The rise in insecticide resistance in Anopheles mosquitoes in the Americas represent a rising challenge to the control and elimination of malaria [51-53]. Although there are several insecticides with residual action available for indoor spraying, some of these have become ineffective due to the development of resistance, while others, although still effective, are no longer accepted due to its high toxicity towards mammals or their dangerous persistence in the environment . Notwithstanding the fact that 57 Anopheles mosquito species have developed some level of resistance, IRS still continues to be the main method used to control vectors in many malaria endemic countries [54-55]. From the start of the NMP in Panama, a diverse set of organochlorines, organophosphates, carbamates and pyrethroid insecticides have been used; creating a selective pressure against the An. (Nys.) albimanus populations . The NMP, similar to other malaria programs around the world, uses as the first line of defense the application of IRS against anopheline mosquito vectors. This approach has contributed, to a certain extent, to the reduction of malaria transmission in Panama (46). With the regional commitment as an initiative to eliminate malaria, the NMP has decided to conduct tests to detect possible failures in the current control methods and in that way improve the prevention strategies. Among these tests are the determination of bio-efficacy and resistance to insecticides (with indoor residual action) in the main malaria endemic regions of Panama.
The results of this study indicate that the quality of insecticide spraying and the nature of the different surfaces or walls in the seven selected communities have an effect on the toxicity and residuality of fenitrothion. Most of the houses in these communities have zinc plates for roofs and unpainted wooden walls. In comparison, the houses from the Guna indigenous communities followed their cultural tradition and were built with palm leaf roofs, walls made from small woody tree trunks placed vertically, without windows and dirt floors . Our study assayed diverse surfaces such as painted wood, unpainted wood, bamboo, Gira palm and tree trunks. Our results show that smooth surfaces do not absorb large quantities of insecticide particles and resulted in the longer duration of fenitrothion toxicity and residual effect. In comparison, porous surfaces that absorb a large quantity of insecticide had a shorter duration of toxicity and residuality. In this case, smooth surfaces such as painted wood and unpainted wood maintained the greatest insecticide toxicity and the longest residuality. Meanwhile, surfaces such as Gira palm, bamboo and tree trunks, which presented more porosity and absorption lost its toxicity and residuality in a short period of time. This effect has been previously observed, with the chemical nature of surfaces affecting the residuality and efficacy of most insecticides . Thus, the persistence and bio-efficacy of insecticides can vary depending on the type of surfaces and materials that are used to build a house in the rural and economically poor indigenous regions .
In similar studies, the application of deltamethrin on different types of surfaces, among them wood, bamboo and brick, found that the smooth surface of a bamboo presented the longest duration of insecticide toxicity and residuality. On the contrary, the rapid absorption by the brick porous surfaces led to a much faster loss of deltamethrin activity . Several other studies have also found similar results with insecticides losing their toxicity much faster on porous surfaces than on smooth surfaces such as wood panels, and ceramic tiles [59-60]. The duration of bio-efficacy should be within the residuality range found in previous studies or according to WHO recommendations. Nevertheless, in some places the residuality can vary from 2-10 months depending on the kind of treated surfaces . Thus, the residual efficacy of the IRS can vary according to the type of wall construction and the quality of insecticide spray application [58,62]. Furthermore, the IRS effectiveness depends on the level of resistance of the main malaria vectors and the insecticide residuality time on the treated surfaces . An application of low quality can contribute to the development of insecticide resistance, an additional burden to the malaria control strategies . If the residual activity is shorter than expected, it can also contribute to an increase in the malaria incidence where the period of malaria transmission exceeds the insecticidal residual effect . The indoor residual spray has demonstrated that it can have an epidemiological impact through the reduction of malaria transmission. This is especially true if the applied insecticide on different surfaces, and with a large cover area per locality/ transmission area, leads to high mortality rates in the anopheline vector population .
In this study we also found that the bio-efficacy depends on the formulation of the applied insecticide, mosquito species susceptibility, the study area, type of treated surface, humidity and temperature. A sustained evaluation of the bio-efficacy of insecticides used in IRS can provide critical technical information on the efficacy against a specific mosquito species, their safety to the exposed human inhabitants, the sprayers and in the methods of insecticide application . According to the recommendations provided by WHO , an ideal insecticide should have a minimum residual effect on mosquito mortality equal to or greater than 80% at 24h post-exposure.
This research provides the NMP with important evidence and technical information on the fenitrothion bio-efficacy against the main malaria vector, An. (Nys.) albimanus (45). In this study the effectiveness of fenitrothion residuality lasted for 20 weeks; primarily on two types of walls, and decreasing with time on the different surfaces until registering a low mortality/bio-efficacy on the eight month. Nevertheless, this insecticide can only be effective in areas where the An. (Nys.) albimanus is still susceptible, with potential variation or reduction in the susceptibility over time. Thus, our results fall within the recommended WHO guidelines . Nevertheless, additional studies are needed to learn the biting behavior and resting, population density of the different mosquito species and the bioecological characterization to better pinpoint the entomological impact of fenitrothion indoor spray application. This study presented some limitations. For instance, it utilized a study design that covered a limited period of time in each of the selected localities and the absence of a control locality that were to be evaluated at the same time. Thus, we should be careful to make causal inferences on the quantitative impact of the intervention. In addition, other factors, not evaluated in this study, might be related to the transmission reduction, such as the environmental factors associated with transmission intensity such as climate, human behavior and other methods of control .
Furthermore, it is necessary that the NMP evaluates alternative insecticides to be used at the time when a decrease in fenitrothion bio-efficacy is observed. The selection of an alternative insecticide with residual action must depend on An. (Nys.) albimanus susceptibility, the half-life of residual action, safety to humans, other animals and the environment. A technical factor of importance to malaria control programs is to determine the period of insecticide toxicity duration and residuality that leads to high mortality, given that this information will allow the development of programs, plans and logistics to determine spray cycles and coverage of the human population that will be protected until the next application cycle. An insecticide with high bio-efficacy leads to a protection against the bite of mosquitoes decreasing the risks of transmission, and in combinations with other strategies lead to a prevention of epidemic outbreaks. Previous studies have indicated that the insecticide bio-efficacy of residual action is very efficient in regions where the vector is completely susceptible to the insecticide applied .
One of the objectives of this study was to determine the level of insecticide resistance in An. (Nys.) albimanus mosquitoes. The mosquitoes used in the susceptibility assays originated from one of the main malaria endemic regions of Panama with characteristics that resembles that of the other selected locations in this study. The susceptibility bioassays indicated that An. (Nys.) albimanus continue to be susceptible to the organophosphate insecticide fenitrothion, carbamate propoxur and malathion. We observed that the level of susceptibility for fenitrothion was greater than for the insecticides malathion and propoxur. Nevertheless, the differences in the mortality rates were not statistically significant. This indicates that, despite the re-introduction of fenitrothion in 2002 for its use in IRS, the mosquito populations have not yet developed insecticide resistance. The results from this work coincide with work conducted with other native populations of An. (Nys.) albimanus from three malaria endemic communities (Pintupo, Aguas Claras and Puente Bayano), and in which they are susceptible to the insecticides organophosphate fenitrothion, malathion, chlorpyrifos and to carbamate propoxur. However, they had resistance to pyrethroids, deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin . These levels of insecticide resistance can contribute to failures in malaria control and lead to a greater malaria transmission .
The susceptibility of An. (Nys.) albimanus to fenitrothion could be due to the moderate frequency of application and to the percentage coverage. Given that malaria in endemic regions is characterized by transmission in small outbreaks or focus [45,70], the IRS applications do not cover all the communities in a specific malaria endemic area or the entire community where the outbreak is currently happening. Furthermore, there is reluctance on the part of homeowners to accept the IRS application especially among indigenous communities. Thus, in general, there is a partial application that does not attain 100% coverage of the houses and communities. This would leave a mosquito population free from exposure to the insecticide and allow these mosquitoes to maintain fenitrothion susceptibility within the population of An. (Nys.) albimanus that is targeted.
Another aspect that should be considered is the biting and resting behavior of the mosquito when affronting the selective pressure of the insecticides applied to surfaces. We have to consider that the socio-environmental changes such as the introduction of effective vector control tools could in turn induce a change in the behavior of Anopheles mosquitoes. Within the context of malaria control, the use of physical barriers (nets on windows and doors) as well as the insecticide action, induce a stress in anopheline mosquitoes interfering with blood feeding or with resting on interior walls until their eggs are fully matured. Studies indicate that behavioral modifications could include a change in feeding times, a switch from feeding exclusively on humans to feeding on other animals or an increase in the exophilic behavior . These behavioral changes could rise due to selection pressure induced by LLINs and IRS and lead to mosquito vectors feeding at earlier times and/or in the peridomicile or extra-domiciles. This could in turn give them a selective advantage giving rise to a phenotypic change in the population [72-73].
Despite the results that indicate fenitrothion susceptible in An. (Nys.) albimanus mosquitoes, the NMP should maintain a constant monitoring and surveillance evaluating the bio efficacy and susceptibility in the mosquito populations that are subjected to selective pressure with fenitrothion. In a similar work conducted in the Yucatan Peninsula, Mexico, the mosquito An. (Nys.) albimanus was resistant to DDT and deltamethrin, but was susceptible to carbamate bendiocarb and to the organophosphate pirimiphos methyl, with the exception of a locality (La Union) who was pirimiphos methyl-resistant . It is important to note that the NMP of Panama, does not have program or technical personnel trained in each of the malaria endemic regions that would routinely monitor and conduct surveillance work on the bio efficacy and insecticide-resistance of anopheline mosquitoes. This risks the usefulness of the insecticides currently being used to control mosquito vectors. The early detection of susceptibility changes in mosquitoes is of great importance to the vector control programs to maintain the effectiveness of current insecticides. Because surveillance and monitoring work on insecticide resistance is limited in Panama, the level of susceptibility is not known locally in the communities with active malaria transmission in the different endemic regions. In general, the magnitude of the problem of insecticide resistance is unknown for most of the malaria endemic regions in Panama . The development and implementation of a monitoring program of insecticide resistance by the NMP, is critical to maintain an effective strategy in the fight against An. (Nys.) albimanus. There is a need for finding the basal level of insecticide resistance for all the mosquito vectors in all the malaria endemic regions of Panama. The absence of this information puts at risk the usefulness of insecticides that are currently being used to control mosquito vectors. Furthermore, this information provides crucial technical information that allows us to apply strategies to prevent or delay the development of resistance, while maintaining an effective malaria control.
An important limitation in this study is that we could only work with one field-derived strain from one of the four endemic region of Panama. However, at the moment of strain selection, we made the effort to select a region with similar ecological, environmental and selective pressure than the sites selected for the bio-efficacy work. Thus, additional studies are needed to follow and select additional sites that would allow us to determine the bio-efficacy and susceptibility of the applied insecticides against An. (Nys.) albimanus.
In conclusion, this study shows that fenitrothion maintains an elevated residuality and toxicity in different types of surfaces, with An. (Nys.) albimanus remaining susceptible to this insecticide. This leads to an increased protection against mosquito bites inside the houseand can contribute to the interruption of malaria transmission in places that maintain a high IRS percentage coverage. This could also allow the maintenance of fenitrothion susceptibility in An. (Nys.)albimanus. Furthermore, this study provides important technical information valuable to the NMP efforts at controlling this important malaria vector. Here we present a base line of fenitrothion insecticide susceptibility and those alternative insecticides that could be used at a time when an insecticide change is needed. Furthermore, we provide a detailed map of the areas with An. (Nys.) albimanus populations that are susceptible or resistant to insecticides. This information could be further used by the NMP to considerably diminish the modifications often conducted in the use of insecticides, reduce the number of control strategy changes and reduce the cost of vector control programs. Our study also recommends that the NMP develop and implement a program that routinely monitors the bio-efficacy and insecticide-resistance to attain an adequate management of insecticide resistance when this technical problem eventually arises.