Despite the fact that several non-chemical control methods have been evaluated so far, insecticides are still the most important tool for controlling mosquitoes and reducing the transmission of mosquito-borne human diseases. However, when mosquito populations are exposed to intensive selection pressure from insecticides, they tend to become resistant. Resistance has been defined as ‘the developed ability in a strain of insects to tolerate doses of toxicants that would prove lethal to the majority of individuals in a normal population of the same species’ . In a susceptible population, individuals with resistant genes to a given insecticide are rare, and usually range between 10–5 and 10–8 [2 and references therein] in number, but widespread use of a toxicant favors the prevalence of the resistant individuals. These individuals multiply fast in the absence of intraspecific competition and, over a number of generations, quickly become the dominant proportion of the population. Hence, the insecticide is no longer effective and the insects are considered to be resistant. According to Busvine , the most important factors that affect or determine the development of resistance to insecticides are the following. (1) The frequency and dominance of resistant genes in the initial population. (2) The intensity of selection, i.e. the size of the population that is exposed to the insecticide and the proportion that gets killed. (3) The number of generations per year, and consequently how frequently the target population is selected in one season. Other factors that may also shape the development of resistance involve the application methods as well as the chemical properties of the formulations.
Types of resistance
Mosquito species that have been under continued selection pressure with one or a range of different insecticides may develop cross- or multiple resistance. ‘Cross-resistance’ means that the strain is not only resistant to one insecticide of a particular class (of a given mode of action) but also (often to a lesser degree) to other insecticides in the same class (with similar mode of action), even when it has never been treated with the other insecticides. However, the phenomenon of ‘multiple resistance’ is considerably more important. With this type of resistance, separate detoxification mechanisms for unrelated insecticides are present, resulting in an insect population that is resistant to different classes of insecticides (with dissimilar modes of action), which makes chemical control of that population extremely difficult.
Normally, an insecticide penetrates through the cuticle (exoskeleton) or digestive or respiratory system of an insect before reaching the site of action. The action site may be a vital enzyme, nerve tissue or receptor protein. Insecticide molecules bind to the site and, when they have attained a threshold concentration, they disrupt vital functions, causing insect death. Resistance may take place at each step of this pathway: during penetration reduced permeability may occur, thus reducing the rate of entry of the insecticide; new or more abundant metabolic enzymes may be selected, causing breakdown of the insecticide more efficiently; or altered target sites may be selected to which the insecticide no longer binds . Of these three types of mechanisms, metabolism and insensitivity at the site of action are the most important. However, a reduction in the rate of penetration aids the other types of mechanism in a synergistic way. In addition, another form of resistance is behavioural resistance, where insect behaviour becomes modified so that the insect no longer comes into sufficient contact with the insecticide deposits. Perhaps the best-documented example of this form of resistance is the development of an early, outdoor feeding phenotype among African anopheline populations in areas of extensive indoor insecticide use. These mosquitoes may circumvent long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS) control through preferential feeding and resting outside human homes and being active earlier in the evening before people have gone to sleep . It is generally considered that the resistance mechanisms already exist within the insect, but continuous insecticide pressure is the factor that actually triggers resistance development [2, 4, 6].
Resistance surveillance and management
Resistance monitoring should be an integral part of any mosquito control program. The susceptibility of populations should be verified before the start of control operations, to provide baseline data for insecticide selection and choice of application technique. Regular surveillance will allow early detection of resistance, so that resistance management strategies can be implemented or, in the case of late detection, evidence of control failure can justify the replacement of the insecticide. The operational criterion of resistance has usually been interpreted as a survival of 20% or more of field-collected individuals tested at the currently known diagnostic concentration of a particular insecticide, using standard World Health Organization (WHO) test kits .
Mosquito resistance to insecticides is of great practical and economic importance. Adverse consequences usually include an increment in control cost because more frequent applications and larger doses of a toxicant are necessary for population suppression. Moreover, research for the development of novel insecticides is very expensive and time consuming. These problems indicate the need to establish an efficient resistance management strategy aimed at preventing and delaying, as much as possible, the development of resistance to insecticides, ensuring a sufficient level of mosquito control. In this context, Georghiou  has suggested the following approaches to resistance management. (1) Management by moderation. This approach aims to maintain the susceptibility genes by using low insecticide rates, infrequent applications and non-persistent compounds. (2) Management by saturation. In this context, an insect’s defence mechanisms are saturated by using sufficient doses of insecticides so that no survivors remain. This procedure is useful during the early stages of selection for resistance genes where these are rare and heterozygous. (3) Management by multiple attack. In this approach insecticides are applied in mixtures or in rotation in order to exert selection pressures below the level that may lead to resistance. The use of mixtures assumes that the resistance mechanism to each insecticide initially exists at such low frequency that two different mechanisms are unlikely to occur together in any single individual within a given population. Consequently, individuals that may survive exposure to one of the insecticides used are killed by the other. Rotation is based on the principle that in most cases resistant mosquitoes have a lower biotic fitness than susceptible individuals, which results in a gradual decline of their frequency when the alternative (other) insecticide is used. Nevertheless, despite the fact that there are numerous available registered insecticides for mosquito control, these can be classified into only a limited range of modes of action, which makes the continuous rotation of insecticides with different modes of action difficult. For instance, in Greece, the control of mosquito larvae is primarily based on the use of insect growth regulators and microbial formulations (and to a lesser extent bacterial metabolite-based neurotoxic insecticides and inert materials). Thus, although there are several commercial formulations, the choices available according to the mode of action for a potential rotation are rather limited. Consequently, the evaluation of other ingredients with a different mode of action is essential, in order to encourage the future registration and use of novel insecticides in area-wide resistance management strategies.
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Ch. Ioannou, Ch. Athanasiou, University of Thessaly