3
Vector Resistance

OVERVIEW

One way to help stem the spread of antimicrobial resistance is to reduce the number of cases of disease that must be treated with therapeutic agents, thereby reducing the selective pressure that often leads to such resistance. Toward this goal, insecticides play a major role in controlling major disease vectors—the arthropods that pass pathogens to humans.

The use of insecticides, however, presents a problem parallel to the use of antimicrobials—the chemical agents themselves frequently promote resistance among the vectors they are intended to control. Indeed, resistance to insecticides has appeared in every major species of arthropod vectors— including mosquitoes, ticks, fleas, lice, and sand flies—and various vectors have developed resistance to every class of pesticide. The focus of this session of the workshop was an examination of the role that vectors play in a variety of diseases and how management efforts are being used to better control vector populations.

Malaria represents a classic example of the difficulties associated with vector-control efforts. In 1955, the World Health Organization (WHO) called for the global eradication of malaria through the use of DDT. However, the mosquitoes that carry the disease soon developed resistance to DDT, and in 1976 WHO shifted its goal from eradication to control. The resistance problems continued with the switch to newer insecticides, such as the organophosphates and pyrethroids. Many experts maintain that new insecticides, coupled with the development and implementation of various



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3 Vector Resistance OVERVIEW One way to help stem the spread of antimicrobial resistance is to reduce the number of cases of disease that must be treated with therapeutic agents, thereby reducing the selective pressure that often leads to such resistance. Toward this goal, insecticides play a major role in controlling major disease vectors—the arthropods that pass pathogens to humans. The use of insecticides, however, presents a problem parallel to the use of antimicrobials—the chemical agents themselves frequently promote resistance among the vectors they are intended to control. Indeed, resistance to insecticides has appeared in every major species of arthropod vectors— including mosquitoes, ticks, fleas, lice, and sand flies—and various vectors have developed resistance to every class of pesticide. The focus of this session of the workshop was an examination of the role that vectors play in a variety of diseases and how management efforts are being used to better control vector populations. Malaria represents a classic example of the difficulties associated with vector-control efforts. In 1955, the World Health Organization (WHO) called for the global eradication of malaria through the use of DDT. However, the mosquitoes that carry the disease soon developed resistance to DDT, and in 1976 WHO shifted its goal from eradication to control. The resistance problems continued with the switch to newer insecticides, such as the organophosphates and pyrethroids. Many experts maintain that new insecticides, coupled with the development and implementation of various

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other control measures, will be needed to regain long-term control over the mosquitoes that spread malaria. Some experts, however, argue that DDT has unfairly been declared an unacceptable hazard, and that it can play an important—and significantly larger—role in controlling not only malaria but also such other widespread diseases as dengue. Research on resistance among vectors now takes two basic approaches. One approach is to study the molecular mechanisms involved. Scientists already have a relatively good understanding of the basic mechanisms underlying resistance to commonly used insecticides, and they are now using recently developed molecular techniques to begin to dissect these mechanisms at the DNA level. The next challenge will be to use this molecular understanding to develop novel vector-control methods that avoid or minimize resistance problems. The second approach to research involves resistance management— that is, developing and implementing control methods that minimize the likelihood that vectors will evolve strong resistance to important insecticides. Two such promising control strategies are alternating the use of different insecticides and applying them in mosaic patterns over a given geographic area. Scientists are now conducting, in Mexico, a large-scale trial of these two strategies, compared to the use of DDT or a pyrethroid insecticide used in the conventional manner. Information from such trials may enable scientists and policy makers to establish rational strategies for long-term insecticide use. Expanding the use of integrated pest management, or IPM, also holds promise for improving vector-control programs. Tackling a pest problem in numerous ways at once—including the use of pesticides in a timely manner at select locations—will likely yield more thorough and longer-lasting control than would result from any single method applied individually. Developing and applying mathematical models can provide a solid basis for identifying new ways to effectively control the emergence of resistance among vector populations—and also to identify methods that are not effective. Among their benefits, models enable researchers to examine trends in data, explore questions of population dynamics, compare various management options, and generate new hypotheses for study. A major roadblock to progress in this area is the lack of interdisciplinary work among modelers studying insect resistance and those studying antimicrobial resistance in pathogens. Indeed, increased scientific collaboration across disciplines could foster scientific progress in numerous areas related to antimicrobial resistance. Likewise, policy makers developing programs to control the spread of antimicrobial resistance among pathogens could benefit by examining the successes and failures of current vector-control programs.

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INSECTICIDE RESISTANCE IN INSECT VECTORS OF HUMAN DISEASE Janet Hemingway, Ph.D. Liverpool School of Tropical Medicine Liverpool, United Kingdom Insecticides play a central role in controlling major vectors of diseases. In 1955 the WHO Assembly proposed the global eradication of malaria with DDT. However, the shift from malaria eradication to control in 1976 was prompted by the appearance of DDT resistance in many mosquito vectors. The resistance problems continued with the switch to newer insecticides such as the organophosphates (OPs), carbamates, and pyrethroids. Operationally, many control programs have now switched from blanket spraying of house interiors to focal use of insecticides on bednets. Focal spraying reduces the amount of insecticide used, but also limits the choice of insecticides to pyrethroids due to the need for rapid insect kill and high mammalian safety margin. Today the major emphasis in research into resistance is on the molecular mechanisms of resistance, and rational resistance management, with a view to controlling the spread and development of resistant vector populations. The level of resistance in insect populations is dependent on the amount and frequency of insecticides used, and the inherent characteristics of the insect species selected. For example, for decades tsetse flies have been controlled by DDT treatment, but resistance has never developed in this species; the same is true of triatomid bugs. In both species the major factor influencing insecticide resistance development is the life cycle of the insect pest, in particular the long life cycles for the bugs, and the production of very small numbers of young by the tsetses. Mosquitoes, in contrast, have all the characteristics suited to rapid resistance development including short life cycles and abundant progeny. Vector Resistance The major mosquito vectors span the Culex, Aedes, and Anopheles genera. Culex are the vectors of filariasis and Japanese encephalitis, Aedes of dengue, dengue hemorrhagic fever, and yellow fever, and Anopheles of malaria. The range of many of these species is still expanding. DDT was first introduced for mosquito control in 1946. In 1947 the first cases of DDT resistance occurred in Aedes (Brown, 1986). Since then over a hundred species of mosquito have become resistant to one or more insecticide (WHO, 1992). Insecticides used for malaria control have in

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cluded ?-benzine hexachloride (BHC), organophosphorus, carbamate, and pyrethroid insecticides. Other insecticide groups, such as the benzylphenyl ureas and Bacillus thuringiensis (Bti), have had limited use against mosquitoes. ?-BHC/dieldrin resistance is still widespread, despite the lack of use of these insecticides for many years. OP resistance, in the form of either broad-spectrum resistance or malathion-specific resistance, occurs in many vectors (Hemingway, 1982, 1983; Hemingway and Georghiou, 1983; Herath et al., 1987). OP resistance is widespread in all the major Culex vectors (Hemingway and Karunaratne, 1998) and pyrethroid resistance occurs in C. quinquefasciatus (Chandre et al., 1998). Pyrethroid resistance is widespread in Ae. aegypti (Hemingway et al., 1989) and cases of OP and carbamate resistance also occur in this species (Mourya et al., 1993). The development of pyrethroid resistance in An. gambiae is particularly important given the recent emphasis on the use of pyrethroid impregnated bednets for malaria control (Vontas et al., 2001). The peridomestic vectors of Leishmania are primarily controlled by insecticides throughout their range. The control of these sandflies is often a by-product of anti-malarial housespraying. The only insecticide resistance reported to date in sandflies is to DDT (El-Sayed et al., 1989). The body louse Pediculus humanus has developed widespread resistance to organochlorines (Brown and Pal, 1971), is malathion-resistant in parts of Africa (WHO, 1992), and has “low-level” resistance to pyrethroids in several regions (Fine, 1963). Resistance to DDT and lindane occurs in the human head lice in Israel, Canada, Denmark, and Malaysia (WHO, 1992). Permethrin has been extensively used for head lice control since the early 1980s. The first reports of control failure with this insecticide were in the early 1990s in Israel (Mumcuoglu et al., 1995), the Czech Republic (Rupes et al., 1994), and France (Chosidow et al., 1994). The Simulium damnosum complex, vectors of onchocerciasis, have been subjected to long-term insecticide-based control in West Africa since 1974. Temephos resistance prompted a switch to chlorphoxim, but resistance to this insecticide occurred within a year (Hemingway et al., 1991). Resistance in Simulium is currently being managed by a rotation of temephos, Bti, and permethrin, the insecticide usage being determined by the rate of water discharge in the major breeding sites of these vectors. The Biochemistry of Resistance Insecticide Metabolism Three major enzyme groups are responsible for metabolically based resistance to organochlorines, OPs, carbamates, and pyrethroids. DDT-

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dehydrochlorinase is a glutathione S-transferase (Clark and Shamaan, 1984). Esterases are involved in OP, carbamate, and pyrethroid resistance. Monooxygenases are involved in the metabolism of pyrethroids, carbamate, and the activation and/or detoxication of OPs. Esterase-Based Resistance: The esterase-based resistance mechanisms have been most extensively studied in Culex mosquitoes and the aphid Myzus persicae. Broad-spectrum OP resistance is conferred by the elevated esterases, which act by rapidly binding and slowly turning over the insecticide (Kadous et al., 1983). Two common esterase loci, esta and estß, are involved in Culex (Vaughan et al., 1997). In C. quinquefasciatus the most common elevated esterase phenotype involves esta21 and estß21 (Vaughan and Hemingway, 1995). Smaller numbers of Culex have elevated estß1 alone, elevated esta1 alone, or co-elevated estß1 and esta3 (DeSilva et al., 1997; Hemingway and Karunaratne, 1998). Differences of up to 1,000-fold in the inhibition kinetic constants of these esterases in resistant and susceptible insects occur for the oxon analogues of various OPs (Karunaratne et al., 1995). The superiority of insecticide binding in enzymes from resistant insects suggests that there has been a positive insecticide selection pressure to maintain elevation of favorable esterase. In contrast to the situation in Culex a number of Anopheles species have a non-elevated esterase mechanism that confers resistance specifically to malathion through increased rates of metabolism (Hemingway, 1982, 1983, 1985; Malcolm and Boddington, 1989). Glutathione S-Transferase-Based Resistance: GSTs are multifunctional enzymes that detoxify a large range of xenobiotics. They catalyze the nucleophilic attack of reduced glutathione (GSH) on the electrophilic centers of lipophilic compounds. Multiple forms of these enzymes have been reported for the mosquito, housefly, Drosophila, sheep blowfly, and grass grub (Clark et al., 1984; Clark et al., 1985; Toung et al., 1990). There are at least three families of insect GST and all have a role in insecticide resistance. In Ae. aegypti at least two GSTs are elevated in DDT-resistant insects (Grant and Matsumura, 1989), while in An. gambiae a large number of different class III GSTs are elevated (Prapanthadara et al., 1993). The Ae. aegypti and An. gambiae GSTs in resistant insects are constitutively over-expressed. The GST-2 of Ae. aegypti is over-expressed in all tissues except the ovaries of resistant insects (Grant and Hammock, 1992). Monooxygenase-Based Resistance: The monooxygenases are a complex family of enzymes involved in the metabolism of xenobiotics. The P450 monooxygenases are the rate-limiting enzyme step in the chain. P450s are involved in the metabolism of virtually all insecticides, leading to activation of OPs, or, more generally, detoxication. Elevated monooxygenase activity

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is associated with pyrethroid resistance in An. stephensi, An. gambiae (Vulule et al., 1994), and C. quinquefasciatus (Kasai et al., 1998). Target Site Resistance The OPs, carbamates, organochlorines, and pyrethroids all target the nervous system. Newer classes of insecticides are now coming onto the market place for vector control, but the high cost of developing and registering them inevitably means that insecticides are developed initially for the agricultural market and then utilized for public health vector control, where their activities and safety profile are appropriate. Compounds targeting the nicotinic acetylcholine receptor have recently made this transition from agriculture into public health. Acetylcholinesterase: The OPs and carbamates target acetylcholinesterase (AChE). Alterations in AChE in resistant insects result in a decreased sensitivity to insecticide inhibition of the enzyme (Hemingway and Georghiou, 1983). The OPs are converted to their oxon analogues, via the action of monooxygenases before acting as AChE inhibitors. GABA Receptors: Resistance to dieldrin occurred in the 1950s, but the involvement of the GABA receptors in this resistance was not elucidated until 1990. The GABA receptor in insects is a widespread inhibitory neurotransmission channel in the central nervous system and in neuromuscular junctions (Bermudez et al., 1991). It is a site of action for pyrethroids and avermectins as well as cyclodienes (Kadous et al., 1983; Bloomquist, 1994). Sodium Channels: The pharmacological effect of DDT and pyrethroids is to cause persistent activation of the sodium channels by delaying the normal voltage-dependent mechanism of inactivation (Soderlund and Bloomquist, 1989). In mosquitoes there have been many reports of suspected ”kdr”-like resistance inferred from cross-resistance between DDT and pyrethroids, which act on the same site within the sodium channel. These reports have been validated by electrophysiological measurements in Ae. aegypti and An. stephensi (Hemingway et al., 1989; Vatandoost et al., 1996). The Molecular Biology of Resistance Mutations in Structural Genes Resistance to malathion is caused by a single Trp251-Leu substitution within the E3 esterase of Lucilia cuprina (Campbell et al., 1998). A similar phenotype has been observed in malathion-resistant Anopheles (Hemingway and Georghiou, 1983). A second Gly137-Asp substitution in E3 confers

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broad cross-resistance to many OPs, but not to malathion (Newcomb et al., 1997). Non-silent point mutations within structural genes are the cause of target site resistance. The mutations reduce insecticide binding without causing a loss of primary function of the target site. The number of possible amino acid substitutions is limited and identical resistance-associated mutations are found across divergent taxa. The degree to which function is impaired by the mutations is reflected in the fitness of resistant individuals in the absence of insecticide selection. This fitness cost is important in the persistence of resistance in the field. An alanine to serine substitution in the channel-lining domain of the GABA receptor confers resistance to cyclodienes (ffrench-Constant et al., 1998). The mutation occurs in a broad range of dieldrin-resistant insects (Thompson et al., 1993), occasionally in an alanine to glycine form. Despite the widespread switch away from cyclodienes for agricultural and public health use, the resistance allele is still found at relatively high frequencies in insect field populations (Aronstein et al., 1995). A reduction in the sensitivity of the voltage gated sodium channel to insecticide binding causes the kdr resistance phenotype. Kdr mutants are more variable than those in the GABA receptors, but are still limited to a small number of regions on this large channel protein. The first mutation to be characterized in kdr insects was a leucine to phenylalanine mutation in the S6 transmembrane segment of domain II in the sodium channel sequence (Martinez-Torres et al., 1998) that produces 10- to 20-fold resistance to DDT and pyrethroids. In “super-kdr” houseflies, this mutation is combined with a second methionine to threonine substitution further up-stream in the same domain resulting in >500-fold resistance (Williamson et al., 1996). A PCR-based diagnostic discriminates between homozygous-susceptible, homozygous-resistant, and heterozygotes with the Leu to Phe mutation (Martinez-Torres et al., 1998). Since kdr is partially recessive the ability to detect heterozygotes is of paramount importance in the early detection and management of resistance in the field. The number of changes may be constrained by the possible modifications that can influence pyrethroid/DDT binding to the sodium channels. However, a note of caution is needed, as there is a tendency to investigate pyrethroid-resistant insects with a PCR approach confined to regions where a kdr mutation has already been seen. Hence changes in other parts of the sodium channel gene could be missed. A different approach to isolating kdr-type mutants has been used in Drosophila, utilizing the relative ease with which large numbers of mutants in the para sodium channel gene can be isolated based on their temperature sensitivity. Two classes of these mutations are in positions equivalent to the kdr and super-kdr mutations in

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different domains. The third class is in a novel position (ffrench-Constant et al., 1998). A range of different amino acid substitutions in the acetylcholinesterase Ace genes of Drosophila and the housefly M. domestica cause resistance (Feyereisen, 1995). Many of these mutations lie close to or within the active site gorge. Five point mutations associated with OP and carbamate resistance have been identified in D. melanogaster. AChE and site directed mutagenesis of the sex-linked AChE from Ae. aegypti have demonstrated that these same mutations also confer resistance in the mosquito enzyme, but none of these mutations have been identified in field-collected insects. Gene Amplification In three Culex species an estß gene is amplified in resistant insects (Vaughan et al., 1995; Whyard et al., 1995; Karunaratne et al., 1998). The commonest amplified esterase-based mechanism involves the co-amplification of two esterases, esta21 and estß21 in C. quinquefasciatus and other members of the C. pipiens complex (Vaughan et al., 1997). Other strains of Culex have amplified esta3 and estß1 co-amplified (DeSilva et al., 1997), while the TEM-R strain has amplified estß1 alone (Mouches et al., 1986). The esta and estß genes have arisen as the result of an ancient gene duplication. The genes are in a head-to-head arrangement approximately 1.7 kb apart in susceptible insects (Vaughan et al., 1997). In resistant insects the amplified esta21/estß21 genes are 2.7 kb apart; the difference is accounted for by expansion with three indels in the intergenic spacer (Vaughan et al., 1997), which have introduced further gene regulators (Hemingway et al., 1998). Amplification of these alleles has occurred once and spread worldwide by migration (Raymond et al., 1991). Transcriptional Regulation Amplified esterases are expressed at different levels. For example, there is four-fold more estß than esta in resistant C. quinquefasciatus, although the genes are present in a 1:1 ratio. This difference in expression is reflected at the protein and mRNA level (Karunaratne et al., 1995). GSTs metabolize DDT and OPs (Hemingway et al., 1985) and moderate the toxic effects of pyrethroid-generated radicals (Vontas et al., 2001). GST-based DDT resistance is common in Anopheles, reflecting decades of heavy use of DDT for malaria control. Molecular characterization of GSTs is most developed in An. gambiae and An. dirus (Hemingway et al., 1998; Prapanthadara et al., 1998). Three classes of insect GSTs are important in insecticide metabolism in insects. Aedes aegypti GST-2, is overexpressed in

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FIGURE 3-1 The class I and class III glutathione S-transferase family of Anopheles gambiae, which include an intronless gene aggst1-2, an alternatively spliced gene aggst1a with four distinct viable transcripts, and the aggst1ß gene with two introns. a DDT-resistant GG strain, where the resistance mutation leads to disruption of a trans-acting repressor (Grant and Hammock, 1992). In An. gambiae multiple class I and class III GST genes are clustered in separate single locations. One gene, aggst1a, is alternatively spliced to produce four distinct mRNA transcipts each of which shares a common 5’ exon but differing 3’ exons (see Figure 3-1) (Ranson et al., 1998). The organizations of the class I GST gene family in insecticide-resistant and -susceptible An. gambiae are similar. Insect P450s belong to six families. Increased transcription of genes belonging to the CYP4, CYP6, and CYP9 families occurs in insecticide-resistant strains. It is not known which enzymes are responsible for insecticide metabolism in mosquitoes. Helicoverpa armigera has different resistance-associated P450s between strains; CYP6B2 is overexpressed in a pyrethroid-resistant strain (Xiao-Ping and Hobbs, 1995), whereas CYP4G8 is overexpressed in another (Pittendrigh et al., 1997). P450-based resistance in M. domestica (housefly) and D. melanogaster is mediated by mutations in trans-acting regulatory genes. CYP6A8 is highly expressed in the DDT-resistant 91-R strain of D. melanogaster but not detectable in the uninduced 91-C susceptible strain (Maitra et al., 1996; Dombrowski et al., 1998). Hybrids between the two strains show low levels of expression suggesting that the 91-C strain carries a repressor that suppresses transcription of CYP6A8 (Liu and Scott, 1997).

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Management of Insecticide-Resistant Vector Populations The use of an insecticide until resistance becomes a limiting factor is rapidly eroding the number of suitable insecticides for insect control. Rotations, mosaics, and mixtures have all been proposed as resistance management tools. Numerous mathematical models have been produced to estimate how these tools should be optimally used (Tabashnik, 1989). However, these models have rarely been tested under field conditions due to the practical difficulties in estimating changes in resistance gene frequencies in large samples of insects (Hemingway et al., 1997). With the advent of different biochemical and molecular techniques for resistance gene frequency estimation, field trials of resistance management strategies have become more feasible. A large-scale trial of the use of rotations or mosaics of insecticides compared to single use of DDT or a pyrethroid is currently under way in Mexico (Penilla et al., 1998). Changes in resistance gene frequencies in An. albimanus are being monitored over a four-year period. Information resulting from such large-scale trials may allow us to establish rational strategies for long-term insecticide use. As our ability to manipulate the insect genome improves and our understanding of the regulation of insecticide resistance mechanisms increases, new strategies should be devised for incorporation into these control programs. MANAGING THE EMERGENCE OF PESTICIDE RESISTANCE IN VECTORS William G. Brogdon, Ph.D. Centers for Disease Control and Prevention, Atlanta, GA A linked four-stage process, involving investment, surveillance, interpretation, and remediation, is described for management of insecticide resistance in vectors. Insecticide resistance has been a problem in all insect groups that serve as vectors of emerging diseases. Although mechanisms by which insecticides become less effective are similar across all vector taxa, each resistance problem is potentially unique and may involve a complex pattern of resistance foci. The main defense against resistance is close surveillance of the susceptibility of vector populations. This requires the investment of programs in surveillance. Once surveillance data have been gathered, it is crucial that the results be interpreted practically, in terms of control efficacy. Only then can strategies for remediation be undertaken. There are fundamental differences between the management of pesticide and that of antimicrobial resistance. Rapid generation times in mi

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crobes can lead to rapid selection of highly resistant populations, even in the course of a single infection. Generation times in vectors are significantly longer, resulting in more gradual selection of resistance. Among the more rapid emergences of insecticide resistance we have observed was the appearance and intensification of fenitrothion resistance over a three-month period at one location in Haiti following an insecticide spray cycle (Brogdon et al., 1988). The focus of antibiotic resistance management is on infections in individual patients or in specific institutions. The focus of insecticide resistance management is on vector populations in a geographic control area. The use of antibiotics follows similar principles throughout the world. The application strategies for insecticides are radically different around the world. For control of malaria, wall spraying and the use of insecticide-impregnated bednets are the most used methods of control. In the United States, ground and aerial application of ultra-low volumes of insecticide are widely used. Changes in antibiotic use have little environmental impact. Changes in insecticide use can have significant environmental impact. Collection, culture, and testing of resistant microbe strains is not technically difficult or expensive. Collection, culture, and testing of resistant insects can be both difficult and expensive. Generally, teams of individuals must go into the field and collect through the night, sometimes for days, to obtain an adequate number of insects for resistance testing. Collections may include adults, larvae, or eggs of vectors. Control failure in antibiotic use can have immediate fatal consequences for the patient. Insecticide control failure may have less obvious impact on disease control. The continued survival of a vector species may be less noticeable against a background of pest species that have been controlled, giving the illusion of adequate vector control. For example, good control of pest species of mosquitoes in Florida gave the impression that control with a particular insecticide was adequate. Further testing revealed that Culex nigripalpus, the principal vector species, did not respond to that insecticide (Brogdon, unpublished data). The most significant feature shared by antimicrobial and insecticide resistance management is that the choice of new chemical options is dangerously limited. It is crucial that legislators and health officials make the investment necessary to manage resistance. Funding allows resistance problems to be detected and assessed through the use of resistance surveillance. Going through the motions of resistance surveillance is a waste of resources unless the data obtained are subjected to professional and timely interpreta tion. Only after the problem has been adequately detected and interpreted can the program develop a strategy for remediation of the problem.

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cused almost completely on toxicological resistance, which, in turn, is based on the erroneous premise that insecticides control disease transmission by killing insects. No example of this fundamental error is more revealing than in the resistance of malaria vector mosquitoes to DDT. A recent probability model (Roberts et al., 2000) quantifies DDT’s mode of action in controlling malaria transmission inside houses. The model shows that greatest impact of DDT residues stems from a repellent action. A contact irritant action is next in order of importance. The model was tested against field data for important malaria vectors in the Americas, Africa, and Asia. The results showed that mortality accounted for less than 10 percent of the total impact of DDT residues against two vectors, from 10 percent to about 36.2 percent against a third vector, and 18.3 percent against a fourth vector. In a separate study, the model was field validated in experimental hut studies against a fifth vector species in southern Belize (Grieco et al., 2000). This model represents the first use of stochastic methods to elucidate actual functions of DDT residues, but it was certainly not the first demonstration of DDT’s powerful repellent and irritant actions. From the very beginning of DDT’s use, it was known as a slow-acting poison (Metcalf et al., 1951) that strongly influenced insect behavior. In 1947 Kennedy published a paper (Kennedy, 1947) entitled “The Excitant and Repellent Effects on Mosquitoes of Sub-Lethal Contacts with DDT.” This paper warrants careful consideration because its 1947 publication date means that the research was actually conducted before wide-scale use of DDT in malaria control (which started in 1946). Kennedy’s experiments demonstrated repellent and irritant actions of DDT against a vector of malaria (Anopheles maculipennis atroparvus) and Ae. aegypti. Kennedy’s work was published five years before the first report of DDT resistance in a malaria vector mosquito. Kennedy introduced his paper as follows: During the war it was necessary to concentrate on the practical development of DDT as an insecticide without waiting for studies of its mode of action. Working assumptions had to be made, especially with regard to the possible repellency of DDT deposits. Buxton (1945) sums up what has been the accepted opinion on this subject, as follows: “DDT does not act as a repellent to any insect, so far as is known” (Kennedy, 1947). He went on to say that “… the existence of contact repellency has been firmly established.” Buxton (1945) mentioned “restlessness” as an early symptom of DDT poisoning. This has been described under semi-field conditions by Gahan and others (1945) and by Metcalf and others (1945), who observed also that mosquitoes excited by contact with DDT no longer stayed in dark corners but made for the windows. However, such repellency has been discounted as of no practical signifi

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cance because, in Buxton’s (1945) words: “So far as is known, once visible symptoms develop, death follows: recovery from an early stage of poisoning does not occur.” “This idea is perhaps even more widespread than the idea that DDT is not repellent” (Kennedy, 1947). Kennedy ended his introductory paragraph with the statement that “Our laboratory observations did not bear out these statements” (Kennedy, 1947). Kennedy then reported on a series of laboratory experiments showing that mosquitoes were strongly repelled by DDT residues (for example, see Figure 3-3), and that the test specimens often recovered completely after showing preliminary symptoms of poisoning. With this documentation he concluded that “DDT must be regarded as acting in two contradictory ways simultaneously. It acts as a lethal agent on the one hand, and as an excitant and thereby sometimes a repellent on the other” (Kennedy, 1947). FIGURE 3-3 Percent of Anopheles maculipennis atroparvus females escaping from DDT-treated and untreated chambers in just three minutes post-exposure. Black bars represent data for DDT-treated chambers; white bars are for control chambers. Percent remaining accounts for those that did not immediately escape from the exposure chamber. SOURCE: Kennedy, 1947.

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Kennedy’s work was conducted at a time when Professor George Macdonald was establishing the mathematical foundations of malaria control. Kennedy commented on Macdonald’s views: Macdonald (in discussion on Buxton, 1945, p. 394) was convinced that the reduction in anopheline infestation of sprayed rooms was due to actual destruction of mosquitoes and “not due to repellent effect, a point of extreme importance.” His evidence was the presence of dead mosquitoes and the lack of any increase in the infestation of adjoining, untreated rooms. Nevertheless he remarked that there was no evidence of any reduction in such untreated rooms for which, as he said, “one might legitimately hope” in view of the passage of mosquitoes from room to room. The solution of this puzzle is surely that there was both destruction and repulsion of the mosquitoes (Kennedy, 1947). Six years later, in 1953, Macdonald and Davidson published what became the founding principles for using insecticides in malaria control. Their analyses were based on field studies from 1947 to 1952. They did not cite Kennedy’s paper and chemical actions favoring mosquito survival were characterized as undesirable. Macdonald and Davidson struggled valiantly to reconcile DDT’s slow killing power with its spectacular ability to reduce malaria. The authors knew of DDT’s complex influence on vector behavior, as revealed in assessments that: “DDT was the most irritant of the three insecticides (DDT, BHC, and Dieldrin), and a very large proportion of the mosquitoes escaped its action.” “The marked irritant properties of DDT were evident in all these treatments, the greater proportion, and sometimes as much as 90 per cent, of the mosquitoes being caught in the window traps.” “The marked irritant effect of DDT on mosquitoes makes adequate dosage … imperative” (Macdonald and Davidson, 1953). In the end, Macdonald and Davidson concluded that the answer to DDT’s “irritant” action was to increase dosage so that mosquitoes would absorb a lethal dose before chemical repulsion. A surprising conclusion considering that Kennedy (1947) reported that “… excitation appears quickly, often in a matter of seconds after the insects are brought into contact with DDT …” (Kennedy, 1947). One might wonder how Macdonald and Davidson, with their comprehensive knowledge of malaria, could so seriously err in understanding DDT’s mode of action? In their defense, key elements of field data were missing, as revealed in their assessment that “In most, if not all, … entomological assessment of the efficiency of the insecticides has been based

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on records of the reduction in numbers of the daytime-resting population of treated shelters. No account has been taken of mosquitoes entering and leaving the shelters during the night” (Macdonald and Davidson, 1953). Macdonald and Davidson stated that, of their tentative conclusions, “the one most clearly brought to light is that a considerable expansion of our knowledge is urgently needed—an expansion in the fields of basic theory, anopheline habits, the physical chemistry of insecticides, and their mode of action” (Macdonald and Davidson, 1953). Unfortunately, rapid acceptance of their model virtually guaranteed that research and expansion of knowledge, particularly regarding chemical mode of action, would not occur. Macdonald and Davidson allowed that DDT killed mosquitoes that entered houses and reduced longevity of malaria vector populations below what is needed to maintain malaria transmission. Acceptance of this concept ended debate and support for research on DDT’s mode of action. With preeminence of the idea that DDT reduced vector population longevity, Kennedy’s research, and that of many others, was relegated to obscurity. The facts that DDT was a powerful repellent, a functional irritant, and only a slow-acting poison were ignored. Those facts have now been resurrected in the probability model, described above. To fully appreciate the probability model, it is important to understand that insects have extraordinary abilities to detect and respond to chemicals; for example, certain species are capable of detecting a single molecule in 1017 molecules of air. Insects have developed these abilities as an adaptation to secondary chemicals that occur in nature. Plants and animals produce the so-called “secondary” chemicals to repel, deter, irritate, or poison herbivores, predators, and parasites (Schoonhoven, 1985). DDT is an organohalogen compound, and organohalogens are produced as secondary chemicals in nature (Gribble, 1999). The similarity of DDT to natural organohalogens is suggested by the finding that males of a species of euglossine bee are attracted to DDT and will travel to houses and collect DDT from sprayed walls (Roberts et al., 1982). The bees store (relative to their size) large amounts of DDT in hind tibial pouches and, remarkably, are not harmed. It is also worth noting that we now know that some natural organohalogens are persistent in the environment, are lipophilic, bioaccumulate, and may be present in greater abundance than synthetic compounds like DDT (Vetter et al., 2001). As a final point, winged adult insects can easily and rapidly move away from a poison (given that the chemical is not useful to the insect) and avoid the biological cost of premature death or metabolic detoxification. When a malaria vector is inside a house it is exposed to human hosts, outdoors it is exposed to a variety of hosts. Thus, it is generally understood that malaria transmission becomes most efficient when vectors and humans come together inside the confined spaces of homes. When the vector is

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outside, it only has access to indirect human host stimuli emanating from inside the house. Once the vector is indoors, host proximity and unobstructed “view” of the human host (no intervening wall) alters the balance between factors that inhibit versus factors that stimulate a vector to bite. These factors and host availability influence probabilities of disease transmission. By definition, a repellent action occurs when the mosquito is repelled without making physical contact with the chemical. In contrast, a mosquito is irritated only after making physical contact with a sprayed surface. Physical contact that results in an irritant response (escape response) can occur after very brief physical contact (as shown by Kennedy, 1947). A toxic action requires more prolonged physical contact with a sprayed surface (Roberts and Andre, 1994). The vector’s actions, for example, moving to a house, entering the house, resting on walls, and biting, can be aligned sequentially with repellent actions that prevent house entering; irritant actions that promote premature exiting and prevent biting indoors; and toxic actions that produce mortality (Roberts et al., 2000). We used the following notations to simulate interactions of repellent, irritant, and toxic actions of insecticides: T: treatment house. C: control house. pe: probability a mosquito will enter the house. pb|e: probability a mosquito will bite, given that it enters the house. pbe: probability a mosquito will enter the house and bite indoors. ps|be: probability a mosquito survives, given that it entered the house and took a blood meal indoors. psbe: probability a mosquito will enter the house, bite indoors, and survive. Applying the multiplication law of probability (Rosner, 1995), we have the following equations pbe = pb|e × pe (1) psbe= ps|be × pb|e × pe (2) As stated above, we assume that probabilities for different vector activities in the control house each equate to unity. This assumption leads to the relationship of and implies that ; see equations (1) and (2). The influence of insecticide on mosquito activities can be described as the probability difference between control and treatment for entering, biting, and surviving. For the act of entering the house, the probability difference is ; and for the biting and surviving,

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and, respectively. Actually, based on conditional probabilities, is the cumulative effect due to insecticide repellency, is the cumulative effect due to repellency plus irritancy, and is the cumulative effect due to repellency, irritancy, and toxicity. The probability of entering, , can be estimated by Using comparative data from the control house, pb|e can be estimated by the observed proportion of mosquitoes that bite or become blood engorged within the treated house, and ps|be can be estimated by the observed proportion of mosquitoes that survived after they entered and fed in the treated house. To model field data, we would further adjust probabilities to account for natural differences in numbers collected in houses as a result of location differences. Our simulations (formulas not presented) partitioned the total impact of a sprayed house by the percent of effect that can be attributed to each chemical action. Overall, the combined effects of repellent and irritant actions () did not drop below a cumulative level of 50 percent effectiveness until probabilities of entering and biting indoors were very high, and (i.e., almost no repellent or irritant action). In experimental hut studies the repellent actions of DDT residues can function at levels above 90 percent compared to unsprayed huts (Grieco et al., 2000; Roberts et al., 2000). No repellent action was detected in a deltamethrin-sprayed hut, but a pronounced irritant action drove mosquitoes out of the deltamethrin-sprayed hut several hours before they exited the control hut. The combined message from model simulations and experimental hut studies is that DDT functions as a repellent, secondarily as an irritant, and lastly as a poison. In contrast, deltamethrin seems to function as an irritant and secondarily as a poison. These findings highlight a simple truth, an insecticide is just one more chemical in the mosquito’s chemical world. The mosquito can detect and respond to an insecticide in many ways, perhaps the least likely being physical contact and death. We have shown that repellent and irritant actions of DDT did not suddenly surface after decades of DDT use; but were actually recognized as major actions of DDT residues from the very start of DDT’s use as a public health insecticide. In conclusion, funding agencies should formally recognize behavioral actions of vectors to insecticides as a priority area for future support.

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