insecticide resistance in the main malaria vector species.

In the Mekong region, cross-country monitoring of insecticide resistance has been conducted through the MALVECASIA network (http://www.itg.be/malvecasia/) to help MCPs in the choice of insecticide to use at regional level. Large differences in insecticide resistance status were observed among species and countries. Anopheles dirus s.s., the main vector in forested malaria foci, was mainly susceptible to permethrin except in central Vietnam where it showed possible resistance to type II pyrethroids [23]. Anopheles minimuss.l. populations were found resistant / tolerant in Vietnam and northern Thailand [189] but almost susceptible in Cambodia and Laos. No kdrmutation has been observed so far in these species [190] and pyrethroid resistance seems to result from increased detoxification by esterases and/or P450 monooxygenases [191]. Indeed, increased mRNA expression of two P450 genes, CYP6P7and CYP6AA3, suspected to metabolize some pyrethroids [76] have been reported in a deltamethrin-resistant population of An. minimusin Thailand [75, 192].

Anopheles epiroticusof the Sundaicus Complex showed to be highly resistant to all pyrethroids in the Mekong Delta [23] but susceptible to DDT, except near Ho Chi Minh City. DDT and pyrethroid-resistant populations ofAn. subpictuswere reported in Vietnam and Cambodia. Biochemical assays suggest an esterase-mediated pyrethroid detoxification in both An. epiroticusand An. subpictuswhereasDDT resistance in An. subpictusmight be conferred to a higher GST activity. In Vietnam and Cambodia, An. vagusand An sinensisshowed various levels of pyrethroid resistance and sequence-analysis of the DIIS6 region of the VGSC revealed the presence of the 1014S kdrallele [193]. Pyrethroid resistant populations of An. sinensiswere also reported in the Republic of Korea (ROK) [194] and in China [195]. In China, cypermethrin resistance in An. sinensiswas associated with the presence of both 1014F and L1014C substitutions, whereas only 1014F and 1014S mutations were found in the ROK and Vietnam, respectively. In Indonesia, molecular analysis carried out in field mosquito samples revealed the presence of the 1014F allele in the four main malaria vectors i.e. An. sundaicus, An. aconitus, An. subpictusand An. vagus[196]. At the present time, it is difficult to speculate on the relative contribution of the kdrmutations versusmetabolic detoxification on pyrethroid and DDT resistance in malaria vectors from the SEA region and more work are needed to establish a clear trend.

Insecticide resistance is known to be widespread in other part of Asia such as India. In this country, resistance has a long history (see section 2) and it represents a big challenge for malaria vector control. Among the Anophelesspecies, An. culicifacies s.l., the major vector of malaria in most parts of the country, has developed strong resistance to pyrethroids [36], DDT [197, 198], dieldrin/HCH [199], and malathion [198]. The 1014F mutation, which generates the kdrphenotype was detected in pyrethroid and DDT resistant An. culicifacies s.l.populations sometimes in co-occurrence with the 1014S mutation [197]. Note that a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC was recently identified in Indian An. culicifaciesand was tightly linked to 1014S substitution [197]. Elevated activities of GST seem to play also an important role in DDT-resistance in this mosquito species [82]. Similarly, strong level of pyrethroid resistance due to the presence of both 1014F and 1014S mutations was found in the urban malaria vector An. stephensiparticularly in the Rajasthan District [200]. Other vectors that are reported to be resistant to pyrethroid, DDT and/or dieldrin/HCH in India are An. annularis, An. subpictusand An. philippinensis[201]. In contrast, An. minimushas still not showed pyrethroid and DDT resistance [202].

The same trend was noted in Sri Lanka where the main malaria vectors species, i.e. An. culificifaciess.l.and An. subpictushave developed DDT, pyrethroid and malathion resistance in several districts [203, 204]. However, the main mechanisms associated with DDT and malathion resistance in An. culicifacies s.l.and An. subpictusare primarily metabolic and involve carboxylesterases (malathion) or monooxygenases and GSTs (for DDT) [205, 206]. An altered acetylcholinesterase conferring organophosphate resistance has been suspected in both vector species [205].

In the delta region of Bangladesh, the An. sundaicusmalaria vector is fully susceptible to DDT but other malaria vectors such as An. philippinensis, An. maculatus s.l.,and An. aconitushave all developed resistance to DDT [207]. Anopheles aconitus,additionally, has been reported to be resistant to dieldrin/HCH. Bhutan records An. maculatus s.l.as resistant to DDT, but there is no record of its resistance to any other insecticides [207]. Two vectors of malaria in Nepal, An. maculatus s.l.and An. aconitus, also have developed resistance to DDT whereas only malathion resistance was reported in An. stephensiin Pakistan [208]. Finally, in Iran and Turkey, An. stephensiand An. sacharovishowed resistance to DDT and dieldrin but both species are mostly susceptible to pyrethroids [209-211].

6.3. Latin America

The countries of the Amazon Basin (Bolivia, Brazil, Colombia, Ecuador, Guyana, Peru, Surinam and Venezuela) carry the greatest burden of malaria in the Americas. The primary vectors of this disease in the Amazon basin are An. darlingiandAn. albimanus. Surprisingly, much less data on insecticide resistance is available for these two mosquito species comparatively to African and/or Asian malaria vector species [212].

In Colombia, DDT resistance was reported in the late 80’s in some populations of An. darlingiin the districts of Quibdó and close to the Atrato River [213, 214]. Successive insecticide susceptibility evaluations revealed resistance to pyrethroids in both An. darlingiand An. albimanusmainly in the Chocó State [215]. In An. darlingi, increased levels of both Multi function Oxidase (MFO) and Non specific Esterase (NSE) were reported in a deltamethrin and DDT-resistant population, hence suggesting a possible involvement of these detoxifying enzymes in cross resistance to DDT and deltamethrin [35]. Note that various levels of resistance to organophosphate and pyrethroids were also reported in the secondary malaria vector An. nuneztovari[216].

In neighboring countries, DDT, permethrin and deltamethrin resistance was found in laboratory colonized populations of An. albimanusfrom Guatemala,whereas full susceptibility was noted in field populations from El Salvador and Belize [217, 218]. The colonies from Guatemala showed significant increase in the specific activity of esterase and/or oxidase as measured by spectrophotometer suggesting their potential involvement in pyrethroid-resistance [34, 217]. In Peru, monitoring campaigns carried out since 2000 showed that An. albimanuswas the only Anopheline species to exhibit pyrethroid-resistance [219].

In Mexico, high level of DDT resistance and low levels of resistance to organophosphate, carbamate and pyrethroid insecticides were detected in field populations ofAn. albimanusin Chiapas, prior to a large-scale resistance management project [220]. Biochemical assays revealed that the DDT resistance was caused by elevated levels of GST activity leading to increased rates of metabolism of DDT to DDE [22], whereas carbamate resistance was attributed to an altered acetylcholinesterase (AChE). More recent studies conducted in the southern Yucatan Peninsula showed high levels of DDT, deltamethrin and pirimiphos-methyl resistance in the An. albimanuspopulations tested [221]. Biochemical tests revealed elevated levels of GST, P450 and esterases activities that could be involved in DDT and pyrethroid-resistance. As for carbamate, pirimiphos-methyl resistance was strongly correlated with the presence of an insensitive acetylcholinesterase.

To our knowledge, it is the main “published” information available on the distribution, levels and mechanisms of resistance (i.e. accessible through Medline and pub med) in malaria vectors in Latin America. It is then essential to strengthen the capacity of all Latin America countries that suffering from malaria to make insecticide monitoring in routine to obtain much accurate information on the insecticide resistance situation in the malaria vectors. This will provide stake holders with useful information for the implementation of more effective and sustainable malaria control programmes in the region.

8.1. Main factors influencing resistance development

The evolution of insecticide resistance is complex and depends on several genetic, biological and operational factors [240-242]. The biological factors relate by the life cycle of the insect (e.g. rate of reproduction, number of generation/offspring, rate of migration and isolation, etc), while the genetic factors include the intrinsic characteristics of the resistant genes (e.g. mono versuspolygenic resistance, dominance, fitness cost and gene interaction). Operational factors concern the treatment itself including the method and frequency of application, dosage and residual activity of the insecticides as well as insecticide coverage.

8.2. Resistance management — Strategies and tactics

Historically, the practice of using an insecticide until resistance becomes a limiting factor has rapidly eroded the number of suitable/available insecticides for vector control. Rotations, mosaics, and mixtures have all been proposed as resistance management tools [260, 261] but there are very few “success stories” in public health. Numerous mathematical models have been produced to estimate how these tools could be optimally used [262-264] but these models have rarely been tested under field conditions due to the practical difficulties in estimating changes in resistance gene frequencies (especially for metabolic resistance) in large samples of insects [220]. With the advent of different molecular techniques for resistance-gene frequency estimation, field trials of resistance management strategies have now become more feasible.