Chemical control of Aedes aegypti continues to be an indispensable alternative to preventing dengue, Zika, and chikungunya outbreaks. The Havana Zoological Garden requires constant vigilance because its special characteristics help in the spread of the causal agents of diseases transmitted by mosquitoes, which put the health of visitors at risk. The goals of this study were to determine the level of susceptibility and insecticide resistance mechanisms in the Ae. aegypti population. Temephos susceptibility in larvae was evaluated with bioassays using the World Health Organization's methodology, and susceptibility of adult mosquitoes was determined by the impregnated bottle bioassay, recommended by the Centers for Disease Control and Prevention. Resistance mechanisms were determined with biochemical assays. Mosquito larvae from the Havana Zoo were found resistant to temephos, which was associated with the activity of the enzymes α- and β-esterases and mixed function oxidases but not glutathione-S-transferase. Adult mosquitoes were susceptible to pyrethroid (lambda-cyhalothrin, deltamethrin, and cypermethrin), organophosphate (chlorpyrifos), and carbamate (bendiocarb). Temephos resistance detected in the mosquito population from the Havana Zoo is an alert for the Vector Control Program, which must take measures to manage their resistance, relying on the surveillance carried out by Cuba's medical entomology laboratories.
Aedes aegypti (L.) is a highly anthropophilic species of mosquito with a great capacity for the transmission of viruses such as dengue (DENV), Zika (ZIKV), yellow fever (YF), and chikungunya (CHIKV) (Ritchie 2014). Other factors that contribute to its spread are the geographic distribution of this vector, increased human migrations, climate change, uncontrolled and unplanned urbanization, increased air travel, accumulation of nonbiodegradable waste, and lack of efficient of public health services and their financing (Gubler et al. 2001, Kraemer et al. 2015).
Currently, there are no effective vaccines or treatments for DENV, ZIKV, and CHIKV, so vector control is the only effective measure to reduce the transmission of these diseases. The use of chemical insecticides is the main control strategy in many parts of the world, but their indiscriminate use has resulted in the inevitable emergence of Ae. aegypti–resistant populations, which make their use and effectiveness difficult (Agramonte and Bernier 2017, Parker et al. 2020). There is evidence of resistance to insecticides in many regions such as Latin America (Rodríguez et al. 2007, Kandel et al. 2019), the Caribbean (Goindin et al. 2017), Southeast Asia (Amelia-Yap et al. 2018), and Africa (Kamgang et al. 2017).
In Cuba, the first dengue epidemic was reported in 1977–1978 caused by DENV-1 (Guzman et al. 2019). Subsequently, the country's first hemorrhagic dengue epidemic was reported in 1981, caused by DENV-2. It was stopped after the National Eradication campaign of Ae. aegypti was created by the Cuban government, which kept the country free of dengue transmission for 14 years because of the reduction of Ae. aegypti populations through the destruction of their breeding sites as well as the application of organophosphate insecticides to control larvae and adults (WHO 2012). However, the eastern part of the country suffered another epidemic of dengue hemorrhagic fever caused by the same serotype in 1997 that lasted for 6 months (WHO 2012). In 2000, a limited outbreak of DENV-4 was reported in Havana, resulting in 138 confirmed cases (Guzman and Kouri 2002). In June 2001, the nationwide dengue case surveillance system identified DENV-3 circulating in Havana that eventually expanded to involve 12,889 serologically confirmed cases, including 78 cases of dengue hemorrhagic fever/dengue shock syndrome and three fatalities (Guzman et al. 2012). Unfortunately, Cuba has been experiencing annual dengue outbreaks since 2006 (Guzman et al. 2019).
Aedes aegypti is the main vector of dengue in Cuba. Since the beginning of the Ae. aegypti eradication campaign in Cuba, in 1981, organophosphate, temephos, and pyrethroids have been the most common insecticides used for Ae. aegypti control (MINSAP 2012). Their intensive use has favored the development of high resistance levels in an Ae. aegypti population in Havana (Bisset et al. 2011; Rodríguez et al. 1999, 2001, 2005, 2020).
The Zoological Garden of Havana (23°06′38″N 82°23′49″W), located in the country's capital, is the main zoo in Cuba. The attractive exhibition of representative species of wild fauna and flora attracts many local and foreign visitors (Quibert et al. 2016). The Havana Zoo is a small ecosystem that favors the reproduction of Ae. aegypti. The use of adulticides is prohibited due to their high toxicity to wildlife, which limits Ae. aegypti control. Environmental sanitation and the application of temephos are the most used measures to combat the vector in this area. The proliferation of Ae. aegypti constitutes a potential risk of arbovirus transmission for visitors to the zoo. This influx of people favors the spread of arboviruses in their communities, where outbreaks or epidemics could occur. The most serious cases of dengue in Havana City are admitted to the Joaquin Albarran Hospital near the Zoological Garden of Havana (unpublished data). Therefore, the zoo is a site of high epidemiological risk, where an effective integrated mosquito control is imperative. The insecticide resistance surveillance is crucial because of the intensive use of temephos to control Ae. aegypti larvae in the zoo.
MATERIALS AND METHODS
Laboratory strains of Ae. aegypti
A strain of Caribbean origin susceptible to insecticides, free of any mechanism of detectable resistance, was supplied by the Centers for Disease Control and Prevention (CDC) of San Juan, Puerto Rico.
A temephos-resistant strain was colonized in 1997 in Santiago de Cuba. The larvae of this strain have been subjected to selection pressure for more than 6 generations (Rodríguez et al. 2002) and kept in the laboratory, selecting resistant larvae with the concentration that causes 90% mortality with temephos.
Larvae of Ae. aegypti, late third or early fourth instars, were collected from 3 tree holes, located near the primate exhibition areas of the Havana Zoo, which are highly frequented by visitors. Collections were made from January to February 2018. Individual collections were combined to form a genetic representative field colony of the site, and F2 progeny specimens were used for laboratory tests. The larvae were identified to species at the Vector Control Department in the Tropical Medicine Institute Pedro Kouri (IPK).
Temephos resistance was evaluated with the World Health Organization (WHO) bioassays (WHO 1981). Five concentrations of the insecticide were applied with 5 replicates each and 1 control per concentration, 25 early fourth instars of uniform size were placed in plastic cups, and all replicates contained 99 ml of tap water and 1 ml of the prepared insecticide solution. Controls were treated using 1 ml of acetone as diluent. Mortality was determined 24 h after application of the insecticide. The results were analyzed using the probit test implemented in SPSS version 11.5. The resistance ratio (RR50) was calculated by comparing the LC50 value of the field population with the Rockefeller strain (Mazzarri and Georghiou 1995).
Biochemical assays can detect the appearance of resistant mosquitoes, providing information about the metabolic resistance mechanisms in a population, as well as the cross-resistance pattern to insecticides used in the area. Insecticide resistance is metabolic when the following enzyme groups are increased: nonspecific esterases (NSEs), mixed function oxidases (MFO), glutathione-S-transferases (GST). Thirty late third or early fourth instars were used per treatment. The individual larvae were placed in microtiter plates and were macerated in 50 μl of 0.02 mol/liter Na2PO4 buffer, pH 7.5 (phosphate buffer), using a plate homogenizer, on an ice pack. The sample was brought up to a volume of 300 μl with Na2PO4 buffer. Analysis of α- and β-esterase (Rodríguez et al. 2001), MFO (Brogdon et al. 1997), and GST (Polson et al. 2011) activities was conducted on a VersaMax™ plate reader (Molecular Devices Corporation, Sunnyvale, CA).
The enzyme activity values of the Havana Zoo populations were compared with the reference strains, using the one-way ANOVA. The differences were considered significant for P < 0.05. Tukey's a posteriori test was applied to determine significant differences between individual means. All statistical tests were performed, using the Statistica 6.0 program (Statsoft, Tulsa, OK).
Polyacrylamide gel electrophoresis for nonspecific esterases (NSEs) characterization
Polyacrylamide gel electrophoresis (PAGE) at pH 8.0 was performed to identify NSEs involved in insecticide resistance to Ops and carbamates (Field et al. 1984). Thirty late third or early fourth instars of Ae. aegypti were homogenized in phosphate buffer (0.01 M, pH 7.5), and xylene cyanol (100 μl of 0.01% in 15% sucrose) was added. The run was carried out at 200 volts for 45 min. The gel was then immersed in 50 ml of phosphate buffer containing 4 ml of each substrate (a-naphtyl acetate and b-naphtyl acetate), and 0.5 g of fast-blue RR dye was added. Gel was immersed in acetic acid (10%) to fix bands.
Bioassays were conducted following the methods recommended by the United States Centers for Disease Control and Prevention (CDC) (CDC 2010). The bioassays allow for the detection and characterization of insecticide resistance in a mosquito population. Information derived from the CDC bottle bioassay may provide initial evidence that an insecticide is losing its effectiveness. Each population was evaluated against the diagnostic dosage, determined previously for the Rockefeller strain for pyrethroids and carbamates insecticides (Rodríguez et al. 2017). A total of 20–25 non-blood-fed females from Havana Zoo colony were introduced into 250 ml glass bottles coated with the diagnostic dose of pyrethroids: lambda-cyhalothrin (6.5 μg/bottle), deltamethrin (6.5 μg/bottle), cypermethrin (13.5 μg/bottle); OP: clorpyriphos (90 μg/bottle); and carbamate: bendiocarb (12.5 μg/bottle). Each test consisted of 4 treatment bottles and 1 control coated with 1 ml of acetone. At least 3 replicates were run for each insecticide/mosquito population combination. All working insecticide solutions were prepared at the time of use from existing stock solutions. This bioassay was run for 120 min, and the number of dead mosquitoes in each bottle was recorded every 15 min. The mortality criteria included mosquitoes with difficulty flying or standing on the bottle's surface. The populations were classified as resistant or susceptible using the updated WHO guidelines (WHO 2016). The temephos-resistant strain (SAN-F6) was not used in the CDC bottle bioassay.
As shown in Table 1, both the resistant SAN-F6 laboratory strain and Ae. aegypti field colony of Havana Zoo showed high resistance to temephos (RR50 > 10x). Furthermore, the LC50 and LC90 values of the Havana Zoo colony differed significantly (P < 0.05) from the Rockefeller and SAN-F6 strains, according to the confidence intervals that did not overlap.
Statistical comparison of enzymatic activity between the Havana Zoo colony with respect to the Rockefeller and SAN-F6 reference strains showed an increased enzymatic activity of α- (Fig. 1a) and β-esterases (Fig. 1b) and MFO (Fig. 1c) associated with temephos resistance. Enzymatic activity of α- and β-esterases and MFO from the Havana Zoo colony differed significantly (P < 0.05) from the Rockefeller strain, but not from the SAN-F6 strain. However, the activity of the GST enzyme from the Havana Zoo colony did not differ significantly (P < 0.05) from the Rockefeller strain, but it did from SAN-F6 (P > 0.05) (Fig. 1d). This shows how high temephos resistance expressed by the Havana Zoo colony derived from a metabolic resistance mechanism involving 3 enzymatic groups in the insecticide detoxification process. Statistical comparison of enzyme activity was performed, using the one-way analysis of variance (ANOVA) for α- [F (2.87) = 92,046] and β-esterases [F (2.87) = 37,771], MFO [F (2.87) = 7.0078], and GST [F (2.87) = 142.813].
Polyacrylamide gel electrophoresis
A zymogram was performed with the Havana Zoo population and laboratory strains (Rockefeller and SAN-F6) to observe esterases involved in temephos resistance (Fig. 2). In the polyacrylamide gel, a strong band of Est-4A was expressed by the Havana Zoo colony and the SAN-F6 strain, while in the Rockefeller strain it was absent (Fig. 2). Twenty-four out of 30 larvae evaluated from the Havana Zoo colony showed EST-4A with a frequency of 80%. This shows EST-4A is the NSE associated with the metabolic resistance of Ae. aegypti to temepfos.
Adult bioassays showed susceptibility to all insecticides evaluated in the Havana Zoo population with a 100% mortality obtained at 30 min of exposure (Fig. 3a, 3b). This shows the absence of cross-resistance between temephos and adulticides evaluated against the Havana Zoo colony.
Temephos has been the most widely used larvicide for mosquito control in Cuba because of its low toxicity, low cost, and high effectiveness. A previous study in Havana uncovered a local population with high resistance to this insecticide induced by its repeated use (Bisset et al. 2011). However, this resistance can be reversed if the exposure of the larvae to the insecticide is stopped (Bisset et al. 2019). Other investigations carried out in the Boyeros municipality of Havana have shown an increase in the last 3 years (Rodríguez et al. 2020) in the levels of resistance to temephos as a consequence of its intensive applications against the high vector population (Bisset et al. 2011). It also has been observed that resistance levels decreased when temephos application was discontinued and replaced with Bacillus turingiensis israelensis de Barjac (Bti) (Rodríguez et al. 2012). Worldwide, this species of mosquito has also been subjected to selective pressure for resistance due to the mismanagement of temephos, causing variations in the susceptibility of mosquito populations, as reported in Colombia (Grisales et al. 2013), Guadalupe Islands, San Martín (Goindin et al. 2017), and Brazil (Valle et al. 2019).
The prolonged use of the insecticide temephos in Havana as well as in other regions already mentioned has been the reason of the high resistance levels of Ae. aegypti populations in these areas. In this study, we found a significant correlation (P < 0.05) of metabolic resistance mechanism to temephos with MFO and α- and β-esterases. These showed an increased enzymatic activity in field-resistant colony when compared to the susceptible Rockefeller strain but did not differ from the resistant SAN-F6 strain. These findings are consistent with the results of other authors that confirm a strong association between resistance to temephos and NSEs (Paiva et al. 2016, Bisset et al. 2019, Rodríguez et al. 2020). Cross-resistance was not shown to the OPs (temephos and chlorpyrifos) and the carbamate (bendiocarb). A high resistance to temephos correlated with MFO was reported in 2 Ae. aegypti strains from Mexico and Peru (Saavedra-Rodriguez et al. 2014). Our study from Havana was not consistent, and the MFO was not associated with temephos resistance in Ae. aegypti populations (Rodríguez et al. 2020). Despite MFO, there are metabolic enzymes involved in temephos resistance, and they usually confer resistance to pyrethroids (Brogdon 1989). However, the GST activity was not associated with increased temephos resistance. Several research studies are not consistent with our result because they have revealed a significant correlation between temephos resistance and GST metabolic enzymes (Bisset et al. 2019, Rodríguez et al. 2020). The GST enzymatic group is also involved in metabolic resistance to dichloro-diphenyl-trichloroethane (DDT) in some Anopheles spp. (Djegbe et al. 2014, Tchigossou et al. 2018) and Ae. aegypti (Fonseca et al. 2011). Electrophoresis shows the presence of the EST-4A esterase band in the Havana Zoo colony and the SAN-F6 strain, but it was not expressed in the Rockefeller strain.
The relationship between esterase activity and temephos resistance in Ae. aegypti was discovered for the first time in 1997 in Cuba (Rodríguez et al. 1999). This enzyme was named EST-4A because of its specificity to react with the substrate α-naphthyl acetate, as well as its mobility in the polyacrylamide gel (Rodríguez et al. 1999). In India, high activity of esterases and MFO enzymes were shown to be associated with temephos resistance (Bharati 2018). Other studies carried out in 4 regions of Brazil indicated that esterase and GST enzymes were related to temephos resistance (Valle et al. 2019). There are also studies that have demonstrated the susceptibility of Ae. aegypti to temephos in the Central Pacific Region of Costa Rica (Calderón-Arguedas et al. 2018).
The fact that the Havana Zoo population of Ae. aegypti was susceptible to all the adulticides evaluated supports the absence of adulticide treatments in this location. Pyrethroids are the main insecticides used to control adult Aedes mosquitoes in Cuba. Besides biochemical activity, one of the main mechanisms of pyrethroid resistance in insects is the target-site insensitivity (Moyes et al. 2017). Nonsynonymous mutations on the knockdown resistance (kdr) region of the voltage-gated sodium channel (VGSC) gene are responsible for the pyrethroid insensitivity (Du et al. 2016). Pyrethroid resistance has become a worldwide concern affecting Ae. aegypti control programs in the USA (Parker et al. 2020), Puerto Rico (Agramonte and Bernier 2017), Mexico (Saavedra-Rodriguez et al. 2019), West Africa (Badolo et al. 2019), Selangor, Malaysia (Leong et al. 2019), India (Saha et al. 2019), Indonesia (Amelia-Yap et al. 2019), Taiwan (Chung et al. 2019), Brazil (Rodrigues de Sá et al. 2019), and Thailand (Mano et al. 2019).
Studies demonstrate that temephos susceptibility can be recovered because its metabolic resistance mechanisms are reversed when its use is stopped (Melo-Santos et al. 2010, Bisset et al. 2019). Biological control of mosquitoes is the best and the only alternative to replace temephos because of its use as an adulticide being prohibited in the zoo environment. A successful strategy carried out in the Boyeros municipality of Havana City showed how resistance levels were decreased when temephos application was discontinued and replaced with Bti (Rodríguez et al. 2012). This rotation policy could be implemented by Ae. aegypti control programs to avoid the evolution of resistance or removal of selection pressure for a sufficient time to allow mosquito populations to regain susceptibility. The National Aedes aegypti Control Program should promote the use of integrated mosquito management strategies to contribute with an effective mosquito control aimed to reduce the development of insecticide resistance in mosquito populations. Susceptibility tests in target mosquito populations should be conducted to identify the most suitable insecticides to be used in each locality and implement an efficient rotation of control strategies to minimize the development of resistance.
We thank Pablo Cárdenas and Maritza Pupo Antunez for reviewing this article.
Vector Control Department, Institute of Tropical Medicine “Pedro Kourí,” Autopista Novia del Mediodía, KM 6½, La Lisa, La Habana, 11400, Cuba.
Florida Medical Entomology Laboratory, University of Florida. 200 9th Street SE, Vero Beach, FL 32962.