Aedes aegypti is one of the most significant vectors in the world, as it can transmit various diseases caused by different arboviruses, such as yellow fever, dengue (with the four serotypes: DEN-1, DEN-2, DEN-3 and DEN-4), chikungunya, and Zika (WHO, 2017 and 2017, February). This mosquito has a high capacity to adapt to domestic and urban environments, given that it is greatly anthropophilic and anthropophagic, which has allowed it to achieve broad worldwide distribution (WHO, 2017).
Dengue and other arboviruses transmitted by Ae. aegypti throughout America spread for several reasons, but one that is undoubtedly very important is because traditional methods are no longer as effective against the Ae. aegypti, mainly due to insecticide resistance. However, the control of dengue, chikungunya and Zika continues to depend mainly on the vector control, in order to reduce mosquito population densities at a level where virus transmission low enough to avoid epidemic outbreaks. Thus, environmental management methods can be used as well as the application of chemical or biological agents. In Mexico, said measures mainly include the elimination of breeding grounds through social promotion campaigns, larval control by means of chemical and biological insecticides in non-disposable breeding sites, as well as the use of spatial application (ULV) and residual spraying insecticides to control adult mosquito populations (CENAPRECE, 2016). Consequently, even with certain levels of resistance detected in mosquito populations, the use of insecticides is still being justified in many species of public health importance (Hemingway et al., 1997, Dzul et al., 2007, Aponte et al., 2013).
Eradication of Aedes aegypti
In an effort to eliminate yellow fever since 1947, intensive campaigns were promoted by the Pan-American Sanitary Agency, whereby resources were destined to implement an Aedes aegypti eradication program (Brathwaite et al., 2012). Eradication was achieved in 1962 in 18 countries throughout the continent and in some islands of the Caribbean (PAHO, 1997), while leaving many countries such as Cuba, the United States, Venezuela and many more countries in the Caribbean unable to achieve eradication, causing epidemics during that period (Brathwaite et al., 2012). In 1962, Mexico was declared free of Ae. aegypti, after eliminating the vector as part of yellow fever eradication.
During the eradication period, major measures included the use of DDT as adulticide. During this campaign and until the mosquito was declared eradicated, no cases of DDT resistance in Ae. aegypti were reported in Mexico although they were found in Trinidad, Puerto Rico, the Dominican Republic and Jamaica (Ibañes-Bernal and Gómez-Dantes, 1995). DDT, the most classic example of organochlorides belonging to the group of aliphatic diphenyls, has its mode of action in sodium channels that are transmembrane proteins located at the level of the axon of the nerves, the latter of which are depolarized by the flow of sodium ions towards the inside when the channel doors are opened, which results in the activation and inactivation of the voltage-gated sodium channel (Narahashi 1992, Zlotkin 1999).
The resistance mechanisms based on target site resistance have been directly linked to the mode of action of insecticides, whereby the importance of knowing where insecticides in use are acting seems evident, seeing that where they take effect is where modifications to the protein structure, which prevent the union of the insecticide, is expected to take place and results in resistance. On the other hand, there are resistance mechanisms not based on sites of action, resulting from enhanced metabolism due to the enzymes that trap and metabolize insecticides in their path to the site of action. Such metabolic resistance mechanisms are not linked to any specific site of action classification and therefore they may confer resistance to insecticides in more than one group with different sites of action (IRAC July 2017). Thus, one of the main DDT resistance mechanisms besides the overproduction of glutathione s-transferases that metabolize it are the mutations of a single nucleotide occurring in the hydrophobic segment 6 of domain II of the voltage-gated sodium channel (Brengues et al., 2003), which reduce the union of insecticides and apparently need to be homozygous mutations to provide potent DDT resistance.
As of 1970, the eradication campaign was officially suspended due to the lack of political willingness; thereafter the countries where the vector had been eradicated were re-infested once more. By 1997, the distribution range of Ae. aegypti encompassed more countries than prior to eradication (Badii et al., 2007).
Vector Control in the Era of Malathion as Adulticide and Temephos as Larvicide
Following the eradication period and before the re-infestation of areas by this vector in 1975 by both Mexican borders, classic dengue reappeared (SSA, 2001), which is why the control was implemented by means of the malathion as adulticide and temephos as larvicide until 1999 (Fernández, 1999). During this period, there were no reports in literature about resistance to both compounds of Ae. aegypti from Mexico. Further, in 1996, Sames et al. reported susceptibility for Ae. aegypti and Ae. albopictus to malathion, chlorpyrifos, resmethrin and permethrin in the border of the valley of Texas and Mexico, and although it is not an area that represents the entire national territory, the Northeast of Mexico was and has been subject to vector control actions. Nevertheless, the lack of reports does not mean that there are no mosquito populations with resistance emerging from those insecticides.
Organophosphates (just like carbamates) are cholinesterase inhibitors. Organophosphates are compounds whose combination with the active site of the acetylcholinesterase (AChE) is many times irreversible, but depending on the enzyme compound, it can dephosphorylate and take days to recover and regain its normal function. This is unlike carbamates that, when combined with the carboxylase enzyme, have a reversible reaction and can take just half an hour to reactivate (Fukuto, 1990). The combination of these insecticides with AChE, therefore, prevents normal enzyme activity, which is the hydrolysis of the neurotransmitter acetylcholine. In a neuronal poisoning with organophosphates and carbamates, insects die from overactivation of nerve impulse after lacking control over acetylcholine depletion.
A mutation caused naturally at random, identified in anophelines and culicines from different parts of the world that confer resistance to organophosphates and carbamates, is the replacement of serine for glycine in position 119 (G119S) in AChE1 (Weil et al., 2003). However, this mutation has not been found in the ace-1 gene of Ae. Aegypti, which is probably because this gene uses a different codon for glycine in this position, requiring two specific mutations for the conversion of glycine to serine (Weil et al., 2004).
Resistance measures may differ in larvae and adult mosquitoes to the same insecticides.
Organophosphates are prone to degradation by chemical or enzymatic hydrolysis, releasing products from detoxification. Enzymatic hydrolysis is mediated by a variety of esterases, also called hydrolases. Hydrolysis can be carried out far from the center of OP phosphorous by the so-called carboxylesterases, just as with malathion, releasing derivative products from the non-toxic carboxylic acid (Fukuto, 1990). These detoxification reactions to monoacids react much faster than the activation reaction P=S to P=O. Therefore, malathion is toxic for insects with low concentrations of carboxylesterases. The primary metabolic pathway for carbamate is oxidative, and it is associated with monooxygenase enzymes, whereby the typical reaction is oxygenation, where an oxygen atom is introduced within the molecule through reactions such as hydroxylation, dealkylation, demethylation or oxidation. Methyl-carbamates are more toxic than OFs because they are direct inhibitors of AChE as they no longer require activation to react with the enzyme (Fukuto, 1990).
Resistance measures may differ in larvae and adults to the same insecticides (Rawlings et al., 1996), indicating that the mechanisms selected in larva are not necessarily going to be manifested in adults. In studies from other countries where resistance to temephos and malathion has been assessed, no correlation has been observed that indicates a possible cross-resistance. An example of this phenomenon is from Brazil, where malathion has been used as an adulticide and temephos has been used as a larvicide; there, Bento et al. (2003) report that resistance to malathion was lower than to temephos. In Guadalupe and Saint Martin, where there was also a strong use of temephos as larvicide and malathion as adulticide – which were removed from use in 2009 and 2010, respectively – Goindin et al. (2017) report high levels of resistance to temephos (RR between 8.9 and 33.1) and low to moderate for malathion (RR between 1.7 and 4.4), thus finding no association regarding resistance to both insecticides. Saavedra-Rodriguez et al. (2014) selected resistance to temephos in five generations of Ae. aegypti from Mexico in the laboratory; bioassays using inhibitors of glutathione s-transferases, esterases and monooxygenases suggested that several esterases, instead of glutathione s-transferases that were quite high, are the primary metabolic mechanism that confers resistance to temephos.
Malathion came out of the official standard at the end of the 1990’s and NOM-032-SSA2-2002 provides the following recommendation:
“9.11.5 Insecticides. The use of permethrin, esbiol and piperonyl butoxide at doses of 10.9, 0.15 and 11.1 grams of active ingredient per hectare respectively; or as an alternative, cyfluthrin at doses of 1 to 2 g/ha. The malathion insecticide in spatial application is kept temporarily out of use for vector control, until a larvicide that does not belong to the same group of organophosphates becomes available.”
Thus, malathion is replaced for a mixture of pyrethroids, arguing that both the adulticide and larvicide belong to the same toxicology group. Although it is an acceptable recommendation of first instance in the selection of insecticides, belonging to the same toxicology group does not necessarily lead to resistance mechanisms developing in the same way, and this is even more so when insecticides are applied at different stages of the insect’s life, such as with these two insecticides.
The Use of Pyrethroids
Pyrethroids came into action in the control program in Mexico by the end of the 1990’s, not in response to a situation of resistance to OPs, specifically the malathion, but in fear of or as a precaution for the development of them (NOM-032-SSA2-2002). Other reasons mentioned, but not cited, include the formulation of the malathion that was used (at a 96% technical degree); in addition to being very odorous and irritating, it was highly corrosive, and there were many complaints about it from the general population. Another aspect that can be attributed as part of the right decision to change the malathion for the formulation of the first pyrethroids used after the malathion (Aqua Reslin Super® (permethrin + esbiol + piperonyl butoxide)) was the practicality of having a water-based spray product for the first time in Mexico. This simplified operational problems such as transportation and storage, as the equivalent of an oil-based 200 L ULV container could be transported in 20 L containers. As of 2009, the mixture was changed for another pyrethroid, d-phenothrin, to which different formulations, also synergized with piperonyl butoxide, have been added.
|In most cases, resistance not only negatively affects the compound that is in use, but it also confers cross-resistance to other chemically-related compounds. This is because products from a same chemical group usually affect a common point of action.|
In most cases, resistance not only negatively affects the compound that is in use, but it also confers cross-resistance to other chemically-related compounds. This is because products from a same chemical group usually affect a common point of action, which is why it is considered that they share the same mode of action, as is the case of pyrethroids. Therefore, there is a high risk that the resistance developed automatically confers cross-resistance to all compounds from the same group (IRAC, 2011). All pyrethroids, just like DDT, act in the sodium channel of nerve cells, where various mutations have been reported, not allowing these insecticides to join its site of action. At the beginning of this past decade, several mutations that confer cross-resistance to Ae. aegypti from several parts of the world were detected. These mutations were not correlated to those that are typically reported in the most commonly studied insect species (Brengues et al., 2002).
Given that both pyrethroids used during this period are of the same type, it is expected that wherever there is resistance to permethrin, resistance to d-phenothrin will also be present. Therefore, except for the difference in formulation (one water-based and the other oil-based), the impact on populations resistant to permethrin would be very similar.
There are several works that report resistance to permethrin in Mexico, both metabolic and by alteration in the site of action. In Mexico, Flores et al. (2006) found different resistance mechanisms (α and β esterases and P450) in Ae. aegypti gathered in five localities in the state of Quintana Roo (Benito Juárez, Cozumel, Isla Mujeres, Lázaro Cárdenas and Solidaridad) that confer resistance to organophosphate and carbamate insecticides and some pyrethroids. Saavedra et al. (2007) found that resistance to permethrin PYR in Isla Mujeres, Quintana-Roo, is associated with the mutation of allele Ile1011Val in Ae. aegypti. This mutation was also found to be combined with mutation Val1016Iso in Ae. aegypti populations in the localities of Puerto Chiapas, Pijijiapan, Huehuetan, Huixtla, Motozintla and Escuintla in the state of Chiapas. Recent studies from our laboratory, in collaboration with William Black from the University of Colorado (not published), reveal that monooxygenases are involved in the permethrin metabolism, as greater quantities of metabolite 3-phenoxybenzoic were found in mosquitoes that survived permethrin than in those who did not survive.
What is interesting about the pyrethroid situation, at least in Mexico, is that resistance to these active ingredients appeared during approximately the same period in which the malathion was previously used, as verified in several reports on the status of permethrin resistance. What is not necessarily interesting but surprising, indeed, is the fact that, despite there being reports of resistance to pyrethroid type I, replacing it for another pyrethroid type I has been suggested. However, regardless of the latter, or maybe exactly because of that idea and to make better decisions in the future, it is appropriate to revise the notes and basic teachings from the work of George P. Georghiou (1925 – 2000), who established and described the factors associated with the development of resistance to insecticides in insects. Some of these factors may perfectly explain the phenomenon of the relatively quick appearance of pyrethroid resistance in an urban and highly domesticated vector as the Ae. aegypti.
At present, the vector control program in Mexico has a more integrated view about the approach of the use of insecticides for vector control, such that the use thereof is regulated based on the susceptibility of the vector populations in different regions of the country. In this sense, in the list of recommended insecticide products, there are active ingredient options that adjust better based on the state of susceptibility/resistance of the vectors.
The development of resistance and current levels reported in a large part of Mexico may be the consequence of not only the pressure of used insecticides, but also of other factors that are involved in resistance development. Said factors were defined by Georghiou and Taylor (1977a, 1977b, 1986) and are divided into genetic, biological and operational. The most relevant genetic factors are frequency, number and dominance of resistance alleles in a specific population. Biological factors include generational time, descendance by generation, migration and fortuitous survival or refuge. Genetic and biological factors are inherent to the species in question and cannot be controlled. Nonetheless, they may be considered within the operational factors, which are controlled, as the influence of operational factors may affect the frequency of alleles that confer resistance (Georghiou and Taylor, 1977b). Out of the nine operational factors listed (Table 1), four of them may be very important in the context of Ae. aegypti resistance, three of them related to “the chemical” and one to “the application.” The first three related to “the chemical” are: 1) the chemical nature of the pesticide, 2) the relationship with the chemical (insecticide) previously used and 3) the persistence of residue and formulation, while the one related to “the application” refers to the manner of application.
First, we will discuss the genetic and biological factors of Ae. aegypti. After the eradication campaign was suspended, DDT was not used for several years, after which, when this species was reintroduced into the country, malathion was used. Therefore, if we consider that KDR could have been pre-selected by DDT, the frequency, number and dominance of selected resistance alleles in the places from which the species was reintroduced, had to return to baseline after the use of an insecticide from another toxicological group. In regard to the biological factors, specifically migration and fortuitous survival or refuge, it is known that Ae. aegypti is a highly anthropophilic and domestic mosquito, and whose dispersion follows the findings from mark-release-recapture studies. These mainly show that released mosquitoes do not move from the house where they were released or at least go to the adjacent house, with a very low percentage dispersing to a maximum of 512 m (Harrington et al., 2005); given that it is a highly domestic species, perhaps the only protected refuge from insecticides is inside homes.
Table 1. (Taken from Georghiou and Taylor, 1986).
In regard to the operational aspects, the main impact of insecticide use in the development of resistance is when the indiscriminate use of a single active ingredient is maintained for very long periods of time (Rodriguez et al., 2006). This is coupled with whether the insecticide previously used is from the same toxicology group or acts in the same site. In the case of Ae. aegypti control in Mexico, after the extended use of DDT in the eradication campaign, in addition to suspending applications when declaring the vector eradicated, eliminating the pressure of selection for a few years, the following insecticide used corresponded to an organophosphate. In theory, the few mosquitoes with resistant alleles, in the case of KDR having been selected with DDT, should have disappeared during the period of the malathion application. This should also occur in populations where there are refuges and migration prone to treated areas (Figure 1). As a result, when pyrethroids are introduced (permethrin + esbiol + piperonyl butoxide) for the first time, Ae. aegypti populations should have had an extremely low R allele rate. Up to now, factors that affect resistance and the principles of an adequate management thereof may be said to be in the process of being fulfilled. However, what happened in the same time frame so that malathion was used without developing resistance to Ae. aegypti populations, but resistance did develop in pyrethroids? Could it be that the R alleles selected during the use of DDT in places from which the species was reintroduced were not so low? Or, was the species never eradicated in Mexico and populations selected with DDT were present when the PYR came into use?
|Figure 1. Schematic representation of a “normal” cycle of pressure of selection by insecticides where there is immigration of susceptible genes.|
What happened is most likely the result of various combined factors. It is known that spatial applications outdoors firstly impact mosquitoes that, at the time of application, are outdoors. Therefore, assuming that the insecticide concentration is appropriate, the fraction of the population outdoors is eliminated. Now, if we assume that the only available refuge is inside houses, then that is where susceptible genes would hide to cut resistance in the following generation. However, given that the intention of spatial applications has been to try to make the insecticide enter domiciles (for which people are advised to open doors and windows), then it would also be eliminating the fraction of the safeguarded population. Nonetheless, it is known that the insecticide concentration that enters houses when it is applied outdoors at ULV is much lower than the advised concentration (Peirich et al., 2000), which is why mosquito populations inside would be exposed to lower concentrations, and only the susceptible fraction of the population would be eliminated, leaving the survival of resistant heterozygotes and homozygotes (Figure 2). Even more so, given the small dispersion of this species, and adding to the fact that applications cover complete areas where no area is left untreated (refuges), it would quickly impact the presence of susceptible genes that may cut resistance by the selection of resistant individuals (Georghiou and Taylor, 1986). Another relevant aspect of pyrethroid formulations recently used is that, in order to have a greater impact on mosquito populations, they are formulated with the PBO synergist. The scope of this synergist is to inhibit enzymes that metabolize the insecticide when entering the insect, which guarantees that the insecticide reaches the site of action. Although the use of synergists to eliminate detoxifying enzymes is considered within the resistance management strategies (Georghiou, 1994), it is true that the removal of this advantage from a population may speed up the selection of a mechanism through alteration in the site of action (in this case, KDR). Once this is established in a population and in lieu of the immigration of susceptible genes, it basically makes this insecticide permanently useless for this population in this specific geographic area. The foregoing, in addition to the method of application as an operational factor that affects the development of the resistance (Georghiou and Taylor, 1986), has perhaps been the main reason for the extremely fast development of resistance to pyrethroids in Ae. aegypti in Mexico and in other parts of the world.
|Figure 2. Possible impact of spatial spraying as an operational factor in the development of insecticide resistance in populations of Aedes aegypti.|
Therefore, it is not surprising that there have been cases of resistance to almost fixed levels in populations of Ae. aegypti in Mexico, reported by diverse authors (Ponce et al., 2009, Aponte et al., 2013).
In conclusion, it is very likely that the development of resistance in Ae. aegypti has been the result of a combination of factors, both intrinsic given the highly domestic characteristics of the species, and operational given the manner of application, the nature and formulation of the insecticide, and the lack of migration of susceptible genes due to the low dispersion of this species, on the one hand, and, on the other, the lack of refuge of said susceptible genes when undertaking applications that broadly cover areas with arbovirus transmission problems.
It would be ideal to have other control tools and depend less on the use of insecticides, or in any case, not risk so much on the spatial spraying outdoors, and continue promoting the improvement of vigilance so that a better system can be achieved, which allows focus on chemical control actions indoors, be it by means of ULV (cold or thermal) or by means of residual spraying using application equipment that allows to do it more quickly.