Insecticide resistance in Culex quinquefasciatus Say, 1823 in Brazil: a review

Culex quinquefasciatus is a successful invasive species broadly distributed in subtropical regions, including Brazil. It is an extremely annoying mosquito due to its nocturnal biting behavior, in high-density populations and it is a potential bridge between sylvatic arbovirus from birds to man in urban territories. Herein, we present a review concerning the methods of chemical control employed against Cx. quinquefasciatus in Brazil since the 1950’s and insecticide resistance data registered in the literature. As there is no specific national programme for Cx. quinquefasciatus control in Brazil, the selection of insecticide resistance is likely due in part to the well-designed chemical campaigns against Aedes aegypti and the elevated employment of insecticides by households and private companies. There are very few publications about insecticide resistance in Cx. quinquefasciatus from Brazil when compared to Ae. aegypti. Nevertheless, resistance to organophosphates, carbamate, DDT, pyrethroids and biolarvicides has been registered in Cx. quinquefasciatus populations from distinct localities of the country. Concerning physiological mechanisms selected for resistance, distinct patterns of esterases, as well as mutations in the acetylcholinesterase (ace-1) and voltage-gated sodium channel (NaV) genes, have been identified in natural populations. Given environmental changes and socioeconomical issues in the cities, in recent years we have been experiencing an increase in the number of disease cases caused by arboviruses, which may involve Cx. quinquefasciatus participation as a key vector. It is urgent to better understand the efficiency and susceptibility status to insecticides, as well as the genetic background of known resistant mechanisms already present in Cx. quinquefasciatus populations for an effective and rapid chemical control when eventually required.

that Cx. quinquefasciatus would also be competent to transmit the protozoan Plasmodium relictum (agent of bird malaria) [13] and Hepatozoon breinli, an intracellular parasite infecting birds, reptiles, amphibians and rodents [14]. An extensive list of viruses, protozoans and nematodes isolated from Cx. quinquefasciatus under natural and laboratory conditions can be found elsewhere [15].
In the absence of effective vaccines available against most of the Culex-transmitted pathogens, the best strategy to avoid transmission relies on the chemical control of the mosquito [16]. At the end of the last century, the World Health Organization (WHO) launched a manual focused on mosquito vector control, including Culex spp., highlighting the necessity of measures to prevent their reproduction and dispersion [17]. Although Culex spp. females preferentially lay their eggs in collections of water, either stagnant or gentle flow, rich in organic matter, Cx. quinquefasciatus is very opportunistic so that any permanent or temporary collection of water may serve as a potential breading site for their larvae [1,18]. Therefore, vector control planning has to focus on breeding-site elimination or treatment by improving the basic sanitary infrastructure of water supply and waste destination, as well as activities to promote community engagement within an environmental agenda. However, given the accelerated and disorganized process of urbanization in the last decades, especially in the tropical, lowincome countries, these tasks are too complex to be fully achieved. Additionally, even in well-developed regions, the density of these mosquitoes may be positively correlated with seasonal high temperatures [19,20]. In this scenario, chemical larvicides or polystyrene granules can be applied to water collections. Insecticide residual spraying (IRS) in the interior of the houses is generally not effective against Cx. quinquefasciatus given its habit of posing on substrates generally not treated with insecticide, such as cloth, curtains and other suspended fabrics, instead of resting on the walls and ceiling [17].
The first official actions in Brazil specifically targeting Cx. quinquefasciatus based on chemical control were during the years 1951-1955, as a first phase of a governmental campaign to control Bancroftian filariasis [21]. This campaign was coordinated by the National Service of Malaria, opting the use of hexachlorobenzene (BHC), dichlorodiphenyltrichloroethane (DDT) and dieldrin as residual action insecticides [22]. The second phase of this campaign was initiated in 1956, with the creation of the National Department of Rural Endemics (DNERu). By 1960, 120,339 midguts were dissected from female mosquitoes caught in filariasis endemic areas, which recognized Cx. quinquefasciatus (at that time named Cx. pipiens fatigans) as the main vector in the country [21]. In the following decade, Brazilian national campaigns against filariasis were coordinated by the Superintendence of Public Health Campaigns (SUCAM). These campaigns aimed at eradicating or controlling the filariasis transmission in endemic areas by treating committed persons with the chemotherapy diethylcarbamazine, as well as decreasing the density of the mosquito by improving the sanitary infrastructure and applying residual insecticides (BHC and dieldrin) against both larvae and adult stages of Cx. quinquefasciatus [22].
The employment of residual insecticides (BHC, DDT and dieldrin) for controlling adult mosquitoes was incipiently effective. Nevertheless, they became ineffective, their use being suspended [22]. Given the lack of an efficient adulticide together with the high cost of larvicide applications in the breading sites, the chemical treatment was discontinued and the national programmes centralized their actions on the treatment of human cases and health educational programmes [22]. Currently, the Brazilian Ministry of Health (MoH) acquires the insecticides and provides them to the states, which supply the municipalities. In turn, the municipalities have autonomy to complement alternative compounds in their territories, as long as they are approved by the WHO and the Brazilian National Agency of Sanitary Surveillance (ANVISA) [23]. There is no specific national programme for combating Culex, as most of the governmental actions against this mosquito are a side-effect of the well-structured programme for Aedes aegypti control. In this sense, most of the insecticide selection pressure geared toward Cx. quinquefasciatus populations in Brazil is substantially derived from that targeting Ae. aegypti [24].
In the last three decades the main larvicides utilized in Brazil under national scale against Ae. aegypti have been the organophosphate temephos, followed by the IGRs (insect growth regulators class) diflubenzuron, novaluron and more recently, pyriproxyfen. Pyrethroids were adopted as adulticides from 2000 until 2013 when the organophosphate malathion began to be implemented, as the only permissible alternative after reports that pyrethroid resistance in Ae. aegypti was apparent all over the country [23,25]. Nevertheless, commercial pyrethroids have intensively been sprayed inside the dwellings as well as under thermo-fogging or ultra-low volume in the peri-domicile by private companies. In addition to neurotoxic insecticides, as recommended by the WHO [26], the bacterium Lysinibacillus sphaericus (Lbs), previously known as Bacillus sphaericus (Bs) [27], is also indicated and largely enlisted as a biolarvicide for Culex control [28]. Lysinibacillus sphaericus began to be exploited on a large scale for Culex control in Brazil since 1989, ever since used as a larvicide by the Filariasis Elimination Programme in Recife/PE and on the border of the Pinheiro River in São Paulo [29,30].
Likewise, as with Aedes and Anopheles mosquitoes, the exacerbated use of insecticides has been selecting resistant Cx. quinquefasciatus populations around the world [31][32][33][34]. Resistance to insecticides is a multi-factorial genetic trait, preceding insecticide exposure. Normally, the frequency of resistant insects in natural populations is very low in an environment free of insecticides, i.e. without a selection pressure. Hence, the continuous application of insecticides favorably selects the resistant individuals, while those susceptible are progressively eliminated, reducing the genetic variability of the target population [35]. Depending on the intensity of the selection pressure over genetically well-structured and isolate populations, resistance may become irreversible due to the lack of susceptible mosquitoes to contribute their genes to the next generations, where migration among other populations is absent or very low [36]. In Brazil, in addition to the chemicals employed in governmental campaigns, the uncontrolled application by households increases during arbovirus outbreaks and also when targeting Culex itself due to its usual high densities and annoying nocturnal biting behavior [37,38]. As the odor of pyrethroids is less noxious to the people, this class of insecticide is largely preferred [39,40]. There is evidence that this excessive household use of chemicals is the main factor contributing to pyrethroid resistance selection in Ae. aegypti [41,42], Cx. quinquefasciatus populations as such being likely to experience a similar phenomenon.
There are four main classes of mechanisms attributed to resistance in a mosquito population: behavioral changes, decrease of cuticular penetration, increase in the metabolic detoxification and alteration in the insecticide target-molecule, these two latter mechanisms being the mostly molecularly elucidated [43][44][45]. An increase in the metabolic detoxification may occur due to an increase in the detoxification power, generally related to the classes of enzymes esterases, glutathione S-transferases (GSTs) and multi-function oxidases P450s, which are able to modify or break up the insecticide molecules before they reach their target. In turn, target-site alterations inhibit the interaction between the insecticide and its action target molecules, rendering the insecticide less effective or even ineffective [46].
In 2011, the Brazilian MoH launched a surveillance and control methods guide against Cx. quinquefasciatus, recommending the use of neurotoxic (pyrethroids, carbamates or organophosphates) and IGR compounds (juvenile hormones analogues and benzophenil ureas, as chitin synthesis inhibitors) in conjunction with the biolarvicide Lbs [5], very similarly as indicated against Ae. aegypti. The compounds currently indicated by the MoH are: organophosphates and pyrethroids for adult control and spinosyns; bacterium biolarvicides; benzophenilureas; juvenile hormone analogues; and organophosphates against larvae [47]. Nevertheless, there are some reports of Cx. quinquefasciatus populations resistant to some of these compounds in the country (see Fig. 1). A list with insecticide resistance data available in the literature, including susceptibility tests, biochemical and molecular assays, distributed per region and year are provided in Table 1.

Organophosphates and carbamates
First reports of organophosphate resistance in Cx. quinquefasciatus from Brazil came from Rio de Janeiro populations evaluated with chlorpyrifos in 1978 [48] and later in 1994 [49]. Resistance to the larvicide temephos was described in populations from Campinas (São Paulo State) [50], Santa Cruz do Capibaribe (Pernambuco State) and Campo Grande [51] and Naviraí [52] (Mato Grosso do Sul State). In addition, a population from Cuiabá (Mato Grosso State) collected in 2000 was classified as "tolerant", while Ae. aegypti collected at the same site and year were susceptible to the insecticide [50]. Resistance to the carbamate propoxur was evidenced in Cx. quinquefasciatus collected in the region of Pinheiros River in the center of São Paulo, in 1995 and 1996 [53]. Also, in São Paulo, resistance was detected to both organophosphates malathion and fenitrothion [53,54] and to malathion (RR 50 of 43.81) in Rio de Janeiro [24].
High levels of resistance to malathion were also observed in Cx. quinquefasciatus from other Latin American countries such as Cuba (RR 50 of 207.91 and 135.97) and to a lesser extent Venezuela (RR 50 of 16.11). The authors considered that the intense employment of the OP themephos, malathion and propoxur and also pyrethroids, for the first dengue outbreaks in the 1980's, contributed to control Ae. aegypti, however inducing resistance in Cx. quinquefasciatus to OPs and propoxur [55,56]. In Brazil, resistance to temephos is currently disseminated in Ae. aegypti populations throughout the country which forced the National Dengue Control Programme to replace this chemical by Insect Growth Regulators (IGRs) compounds [25]. This scenario of disseminated resistance to temephos might as well be extended to Cx. quinquefasciatus. For example, more than 40,000 kg of temephos were applied in the city of Santa Cruz do Capibaribe alone against Ae. aegypti during 2007-2010 which was likely the source of pressure that selected resistant Cx. quinquefasciatus in the region [51].
Organophosphates and carbamates both target the acetylcholinesterase enzyme (AchE), causing the accumulation of the neurotransmitter acetylcholine in a synaptic shift, which inhibits the interruption of a nervous impulse and therefore, kills the insect. The metabolic enzyme participation also plays an important role in organophosphate resistance in Culex. For instance, a gene or a set of esterase genes suffered several duplications, causing the increase of their codified enzymes and consequently, more sequestration of the insecticide molecule [57][58][59]. The register of detoxifying enzyme quantification associated with insecticide resistance in Cx. quinquefsciatus from Brazil was noted in populations from Fortaleza [60], São Paulo [53], Santa Cruz do Capibaribe [51] and Rio de Janeiro [24,49].
A single nucleotide polymorphism (SNP) in the acetylcholinesterase gene (ace-1) with the substitution of a Gly by a Ser in the 119 codon (G119S, ace-1 R allele) is a target site mechanism mostly displayed in Culex populations resistant to organophosphates. The same G119S SNP is also found in other insects, including Anopheles mosquitoes [61][62][63]. Other mutations (F290V and F331W) were also described in the Culex ace-1 gene, also possibly related to resistance to organophosphates [64][65][66][67]. The G119S was the only ace-1 mutation found in Cx. quinquefasciatus from Brazil, in the localities of São Paulo [53], Recife [64], Santa Cruz do Capibaribe [51] and Rio de Janeiro [68].

Organoclhorines and pyrethroids
The organochlorine DDT and the pyrethroids act in the voltage-gated sodium channel (Na V ) in the neuron membranes, prolonging its open state. This interaction results in a repetitive firing of the nervous impulse, leading the insect to involuntary muscle spasms, exhaustion and death, a phenomenon known as knockdown effect [69]. Organochlorine may also inhibit the gamma-aminobutyric acid (GABA)-gated chloride channel. This is the case of cyclodienes, such as dieldrin which antagonizes the effects of the GABA receptor by preventing chloride ions from entering the neurons, thus inhibiting the return to resting state after an impulse transmission. A classical mutation (A302S) in the GABA receptor induces resistance to dieldrin, thus being referred to as RDL in several insects including Cx. quinquefasciatus [70,71]. No substitution in the GABA gene has been reported in populations from Brazil. Increase in the action of detoxifying enzymes is usually related to DDT-and pyrethroid resistance, especially GST and monoxygenases P450 classes [72,73]. However, given the diversity of multiple genes in these classes, it is difficult to find the same specific molecular marker for metabolic resistance across distinct         In the case of the target-site alterations, however, the same SNPs are found in distinct species, as is the case of the substitution Leu by Phe in the 1014 site of the Na V gene, known as the classical kdr (knockdown resistance) mutation [74]. There is one report of a Cx. quinquefasciatus population from Rio de Janeiro collected in 1994 resistant to DDT [49]. The use of this compound had been discontinued against mosquitoes since 1971 and finally prohibited in Brazil in 1985 [75]. Therefore, this could result from a persisted resistance selected by DDT itself or cross-resistance by the use of other compounds. Resistance to the pyrethoids cyfluthrin and cypermethrin was identified in a population from Campinas collected in 1999, whilst simultaneously collected Ae. aegypti were susceptible [50]. In addition, there was resistance to deltamethrin in a laboratory strain from Divinópolis [76]. Resistance to deltamethrin emerged and spread very rapidly in Ae. aegypti since its introduction by national campaigns against the dengue vector in 2000. One decade later, high levels of resistance to this chemical were acquired throughout the country, especially during dengue epidemic seasons, probably with an important contribution of the use of household insecticide sprays, all pyrethroid-based products [25,41]. This environment with constant insecticide application near and inside the houses has likewise been selecting resistance to pyrethroids in Cx. quinquefasciatus.
To date, there has only been one report for a kdr mutation in Cx. quinquefasciatus from Brazil in which the classical L1014F was detected, yet under low frequencies (4-7%) in samples from Campo Grande, Rio de Janeiro, Niterói and Belo Horizonte [77]. In these localities, resistance to deltamethrin was apparent in Ae. aegypti probably related to kdr mutations [78]. Similarly, L1014F was detected in Cx. quinquefasciatus from Mexico, indirectly exposed to pyrethroids targeting Ae. aegypti during dengue control campaigns [79]. Other substitutions were found in the same 1014 aminoacid position (L1014S, L1014C) in the Cx. pipiens complex [80]. However, none were ever observed in Cx quinquefasciatus from Brazil, other than L1014F.

Biolarvicides
Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lbs) are bacteria that produce endotoxins that are activated by mosquito larvae intestinal proteases and bind to specific receptors in the intestinal epithelium, causing degeneration and consequently, killing the larvae [81,82]. The Lbs is more suitable for controlling Cx. quinquefasciatus because it presents higher activity in polluted water when compared to Bti [83].
The cqm1 gene encodes an epithelial protein to which the toxin binds. Some mutations in the cqm1 gene were associated with Lbs resistance. For example, a deletion of 19 nucleotides (cqm1 REC ) and the substitution of a guanine (G) to an adenine (A) in the codon 1324 (cqm1 REC-2 ), both present in Cx. quinquefasciatus field populations from Recife [84,85]. Resistance to Lbs was found in a Cx. quinquefasciatus populations from Coque, an urban area of Recife, only two years after its implementation in 1991, reaching a resistance ratio 10-fold higher than the susceptible control [86]. However, this resistance was later reversed with the interruption of the biolarvicide application in that locality [87]. In other areas of Recife city, Lsb continued to be enlisted and several studies have been evaluating the levels of susceptibility to this bioinsecticide in the city as well as identifying new molecular markers [88][89][90]. These studies have shown that the frequency of resistant individuals in Recife city remains at low levels, even with the continued application of the bacterial insecticide, either alone or in combination with other larvicides such as Bti [91][92][93][94]. In a recent study in Colombia, Lbs proved to be efficient against both Cx. quinquefasciatus and Ae. aegypti field populations, suggesting it as an interesting alternative to chemical insecticides [28].

Conclusions
In accordance with the WHO Global Vector Control Response [95], the emergence and spread of vector-transmitted diseases is likely to be intensified in the following years, especially those with the participation of urban mosquitoes such as Culex spp., given their strong adaptation to climate changes and inefficient urban sanitary infrastructures. Therefore, as the application of insecticides is a primarily action against Cx. quinquefasciatus in Brazil, it is urgent to investigate the status of susceptibility/resistance of natural populations to all the chemical compounds available for use. Effective vector control in Brazil is a complex and multifactorial task considering the continental dimensions together with the greatly heterogeneous ecological and demographic aspects [96]. Future successful campaigns based on the use of chemicals have to implement a constant monitoring of insecticide effectiveness, employing integrated methods against all targeted species and considering a plan well adapted to regional peculiarities.