Effectiveness of ALPHABAN 40 WP (Cypermethrin) insecticide in Blattella germanica and Aedes aegypti control in Cuba

Disease vector control in Cuba is carried out primarily with chemical insecticides, with those based on cypermethrin being the most commonly used .In Cuba there are 30 registered formulations whose active ingredient is the cypermethrin molecule. ALPHABAN 40 WP (cypermethrin) formulation from PELGAR INTERNATIONAL LTD, UNIT 13 NEWMAN LANE, ALTON, HAMPSHIRE, GU34 2QR. UK is a high-potency wettable powder containing the potent synthetic pyrethroid. Cypermethrin is has excellent insecticidal qualities and a good knockdown effect. Before starting the field evaluation, the susceptibility status to cypermethrin was evaluated using the impregnated bottle methodology on a population of Aedes aegypti mosquitoes in the selected area, which was resistant (90% mortality). In the following study, we set ourselves the objective of evaluating the effectiveness of ALPHABAN 40 WP in Aedes aegypti control through surface bioassays and in Blattella. Germanic through residual treatment. The residual effect on different surfaces in tile, concrete and wood was evaluated with the mosquito strain collected for 10 weeks. Mortality fluctuated from the first weeks (, but remained high > 80%), on all surfaces until week 6. The highest mortality rates occurred on plastic and metal during the 10 weeks (100%). In the case of Blattella germanica, sticky traps (Insect Trap and Monitor) were used to monitor the infestation index in cockroaches, the results of which show up to a 95% reduction in the eighth week.

ALPHABAN 40 WP (Cypermethrin) has powerful dislodging and knockdown effects against crawling and flying insects, and, thanks to its long residual life, provides good control even on difficult or absorbent surfaces [1]. ALPHABAN 40 WP has a contact effect against insects and provides systematic protection against them. Wettable powders (WP) are formulated to provide exceptional residual control. The active ingredient is mixed or adsorbed onto a mineral filler (depending on whether the active is liquid or solid at room temperature). Dispersants and surfactants are added so that the formulation can be diluted, suspended or dispersed in water for easy application. This type of formulation is especially useful for the treatment of highly absorbent surfaces such as brick, clay or some woods. After application, the active ingredient (and filler) remains on the surface as a microscopic powder with insecticidal activity, which is easily picked up when an insect passes over it. Cypermethrin is a non-systemic and non-volatile insecticide that acts by contact and ingestion offers effective insect control, with no activity on mites and low toxicity to mammals. It is very effective on Lepidoptera, Coleoptera and Hemiptera., is also used to control flies and other insects in the habitats of domestic animals and pests that affect public health (mosquitoes and cockroaches),its spectrum of action covers the entire range of crawling and flying insects such as: cockroaches, flies, mosquitoes, fleas, bedbugs, ants, wasps, crickets and spiders; hence its performance in the control of arthropods in all types of environments, such as homes, factories, warehouses, hospitals, slaughterhouses, industries, buildings, restaurants, hotels, market places, holiday centers, etc.

The mechanism of action of cypermethrin is that it rapidly penetrates the cuticle of insects and disrupts nerve conduction by delaying the closure of sodium channels in nerve axons. This initiates a repetitive impulse and causes loss of muscle control resulting in rapid knockdown of the insects. Disorientation and cessation of feeding activity occur within minutes, followed by paralysis and death.

Susceptibility and/or Resistance study to cypermethrin under laboratory conditions using the impregnated bottle methodology.

To determine susceptibility to the active ingredient cypermethrin, a fundamental requirement for defining the application of the treatment, mosquitoes from the Varadero-Peninsula population and the Rockefeller reference susceptible strain raised in the laboratory were used. The bioassays using impregnated bottles were carried out following the CDC 2010 protocol, modified by the use of 250 ml capacity glass bottles with ground-in lids. The bottles were impregnated with 1 mL of cypermethrin at 13.5 µg/mL, the dose recommended by Rodriguez MM, et al. [2]. The solutions were applied inside each bottle and rotated until the solvent, acetone, had completely evaporated. The control was impregnated with 1 ml of acetone. The bottles were covered with aluminum foil after impregnation and the lid was placed on them. After 24 hours, bioassays were performed. One control and four replicas were used for each concentration to be evaluated. For the bioassays, 15 females from one to three days old were placed in each bottle, starting with the control. For 1 hour, the individuals were observed and the number of those knocked down was confirmed every 5 minutes. After 60 minutes, those mosquitoes that were unable to fly when the bottle was gently moved or those that remained motionless at the bottom of the bottle were considered dead.

For the application of the Alphaban 40 WP formulation, a mixture was prepared at a rate of 6 g/l of water, for a dose of 20-25 mg of a.i/m2 to be distributed at a rate of 30-50 ml/m2. The application was carried out with the Guarany 1-gallon pre-compression sprayer. The equipment had 80-02 flat nozzles that allow a flow rate of the mixture within the range recommended by the WHO [3], of 760 ml/min ± 15 ml. Spraying was carried out in vertical strips 75 cm wide, with an overlap of 5 cm, from top to bottom, keeping the tip of the sprayer 45 cm from the surfaces to be treated, to ensure the correct width of the strip according to the WHO methodology [3]. After treatment, 2 houses from the blocks that were treated indoors and 2 control houses were used to carry out the evaluation of the deposition of insecticides on the walls Various surfaces were selected, such as tile, wood, plastic, metal, masonry where the cones were placed. The first bioassay was carried out 24 hours after the surfaces were impregnated and on a weekly basis after the treatment. Batches of 20 females from 3 to 6 days of emergence were used, from Ae. aegypti strain from Cárdenas, City, established in the insectarium in Mosquito Control Center based in Varadero as an exception, during the evaluation stage, for which all biological security measures were adopted. Four replicates were evaluated for each type of surface. The exposed females remained in contact with the treated surfaces for one hour. After this time, they were extracted using a capture device (glass exhauster) starting with the controls and then the cones on the treated surfaces to avoid contamination. The mosquitoes were transferred to clean glasses, which were covered with double mesh cloth placed on top and tied with an elastic band. The knockdown (KN = Knock Down) corresponding to each of the treated surfaces was recorded, including those used as a control that were not exposed to the insecticide. A previously moistened cotton fragment was placed on the upper external part of each glass. Mortality was read after 24 hours, and the test was repeated every 7 days for 10 weeks, and was discontinued when the knockdown was less than 80%, according to the WHO criteria [4]. For each weekly bioassay, the change of location on each type of surface where the cones would be fixed was taken into account. It was not necessary to apply the Abbott formula to correct the mortality of the bioassays, since the mortality was less than 5% [5]. The temperature did not exceed 25º C and the relative humidity was > 50%

To determine the infestation rates of cockroaches in the selected location before applying the insecticide, small sticky traps (Insect Trap and Monitor) were used to monitor the infestation rate, which were placed according to the infestation detected by visual inspection in the sites of preference of the species at a rate of 1 per 16 m2. , the traps were checked 48 hours after they were set, and the number of specimens captured per unit and location was counted. The aforementioned methodology was followed for the post-treatment surveys. To carry out the bioassays, the manufacturer's recommended dose for high infestation by crawling insects (cockroaches, fleas, ants) of 30 gr/5 liters of water was used. This was applied with a 1-gallon Guarany pre-compression sprayer, conical nozzle, and a pressure of 25-40 pounds/inch 2 in the kitchen of a home, in breeding places of cockroaches such as cracks in the walls, oven, behind cooking cabinet, plateau. After treatment, sampling was carried out weekly using sticky traps.

Susceptibility and/or Resistance study to cypermethrin under laboratory conditions using the impregnated bottle methodology.

When carrying out susceptibility and/or resistance tests using the impregnated bottle methodology, we found that the Varadero Peninsula mosquito population showed resistance to cypermethrin at a dose of 13.5 ug/mL with values of 90% kill after 30 minutes as shown in (Figure 1). Effectiveness of Alphaban 40WP formulations using the biological procedure to evaluated the insecticides in the container walls against Ae. Aegypti.

Figure 1: Susceptibility tests performed on Rockefeller susceptible strain and Varadero, strain collected in 2022 using impregnated bottles with cypermethrin at 13.5 µg/mL.

After the intervention with Aphaban 40 WP, the residual effect on different post-treatment surfaces was evaluated for 10 weeks using Ae.aegypti mosquitoes. Figure 2A shows the high percentage of mortality obtained after that time. The residuality of the tile followed by concrete and wood fluctuated from the first weeks, but remained high (> 80%) until week 6 for tile, concrete and wood until week 8. Regarding mortality, however, on all surfaces it remained above 80% until week 9. The highest mortality rates occurred in plastic and metal during the 10 weeks of the evaluation (Figure 2B). It is important to highlight that most of the water storage containers for human consumption in Cuba are made of these materials.

Figure 2: Percentage of knockdown.
(A) Mortality
(B) Obtained by exposing Aedes aegypti females from Cárdenas population on surfaces impregnated in situ with Alphaban 40 WP for 10 weeks post-treatment. Yellow line highlights WHO criterion for discontinuing study (knockdown < 80 %).

Effectiveness of Alphaban 40WP formulations in Blattella germanica control. Table 1 shows the infestation averages and percentage reductions of B.germanica in post-treatment surveys in the home where the treatment with Alphaban 40 WP was carried out. In the data from the pre-treatment surveys, it can be seen that the infestation rates were very high; after treatment, there was a reduction in the infestation averages and an increase in the percentage reductions compared to the pre-treatment, as the weeks went by.

Throughout history, four common classes of chemical insecticides have been used to control Ae. aegypti: organochlorines, organophosphates, carbamates, and pyrethroids [6]. These are known for their toxicity, persistence in the environment, and bioaccumulation in the food chain. This last property was one of the main reasons why they were replaced by organophosphate pesticides, since they could be more easily degraded in the environment [6]. Organophosphate insecticides do not bioaccumulate in organism tissues or in the environment [7]. Carbamates share the same mode of action with Organophosphates inhibiting acetylcholinesterase activity, although this effect can be more easily reversed and insects can be recovered at low doses [8]. The fourth group comprises the pyrethroids, which entered the market in 1980 [9]. They are considered safe due to their high insecticidal properties at low application rates, short persistence in the environment, no bioaccumulation and low toxicity to mammals, reasons that support their extensive use [10]. This group includes cypermethrin, which is the active ingredient of the Alphaban 40 WP formulation. The cypermethrin molecule has been used in numerous types of formulations either alone or in combination with other molecules.

Susceptibility and/or Resistance study to cypermethrin under laboratory conditions using the impregnated bottle methodology. Interventions based on the application of insecticides can be unsuccessful when the susceptibility and/or resistance status is not taken into account. Taking this aspect into account, monitoring the insecticide that will be applied in the intervention is essential, this will result in its success or failure [11]. Pyrethroids began to be applied in Cuba in 1986 and especially cypermethrin in recent years. The cypermethrin molecule is the active ingredient of numerous formulations registered in Cuba and as such it has been used regularly in our country, as a spatial treatment [12] so there is a sustained application of this insecticide within control policies in our National Vector Control Program.

In populations collected in the field, [13] evaluated the status of resistance to insecticides and its mechanisms in Ae. aegypti adult mosquitoes, from a strain from Boyeros municipality, Havana, which was susceptible to cypermethrin. In the same year, Rodríguez MMJ, et al. [14] evaluated different insecticides in Ae.aegypti adult mosquitoes from Pinar del Río municipality, resulting in susceptibility to cypermethrin (100% mortality). There are several studies that suggest great variability in terms of susceptibility state to this pyrethroid in Ae. aegypti populations for example in Cuba we have diverse results. Montada DD, et al. [15], in studies carried out with Ae.aegypti adults from a strain from Playa municipality, Havana, did not find resistance (FR < 5) to cypermethrin insecticide. The same author [16] in a similar study carried out with adult mosquitoes from Ae. aegypti population, from Santiago de Cuba, presented moderate resistance (FR = 7.2), while in 2009 in Ae. aegypti populations from three Havana municipalities, a study was carried out on adults whose results showed that they were resistant to cypermethrin insecticide. These results show us the variability of responses of this mosquito to a certain insecticide such as cypermethrin, a response that depends on various factors such as place of origin and collection of the strain under study and frequency of treatment in the locality [17]. Taking this background into account, it is not illogical to find that the population study was resistant. It is important to clarify that the mortality percentage obtained at other times would be considered close surveillance. The application for control adult mosquitoes with formulations containing cypermethrin in that location is not carried out as a routine measure by the Control Program, so the mosquito population has not been subjected to selection pressure.

Effectiveness of Alphaban 40WP formulations using the biological procedure to evaluated the insecticides in the container walls against Ae. Aegypti. The background in Cuba shows treatments with adulticides were used routinely in Cuba from 1981 to 1986. First malathion as Ultra-Low Volume (ULV) spraying [18]. Then since 1986, the pyrethroid lambdacyhalothrin [19] and later cypermethrin until the present. In recent years, the control strategy is based on the application of different adulticidal formulations where cypermethrin constitutes the active ingredient [19-21] in addition to the application of residual products with other groups of insecticides [22-25]. Aedes aegypti adults normally rest indoors (endophilia), where they frequently and almost exclusively feed on human blood (anthropophilia) [26,27].This explains why space fumigation outside the dwellings have little efficacy against this vector [28,29]. Indoor insecticide applications may have a more direct impact on resting adult mosquitoes [30]. This is particularly used during epidemics because it quickly kills adults that are presumably flying. It often happens that up to three applications are required to achieve maximum efficacy even if the durability is short [31].

Indoor application of residual insecticides can provide longer-term protection after a single application; however, the time spent can increase, since it is necessary to remove all the furniture and belongings of the dwellers from the house [32]. This reason makes the work more intense and less accepted by the community [33], which hinders its generalization for vector control specifically on Ae. aegypti. The effectiveness of a residual action intervention depends on several factors: a) the susceptibility of the vector mosquitoes to the sprayed insecticides, b) the type of formulation, the quality of the spray, the spray coverage, c) the nature of the surface and d) the cooperation of the inhabitants in not removing the spray from the surfaces through washing or painting [34-38]. Regarding the formulation; Alphaban 40 WP, with few practical results published in the literature, is a potentiated suspension. Usually, this type of intervention is evaluated through cone tests to determine the residuality in the different types of surface and thus the efficacy [3], although other methodologies could be followed [31]. According to some authors, these bioassays should ideally be carried out on impregnated surfaces in real human rooms, since bioassays under controlled conditions can show a longer residual effect [35]. In our study, using the cone methodology on treated surfaces in a house, a high percentage of mortality was obtained in mosquitoes exposed to metal and plastic surfaces for 10 weeks, followed by concrete, where mortality was above 90% from week 4 to 10. Dzib Florez, 2019 [34] in tests carried out with Ae.aegypti, under controlled conditions with domestic sprays containing pyrethroids, durability lasted between 2 and 3 weeks. The wood and concrete surfaces evaluated with these sprays provided the longest residual time, results that partially coincide with ours.

In most studies, the mortality rates in the exposed groups are established as satisfactory when this indicator are greater than or equal to 80%, according to the criteria of Rodríguez MMJ, et al. [14]. In the case of the study carried out by Ibrahim KT, et al. [38], the knockdown and mortality rates at 24 hours were similar in the time intervals evaluated. In bioassays carried out by Corrêa APSA, et al. [39], using deltamethrin on two types of surfaces, the mortality rates of Anopheles (Nyssorhynchus) marajoara remained above 80% for the 8 months of the experiment on painted wood and cement surfaces, mortality was higher for wood than for cement. In our study, the knockdown and mortality values at 24 hours remained similar until week 4 [40]. From week 6, mortality continued to be higher than 80% on most surfaces, except for metal and plastic, which remained on this indicator until the trial was discontinued at week 10 [41]. These results differ from studies by Ab Hamid NN, et al. [36], in the cement walls those that showed high mortality up to week 16.

The factors that may have influenced in our study, the low durability on some surfaces, may be from the smooth and poorly adherent texture of the tile to the porosity of the concrete. Some authors suggest that the rough and porous surface of the concrete walls are highly alkaline, tend to absorb the insecticide, degrade the active principle more quickly and reduce the residual effect [36,37], to which is incorporated the effect of temperatures and rainfall on the exterior walls of the houses. Effectiveness of Alphaban 40WP formulations in Blattella germanica control. Despite the fact that various intensive control activities against the German cockroach, both by government entities and by the affected population are being implemented, Blattella germanica is an important pest in public and medical health, and continues to infest buildings such as houses and restaurants at high and low population densities.

Many conventional insecticides are not effective in controlling massive infestations of Blattella germanica, due to its short life cycle of three to six months depending on the temperature. Therefore, it is very difficult to control this species in comparison with other species of cockroaches. On the other hand, according to literature Blattella germanica shows resistance to insecticides such as malathion and propoxur. In addition, this species shows a slight tolerance to sumithion and tetramethorin has found that field varieties of Blattella germanica show high to moderate resistance to cypermethrin. Our results show greater efficacy after 8 days of treatment. Previous studies have revealed that Blattella germanica massively infests kitchens in hospitals, apartments, hotels, private homes, and restaurants for breeding and feeding purposes, being able to visit other places to find its food. These results were similar to ours, since higher densities of Blattella germanica were found in the kitchen.

Different methods have been used for the census of cockroaches such as visual counting and sticky traps. The visual count is carried out after 22:30, so the lights go out at 21:30 p.m. for one hour. Then the light is turned on and the cockroaches that are moving on the tables and feeding areas are observed and counted for 5 min. This method allows dividing cockroaches into three categories of infestation: high (>75), medium (25-75) and low (<25) density of cockroaches. On the other hand, we propose to census cockroaches, by placing sticky traps in sufficient quantities close to their shelters to estimate the density and type of cockroach present.

Finally, we must say that in Cuba for more than 30 years various formulations with cypermethrin as an active ingredient have been used, which is why in some localities depending on the intensity of their use moderate resistance has been detected, leading to treatments with these formulations in these localities not being effective, which in turn means unnecessary environmental contamination. Therefore, these results show us the need to carry out a monitoring program on the susceptibility to this molecule in species as important as those studied, in order to carry out effective treatments and avoid environmental contamination and ecological damage unnecessarily. According to the results obtained, we can conclude that the use of the Alphaban 40 WP (cypermethrin) formulation can be used to control the Ae. aegypti mosquito and suppress the populations of Blattella germanica.

To Pelgar International for its Technical assistance and its logistical support.

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