First report of natural Wolbachia infections in mosquitoes from Cuba.

Mosquitoes are extensively responsible for the transmission of pathogens. Novel strategies using Wolbachia could transform that scenario, since these bacteria manipulate mosquito reproduction, and can confer a pathogen transmission-blocking phenotype in culicids. Here, we screened the Wolbachia surface protein region by PCR in eight Cuban mosquito species. We confirmed the natural infections by sequencing and assessed the phylogenetic relationships among the Wolbachia strains detected. We identified four Wolbachia hosts: Aedes albopictus, Culex quinquefasciatus, Mansonia titillans, and Aedes mediovittatus (first report worldwide). Knowledge of Wolbachia strains and their natural hosts is essential for future operationalization of this vector control strategy in Cuba.

Aedes albopictus (Skuse) dispersion in Havana City, Cuba, 1995-2018.

Various arboviruses are transmitted to humans by mosquitoes, particularly Aedes aegypti and Aedes albopictus, two invasive and frequently sympatric species. The objective of this study was to evaluate the dispersion and the behavior of Ae. albopictus in relation to houses and its association with other mosquitoes in the province of Havana, Cuba. All water-containing deposits in the houses or vacant lots in urban and peri­urban municipalities of the province of Havana were sampled during the two study periods: 1995-1999 and 2010-2018. The following patterns in the presence of Ae. albopictus in the study area were observed: a persistent absence of Ae. albopictus in one of the municipalities; a rapid dispersion in the second period, staring from the absence of vector in the first period, in tow other municipalities; and a sustained decrease in the dispersion of Ae.albopictus in two other municipalities. The peripheral municipalities noted the highest presence of Ae. albopictus, but few associations with other mosquitoes. However, overall, we found an increase in this association when comparing the period 2010-2018 with the first period. Ae. albopictus was present in 8% (2016) to 21.5% (2013) inside the houses with an average of 15%, which evidences an initial domiciliation of the species. The results obtained in this work show an initiation of domiciliation of Ae. albopictus in the urban area of the province of Havana. This is important to alert the National Control Program to strengthen the entomological monitoring of Ae. albopictus, and not only Ae. aegypti. The follow-up of this domiciliation is important to guide control efforts, knowing its role as a vector of different arboviruses.

Vector competence of Aedes aegypti from Havana, Cuba, for dengue virus type 1, chikungunya, and Zika viruses.

BACKGROUND: Like many countries from the Americas, Cuba is threatened by Aedes aegypti-associated arboviruses such as dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. Curiously, when CHIKV was actively circulating in the region in 2013-2014, no autochthonous transmission of this virus was detected in Havana, Cuba, despite the importation of chikungunya cases into this city. To investigate if the transmission ability of local mosquito populations could explain this epidemiological scenario, we evaluated for the first time the vector competence of two Ae. aegypti populations (Pasteur and Párraga) collected from Havana for dengue virus type 1 (DENV-1), CHIKV, and ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Mosquito populations were fed separately using blood containing ZIKV, DENV-1, or CHIKV. Infection, dissemination, and transmission rates, were estimated at 3 (exclusively for CHIKV), 7, and 14 days post exposure (dpe) for each Ae. aegypti population-virus combination. Both mosquito populations were susceptible to DENV-1 and ZIKV, with viral infection and dissemination rates ranging from 24-97% and 6-67% respectively. In addition, CHIKV disseminated in both populations and was subsequently transmitted. Transmission rates were low (<30%) regardless of the mosquito population/virus combination and no ZIKV was detected in saliva of females from the Pasteur population at any dpe. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated the ability of Ae. aegypti from Cuba to transmit DENV, ZIKV, and CHIKV. These results, along with the widespread distribution and high abundance of this species in the urban settings throughout the island, highlight the importance of Ae. aegypti control and arbovirus surveillance to prevent future outbreaks.

Reversal of Resistance to the Larvicide Temephos in an Aedes aegypti (Diptera: Culicidae) Laboratory Strain From Cuba.

The objective of this investigation was to know whether the organophosphate temephos resistance developed in larvae from a laboratory strain of Aedes aegypti (Linnaeus, 1762) from Cuba could be reversed. The resistant laboratory strain of Ae. aegypti, named SAN-F6, was left without temephos selection pressure for 12 generations. The level of temephos resistance was determined using WHO bioassays and mechanisms of metabolic resistance were determined based on enzyme activity levels detected by biochemical assays. Bioassays and biochemical assays were conducted on the SAN-F6 parental strain and every three reversal generations (SANRevF3, SANRevF6, SANRevF9, and SANRevF12) without temephos selection pressure. After 19 yr of keeping the SAN-F6 strain under selection pressure with the LC90 of temephos, the resistance ratio (RR50) was 47.5×. Biochemical assays indicated that esterase and glutathione S-transferase are still responsible for temephos resistance in this strain, but not mixed-function oxidase. Experiments on resistance reversal showed that temephos susceptibility could be recovered as α esterase activity levels decreased. The SAN-F6 strain has provided an essential basis for studies of temephos resistance in Cuba. It was demonstrated that the resistance developed to the larvicide temephos in Ae. aegypti from this Cuban lab strain is a reversible phenomenon, which suggests that similar outcomes might be expected in field populations. As such, the use of temephos alternated with other larvicides recommended by WHO such as Bti or pyriproxyfen is recommended to maintain the effectiveness of temephos and to achieve more effective control of Ae. aegypti.

Vector-borne transmission and evolution of Zika virus.

Zika virus (ZIKV), discovered in the Zika Forest of Uganda in 1947, is a mosquito-borne flavivirus related to yellow fever, dengue and West Nile viruses. From its discovery until 2007, only sporadic ZIKV cases were reported, with mild clinical manifestations in patients. Therefore, little attention was given to this virus before epidemics in the South Pacific and the Americas that began in 2013. Despite a growing number of ZIKV studies in the past three years, many aspects of the virus remain poorly characterized, particularly the spectrum of species involved in its transmission cycles. Here, we review the mosquito and vertebrate host species potentially involved in ZIKV vector-borne transmission worldwide. We also provide an evidence-supported analysis regarding the possibility of ZIKV spillback from an urban cycle to a zoonotic cycle outside Africa, and we review hypotheses regarding recent emergence and evolution of ZIKV. Finally, we identify critical remaining gaps in the current knowledge of ZIKV vector-borne transmission.

Spatio-temporal distribution of vertically transmitted dengue viruses by Aedes aegypti (Diptera: Culicidae) from Arroyo Naranjo, Havana, Cuba.

OBJECTIVE: To study the distribution of vertical transmission of dengue viruses in field-collected Aedes aegypti larvae in the municipality of Arroyo Naranjo in Havana, Cuba. METHODS: Aedes aegypti larvae and pupae were collected monthly between September 2013 and July 2014 in the seven Municipal Health Areas of Arroyo Naranjo. Pools formed of 30-55 larvae were examined through PCR and sequencing to detect the presence of each serotype. RESULTS: We analysed 111 pools of larvae and pupae (4102 individuals) of which 37 tested positive for at least one DENV. More than one DENV type was observed in 10 of the 37 positive pools. Infected pools were detected every month, except in January, suggesting a sustained circulation of DENV in the vector populations. DENV-1 and DENV-3 were the most frequent and dispersed, though all four DENV types were detected. Nucleotide sequencing from positive pools confirmed RT-PCR results for DENV-1 (genotype V), DENV-3 (genotype III) and DENV-4 (genotype II). DENV-2 was detected by RT-PCR but could not be confirmed by nucleotide sequencing. CONCLUSION: Our study of the distribution of natural vertical transmission of dengue virus types highlights extrinsic virus activity patterns in the area and could be used as a new surveillance tool.

First record of natural vertical transmission of dengue virus in Aedes aegypti from Cuba.

While horizontal transmission (human-mosquito-human) of dengue viruses largely determines the epidemiology of the disease, vertical transmission (infected female mosquito- infected offspring) has been suggested as a mechanism that ensures maintenance of the virus during adverse conditions for horizontal transmission to occur. The purpose of this study was to analyze the natural infection of larval stages of Aedes aegypti (Diptera: Culicidae) with the dengue virus (DENV) in Cuba. Here, we report vertical transmission of DENV-3 genotype III in natural populations of Ae. aegypti through RT-PCR detection and serotyping plus sequencing. Our report constitutes the first record of vertical transmission of DENV in Ae. aegypti from Cuba with details of its serotype and genotype.

Inheritance of Resistance to Deltamethrin in Aedes aegypti (Diptera: Culicidae) From Cuba.

The development of pyrethroid resistance in Aedes aegypti (L) (Diptera: Culicidae) is a serious concern because major A. aegypti control programs are predominantly based on pyrethroid use during epidemic disease outbreaks. Research about the genetic basis for pyrethroid resistance and how it is transmitted among mosquito populations is needed. The objective of this study was to determine how deltamethrin resistance is inherited in the Cuban A. aegypti-resistant reference strain. Here, a field population of A. aegypti from Santiago de Cuba (SAN-F14), subjected to 14 generations of selection for high deltamethrin resistance level (91.25×), was used to prepare reciprocal F1 and backcross progeny with the insecticide-susceptible Rockefeller strain. Bioassays with larvae were performed according to World Health Organization guidelines. The activities of metabolic enzymes were assayed through synergist and biochemical tests. The null hypothesis of the parallelism test between the two probit regression lines of the reciprocal F1 (susceptible females × resistant males and vice versa) was not rejected at the 5% significance level (P = 0.42), indicating autosomal inheritance. The LC50 response of both F1 progenies to deltamethrin was elevated but less than the highly resistant SAN-F14 strain. DLC values for the F1 progenies were 0.91 and 0.87, respectively, suggesting that deltamethrin resistance in the SAN-F14 strain is inherited as an autosomal incompletely dominant trait, involving at least two factors, which implies a faster development of deltamethrin resistance in larvae and lost product effectiveness. Metabolic enzymes including esterases and cytochrome P-450 monooxygenases but not glutathione-S-transferases were involved in deltamethrin resistance in larvae.

Caracterización parcial de la actividad de esterasas en una cepa de Aedes aegypti resistente a temefos / Partial characterization of esterase activity in a temephos-resistant Aedes aegypti strain

Introducción: las enzimas esterasas han sido identificadas como mecanismo de resistencia a temefos en Aedes aegypti de Cuba, larvicida más utilizado en el mundo. Objetivo: caracterizar parcialmente la actividad de esterasas en larvas expuestas y no expuestas a dosis subletales de temefos en una cepa de Aedes aegypti resistente a este insecticida. Métodos: se utilizó una cepa de Aedes aegypti de referencia susceptible (Rockefeller) y otra resistente a temefos (SANtemF11). Se expusieron las larvas de la cepa SANtemF11 a la concentración letal 90 (CL90) de temefos (1 ppm), 10 % de larvas sobrevivientes a las 24 h (SANtem [24 h]) se transfirieron a agua limpia y sin exposición a insecticidas por otras 24 h (SANtem [48 h]). Se caracterizó de modo parcial, en estas larvas, la actividad de esterasas a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. Se estimó por duodecil sulfato de sodio (SDS-PAGE) el peso molecular de la esterasa (Est. A4). Resultados: la actividad de esterasas en la cepa SANtemF11 resultó significativamente mayor que en Rockefeller. Se observó una disminución significativa de la actividad de esterasas en las larvas sobrevivientes (SANtemF11 [24 h]), la cual se recuperó 24 h después sin exposición a temefos. En el zimograma se observó que en 10 % de las larvas sobrevivientes a temefos, solo apareció incrementada la banda de esterasa A4, en comparación con las observadas en SANtemF11. El peso molecular estimado de la esterasa A4 fue de 58 kDa. Conclusiones: la presencia de una banda específica de esterasa (58 kDa), en las larvas sobrevivientes a la selección con temefos, confirma su papel en la resistencia a este insecticida. Diagnosticar la función de las esterasas en la resistencia a temefos, a través de ensayos bioquímicos, no debe realizarse en larvas expuestas a dosis subletales de este insecticida, para evitar falsos negativos.

Introduction: the esterase enzymes have been defined as the mechanism of resistance to temephos in Aeges aegypti in Cuba, which is the most used larvacide worldwide. Objective: to partially characterize the activity of esterases in exposed and non-exposed larvae at sublethal doses of temephos in an Aedes aegypti strain that is resistant to this product. Methods: a susceptible reference Aedes aegypti strain (Rockefeller) and another temephos-resistant strain (SANtemFII) were used. The larvae from SANtemF11 strain were exposed to lethal concentration 90 (LC90) of temephos (1 ppm); 10 % of the surviving larvae after 24 hours (SANtem[24 h] was moved to clean water, with no exposure to insecticide for 24 hours (SANtem [48 h]). The activity of esterases was partially characterized in these larvae through biochemical assays and gel-polyacrylamide electrophoresis. The molecular weight of esterase A 4 (ESt. A4) was estimated with the support of sodium duodecyl sulophate (SDS-PAGE). Results: the activity of esterases in SANtemF11 was significantly higher than in Rockefeller strain. Significant reduction of the activity of esterases in surviving larvae was observed (SANtemF11 [24 h], but it increased 24 h later without exposure to temephos. The zymogram showed that 10% of larvae that survived from temephos action, just the esterase A4 band increased if compared with those of SAntemF11. The estimated molecular weight of esterase A4 was 58 kDa. Conclusions: the presence of a specific band of esterase (58 kDa) in surviving larvae confirmed the role of these enzymes in insecticidal resistance. The diagnosis of the function of the esterases in resistance to temephos through biochemical tests should not be made in larvae exposed to sublethal doses of this insecticide, in order to avoid false negatives.

Caracterización parcial de la actividad de esterasas en una cepa de Aedes aegypti resistente a temefos / Partial characterization of esterase activity in a temephos-resistant Aedes aegypti strain

Introducción: las enzimas esterasas han sido identificadas como mecanismo de resistencia a temefos en Aedes aegypti de Cuba, larvicida más utilizado en el mundo. Objetivo: caracterizar parcialmente la actividad de esterasas en larvas expuestas y no expuestas a dosis subletales de temefos en una cepa de Aedes aegypti resistente a este insecticida. Métodos: se utilizó una cepa de Aedes aegypti de referencia susceptible (Rockefeller) y otra resistente a temefos (SANtemF11). Se expusieron las larvas de la cepa SANtemF11 a la concentración letal 90 (CL90) de temefos (1 ppm), 10 por ciento de larvas sobrevivientes a las 24 h (SANtem [24 h]) se transfirieron a agua limpia y sin exposición a insecticidas por otras 24 h (SANtem [48 h]). Se caracterizó de modo parcial, en estas larvas, la actividad de esterasas a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. Se estimó por duodecil sulfato de sodio (SDS-PAGE) el peso molecular de la esterasa (Est. A4). Resultados: la actividad de esterasas en la cepa SANtemF11 resultó significativamente mayor que en Rockefeller. Se observó una disminución significativa de la actividad de esterasas en las larvas sobrevivientes (SANtemF11 [24 h]), la cual se recuperó 24 h después sin exposición a temefos. En el zimograma se observó que en 10 % de las larvas sobrevivientes a temefos, solo apareció incrementada la banda de esterasa A4, en comparación con las observadas en SANtemF11. El peso molecular estimado de la esterasa A4 fue de 58 kDa. Conclusiones: la presencia de una banda específica de esterasa (58 kDa), en las larvas sobrevivientes a la selección con temefos, confirma su papel en la resistencia a este insecticida. Diagnosticar la función de las esterasas en la resistencia a temefos, a través de ensayos bioquímicos, no debe realizarse en larvas expuestas a dosis subletales de este insecticida, para evitar falsos negativos(AU)

Introduction: the esterase enzymes have been defined as the mechanism of resistance to temephos in Aeges aegypti in Cuba, which is the most used larvacide worldwide. Objective: to partially characterize the activity of esterases in exposed and non-exposed larvae at sublethal doses of temephos in an Aedes aegypti strain that is resistant to this product. Methods: a susceptible reference Aedes aegypti strain (Rockefeller) and another temephos-resistant strain (SANtemFII) were used. The larvae from SANtemF11 strain were exposed to lethal concentration 90 (LC90) of temephos (1 ppm); 10 % of the surviving larvae after 24 hours (SANtem[24 h] was moved to clean water, with no exposure to insecticide for 24 hours (SANtem [48 h]). The activity of esterases was partially characterized in these larvae through biochemical assays and gel-polyacrylamide electrophoresis. The molecular weight of esterase A 4 (ESt. A4) was estimated with the support of sodium duodecyl sulophate (SDS-PAGE). Results: the activity of esterases in SANtemF11 was significantly higher than in Rockefeller strain. Significant reduction of the activity of esterases in surviving larvae was observed (SANtemF11 [24 h], but it increased 24 h later without exposure to temephos. The zymogram showed that 10% of larvae that survived from temephos action, just the esterase A4 band increased if compared with those of SAntemF11. The estimated molecular weight of esterase A4 was 58 kDa. Conclusions: the presence of a specific band of esterase (58 kDa) in surviving larvae confirmed the role of these enzymes in insecticidal resistance. The diagnosis of the function of the esterases in resistance to temephos through biochemical tests should not be made in larvae exposed to sublethal doses of this insecticide, in order to avoid false negatives(AU)

Resistencia a insecticidas organofosforados en Aedes aegypti (Diptera: Culicidae) de Santiago de Cuba, 1997-2009 / Resistance to organophosphorus insecticides found in Aedes aegypti (Diptera: Culicidae) from Santiago de Cuba, 1997-2009

INTRODUCCIÓN: la resistencia a insecticidas organofosforados en Santiago de Cuba fue diagnosticada en Aedes aegypti (Linnaeus, 1762) en 1997, y alguno de ellos se han continuado utilizando hasta la fecha, de ahí la necesidad de conocer cómo ha variado la resistencia desde entonces, hasta fechas más recientes, año 2009. OBJETIVO: evaluar la resistencia a insecticidas organofosforados en larvas de Santiago de Cuba, colectadas en 2009 y su variación con respecto a 1997. Determinar la frecuencia en que aparece el mecanismo de resistencia, basado en la alta actividad de esterasas y su clasificación. MÉTODOS: se evaluó la resistencia a los insecticidas organofosforados, malation, pirimifos metil, fenitrotion, fention, temefos y clorpirifos en larvas mediante la metodología recomendada por la Organización Mundial de la Salud. El mecanismo de esterasas se determinó a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. RESULTADOS: las larvas de la cepa de Santiago de Cuba resultaron susceptibles a malation, pirimifos metil y fenitrotion y no hubo variación con los resultados obtenidos en una cepa de Santiago de Cuba de 1997, se observó moderada resistencia a fention y alta a temefos y clorpirifos. Al comparar estos resultados con los obtenidos en 1997, se observó un incremento de la resistencia a los tres insecticidas en el período 1997-2009. En la cepa de Santiago de Cuba de 2009 se demostró que las esterasas se encontraron con una alta actividad a una frecuencia de 0,7. Se observó la presencia de una esterasa tipo B amplificada, con un valor de movilidad relativa de 0,95 cm, la cual no se encontró en la cepa susceptible de referencia. CONCLUSIONES: la resistencia a insecticidas y sus mecanismos es un fenómeno sumamente variable, aun en la misma especie sometida a distintas intensidades de aplicación de insecticidas, de ahí que su monitoreo constante de forma local y en el tiempo sea una necesidad para un programa de control de vectores.

INTRODUCTION: resistance to organophosphorus insecticides was diagnosed in Aedes aegypti (Linnaeus, 1762) from Santiago de Cuba in 1997 and some of them are still used up to date; hence the need of ascertaining how the insecticidal resistance has changed in recent times, particularly in 2009. OBJECTIVE: to evaluate the resistance to organophosporus insecticides in larvae from Santiago de Cuba collected in 2009, and its variation in comparison with that observed in 1997; and to determine the frequency of occurrence of resistance mechanisms on the basis of high esterase activity and its classification. METHODS: resistance to organophosphorus insecticides such as malathion, pirimiphos, methyl, phenitrotion, phention, temephos and clorpiriphos in larvae by using the WHO recommended methodology. The esterase mechanism was identified through biochemical assays and polyacrylamide gel electrophoresis. RESULTS: larvae from the Santiago de Cuba strain were susceptible to malathion, pirimiphos, methyl and phenitrothion; there was no variation with the results achieved in a Santiago de Cuba strain in 1997, moderate resistance to phenthion and high resistance to temephos and chlorpiriphos were observed. When comparing these results with those of 1997, it was noted that resistance to the three insecticides increased in the 1997-2009 period. In the Santiago de Cuba strain 2009, it was shown that esterase activity was very high at a rate of 0,7. The presence of an amplified type B esterase with relative mobility of 0.95 cm was detected, which did not exist in the reference strain. CONCLUSIONS: resistance to insecticides and its mechanisms are highly variable, even in the same species subjected to various intensities in the insecticidal use, therefore, it is necessary to constantly monitor both aspects at local level in the course of time, with a view to an effective vector control program.

Resistencia a insecticidas organofosforados en Aedes aegypti (Diptera: Culicidae) de Santiago de Cuba, 1997-2009 / Resistance to organophosphorus insecticides found in Aedes aegypti (Diptera: Culicidae) from Santiago de Cuba, 1997-2009

INTRODUCCIÓN: la resistencia a insecticidas organofosforados en Santiago de Cuba fue diagnosticada en Aedes aegypti (Linnaeus, 1762) en 1997, y alguno de ellos se han continuado utilizando hasta la fecha, de ahí la necesidad de conocer cómo ha variado la resistencia desde entonces, hasta fechas más recientes, año 2009. OBJETIVO: evaluar la resistencia a insecticidas organofosforados en larvas de Santiago de Cuba, colectadas en 2009 y su variación con respecto a 1997. Determinar la frecuencia en que aparece el mecanismo de resistencia, basado en la alta actividad de esterasas y su clasificación. MÉTODOS: se evaluó la resistencia a los insecticidas organofosforados, malation, pirimifos metil, fenitrotion, fention, temefos y clorpirifos en larvas mediante la metodología recomendada por la Organización Mundial de la Salud. El mecanismo de esterasas se determinó a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. RESULTADOS: las larvas de la cepa de Santiago de Cuba resultaron susceptibles a malation, pirimifos metil y fenitrotion y no hubo variación con los resultados obtenidos en una cepa de Santiago de Cuba de 1997, se observó moderada resistencia a fention y alta a temefos y clorpirifos. Al comparar estos resultados con los obtenidos en 1997, se observó un incremento de la resistencia a los tres insecticidas en el período 1997-2009. En la cepa de Santiago de Cuba de 2009 se demostró que las esterasas se encontraron con una alta actividad a una frecuencia de 0,7. Se observó la presencia de una esterasa tipo B amplificada, con un valor de movilidad relativa de 0,95 cm, la cual no se encontró en la cepa susceptible de referencia. CONCLUSIONES: la resistencia a insecticidas y sus mecanismos es un fenómeno sumamente variable, aun en la misma especie sometida a distintas intensidades de aplicación de insecticidas, de ahí que su monitoreo constante de forma local y en el tiempo sea una necesidad para un programa de control de vectores (AU)

INTRODUCTION: resistance to organophosphorus insecticides was diagnosed in Aedes aegypti (Linnaeus, 1762) from Santiago de Cuba in 1997 and some of them are still used up to date; hence the need of ascertaining how the insecticidal resistance has changed in recent times, particularly in 2009. OBJECTIVE: to evaluate the resistance to organophosporus insecticides in larvae from Santiago de Cuba collected in 2009, and its variation in comparison with that observed in 1997; and to determine the frequency of occurrence of resistance mechanisms on the basis of high esterase activity and its classification. METHODS: resistance to organophosphorus insecticides such as malathion, pirimiphos, methyl, phenitrotion, phention, temephos and clorpiriphos in larvae by using the WHO recommended methodology. The esterase mechanism was identified through biochemical assays and polyacrylamide gel electrophoresis. RESULTS: larvae from the Santiago de Cuba strain were susceptible to malathion, pirimiphos, methyl and phenitrothion; there was no variation with the results achieved in a Santiago de Cuba strain in 1997, moderate resistance to phenthion and high resistance to temephos and chlorpiriphos were observed. When comparing these results with those of 1997, it was noted that resistance to the three insecticides increased in the 1997-2009 period. In the Santiago de Cuba strain 2009, it was shown that esterase activity was very high at a rate of 0,7. The presence of an amplified type B esterase with relative mobility of 0.95 cm was detected, which did not exist in the reference strain. CONCLUSIONS: resistance to insecticides and its mechanisms are highly variable, even in the same species subjected to various intensities in the insecticidal use, therefore, it is necessary to constantly monitor both aspects at local level in the course of time, with a view to an effective vector control program (AU)

Resistencia a insecticidas organofosforados en Aedes aegypti (Diptera: Culicidae) de Santiago de Cuba, 1997-2009 / Resistance to organophosphorus insecticides found in Aedes aegypti (Diptera: Culicidae) from Santiago de Cuba, 1997-2009

INTRODUCCIÓN: la resistencia a insecticidas organofosforados en Santiago de Cuba fue diagnosticada en Aedes aegypti (Linnaeus, 1762) en 1997, y alguno de ellos se han continuado utilizando hasta la fecha, de ahí la necesidad de conocer cómo ha variado la resistencia desde entonces, hasta fechas más recientes, año 2009. OBJETIVO: evaluar la resistencia a insecticidas organofosforados en larvas de Santiago de Cuba, colectadas en 2009 y su variación con respecto a 1997. Determinar la frecuencia en que aparece el mecanismo de resistencia, basado en la alta actividad de esterasas y su clasificación. MÉTODOS: se evaluó la resistencia a los insecticidas organofosforados, malation, pirimifos metil, fenitrotion, fention, temefos y clorpirifos en larvas mediante la metodología recomendada por la Organización Mundial de la Salud. El mecanismo de esterasas se determinó a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. RESULTADOS: las larvas de la cepa de Santiago de Cuba resultaron susceptibles a malation, pirimifos metil y fenitrotion y no hubo variación con los resultados obtenidos en una cepa de Santiago de Cuba de 1997, se observó moderada resistencia a fention y alta a temefos y clorpirifos. Al comparar estos resultados con los obtenidos en 1997, se observó un incremento de la resistencia a los tres insecticidas en el período 1997-2009. En la cepa de Santiago de Cuba de 2009 se demostró que las esterasas se encontraron con una alta actividad a una frecuencia de 0,7. Se observó la presencia de una esterasa tipo B amplificada, con un valor de movilidad relativa de 0,95 cm, la cual no se encontró en la cepa susceptible de referencia. CONCLUSIONES: la resistencia a insecticidas y sus mecanismos es un fenómeno sumamente variable, aun en la misma especie sometida a distintas intensidades de aplicación de insecticidas, de ahí que su monitoreo constante de forma local y en el tiempo sea una necesidad para un programa de control de vectores (AU)

INTRODUCTION: resistance to organophosphorus insecticides was diagnosed in Aedes aegypti (Linnaeus, 1762) from Santiago de Cuba in 1997 and some of them are still used up to date; hence the need of ascertaining how the insecticidal resistance has changed in recent times, particularly in 2009. OBJECTIVE: to evaluate the resistance to organophosporus insecticides in larvae from Santiago de Cuba collected in 2009, and its variation in comparison with that observed in 1997; and to determine the frequency of occurrence of resistance mechanisms on the basis of high esterase activity and its classification. METHODS: resistance to organophosphorus insecticides such as malathion, pirimiphos, methyl, phenitrotion, phention, temephos and clorpiriphos in larvae by using the WHO recommended methodology. The esterase mechanism was identified through biochemical assays and polyacrylamide gel electrophoresis. RESULTS: larvae from the Santiago de Cuba strain were susceptible to malathion, pirimiphos, methyl and phenitrothion; there was no variation with the results achieved in a Santiago de Cuba strain in 1997, moderate resistance to phenthion and high resistance to temephos and chlorpiriphos were observed. When comparing these results with those of 1997, it was noted that resistance to the three insecticides increased in the 1997-2009 period. In the Santiago de Cuba strain 2009, it was shown that esterase activity was very high at a rate of 0,7. The presence of an amplified type B esterase with relative mobility of 0.95 cm was detected, which did not exist in the reference strain. CONCLUSIONS: resistance to insecticides and its mechanisms are highly variable, even in the same species subjected to various intensities in the insecticidal use, therefore, it is necessary to constantly monitor both aspects at local level in the course of time, with a view to an effective vector control program (AU)

[Resistance to organophosphorus insecticides found in Aedes aegypti (Diptera: Culicidae) from Santiago de Cuba, 1997-2009]. / Resistencia a insecticidas organofosforados en Aedes aegypti (Diptera: Culicidae) de Santiago de Cuba, 1997-2009.

INTRODUCTION: resistance to organophosphorus insecticides was diagnosed in Aedes aegypti (Linnaeus, 1762) from Santiago de Cuba in 1997 and some of them are still used up to date; hence the need of ascertaining how the insecticidal resistance has changed in recent times, particularly in 2009. OBJECTIVE: to evaluate the resistance to organophosporus insecticides in larvae from Santiago de Cuba collected in 2009, and its variation in comparison with that observed in 1997; and to determine the frequency of occurrence of resistance mechanisms on the basis of high esterase activity and its classification. METHODS: resistance to organophosphorus insecticides such as malathion, pirimiphos, methyl, phenitrotion, phention, temephos and clorpiriphos in larvae by using the WHO recommended methodology. The esterase mechanism was identified through biochemical assays and polyacrylamide gel electrophoresis. RESULTS: larvae from the Santiago de Cuba strain were susceptible to malathion, pirimiphos, methyl and phenitrothion; there was no variation with the results achieved in a Santiago de Cuba strain in 1997, moderate resistance to phenthion and high resistance to temephos and chlorpiriphos were observed. When comparing these results with those of 1997, it was noted that resistance to the three insecticides increased in the 1997-2009 period. In the Santiago de Cuba strain 2009, it was shown that esterase activity was very high at a rate of 0.7. The presence of an amplified type B esterase with relative mobility of 0.95 cm was detected, which did not exist in the reference strain. CONCLUSIONS: resistance to insecticides and its mechanisms are highly variable, even in the same species subjected to various intensities in the insecticidal use, therefore, it is necessary to constantly monitor both aspects at local level in the course of time, with a view to an effective vector control program.

Estado de la resistencia a insecticidas y sus mecanismos en Aedes aegypti en el municipio Boyeros / Situation of the insecticidal resistance and its mechanisms in Aedes aegypti in Boyeros municipality

INTRODUCCIÓN: el control de Aedes aegypti continúa siendo la única medida disponible para poder disminuir la transmisión de dengue. Desafortunadamente Ae. aegypti ha demostrado la habilidad de desarrollar resistencia a una gran variedad de tóxicos. OBJETIVO: evaluar la resistencia a insecticidas químicos en larvas y adultos del municipio Boyeros, Ciudad de La Habana, así como los mecanismos que contribuyeron a esta. MÉTODOS: se evaluó la resistencia a insecticidas químicos en larvas y adultos a través de metodologías de la OMS. Los mecanismos de resistencia se determinaron a través de sinergistas y pruebas bioquímicas. Se realizó electroforesis en gel de poliacrilamida para la visualización de enzimas esterasas. RESULTADOS: en larvas se observó susceptibilidad a los insecticidas organofosforados evaluados. Resistencia se observó a los piretroides cipermetrina y deltametrina. Los bioensayos en larvas con el producto comercial de temefos mostraron 100 por ciento de mortalidad con recambio diario de agua hasta 10 d. Se demostró que ni las esterasas, ni la enzima glutatión transferasa, desempeñaron un papel importante en la resistencia a insecticidas en larvas. Se observó la presencia de la esterasa A4 amplificada a baja frecuencia en las muestras estudiadas. En el estado adulto, la cepa Boyeros resultó resistente a los piretroides ciflutrina y lambdacialotrina, en verificación a deltametrina, y resultó susceptible a cipermetrina; también resultó ser resistente al organofosforado clorpirifos y al organoclorado DDT. CONCLUSIONES: estos resultados corroboran que aun el piretroide cipermetrina, a pesar de su uso en el municipio Boyeros, continúa siendo efectivo para el control de Ae. aegypti.

INTRODUCTION: the control of Aedes aegypti remains the only available measure to reduce dengue transmission. Unfortunately, this vector has proved that it is capable of developing resistance to a great variety of toxic substances. OBJECTIVE: to evaluate the resistance to chemical insecticides in larvae and adult vectors in Boyeros municipality, City of Havana as well as those mechanisms supporting it. METHODS: insecticide resistance of mosquito larvae and adults was evaluated with the WHO methodologies. The resistance mechanisms were determined through synergy and biochemical tests. Polyacrylamid gel electropheresis was applied to visualize esterases. RESULTS: larvae were susceptible to the evaluated organophosphate insecticides whereas resistance to pyrethroids, cypermethrin and deltamethrin was observed. Bioassays performed in larvae with temephos-made commercial product showed 100 percent mortality up to 10 days, with daily change of water. It was proved that neither esterases nor glutathione transferase played an important role in larval insecticide resistance. Low frequency amplified esterase A4 was present in the studied samples. In adult stage, Boyeros strain was resistant to pyrethroids ciflutrhine and Lambdacyalothrine, in verification to deltamethrine and susceptible to cypermethrine; it was also resistant to organophosphate chlorpiriphos and organochlorate DDT. CONCLUSIONS: these results confirm that although the pyrethroid cipermethrine has been widely used in Boyeros municipality, it continues being effective for Ae. aegypti control.

Estado de la resistencia a insecticidas y sus mecanismos en Aedes aegypti en el municipio Boyeros / Situation of the insecticidal resistance and its mechanisms in Aedes aegypti in Boyeros municipality

INTRODUCCIÓN: el control de Aedes aegypti continúa siendo la única medida disponible para poder disminuir la transmisión de dengue. Desafortunadamente Ae. aegypti ha demostrado la habilidad de desarrollar resistencia a una gran variedad de tóxicos. OBJETIVO: evaluar la resistencia a insecticidas químicos en larvas y adultos del municipio Boyeros, Ciudad de La Habana, así como los mecanismos que contribuyeron a esta. MÉTODOS: se evaluó la resistencia a insecticidas químicos en larvas y adultos a través de metodologías de la OMS. Los mecanismos de resistencia se determinaron a través de sinergistas y pruebas bioquímicas. Se realizó electroforesis en gel de poliacrilamida para la visualización de enzimas esterasas. RESULTADOS: en larvas se observó susceptibilidad a los insecticidas organofosforados evaluados. Resistencia se observó a los piretroides cipermetrina y deltametrina. Los bioensayos en larvas con el producto comercial de temefos mostraron 100 por ciento de mortalidad con recambio diario de agua hasta 10 d. Se demostró que ni las esterasas, ni la enzima glutatión transferasa, desempeñaron un papel importante en la resistencia a insecticidas en larvas. Se observó la presencia de la esterasa A4 amplificada a baja frecuencia en las muestras estudiadas. En el estado adulto, la cepa Boyeros resultó resistente a los piretroides ciflutrina y lambdacialotrina, en verificación a deltametrina, y resultó susceptible a cipermetrina; también resultó ser resistente al organofosforado clorpirifos y al organoclorado DDT. CONCLUSIONES: estos resultados corroboran que aun el piretroide cipermetrina, a pesar de su uso en el municipio Boyeros, continúa siendo efectivo para el control de Ae. aegypti(AU)

INTRODUCTION: the control of Aedes aegypti remains the only available measure to reduce dengue transmission. Unfortunately, this vector has proved that it is capable of developing resistance to a great variety of toxic substances. OBJECTIVE: to evaluate the resistance to chemical insecticides in larvae and adult vectors in Boyeros municipality, City of Havana as well as those mechanisms supporting it. METHODS: insecticide resistance of mosquito larvae and adults was evaluated with the WHO methodologies. The resistance mechanisms were determined through synergy and biochemical tests. Polyacrylamid gel electropheresis was applied to visualize esterases. RESULTS: larvae were susceptible to the evaluated organophosphate insecticides whereas resistance to pyrethroids, cypermethrin and deltamethrin was observed. Bioassays performed in larvae with temephos-made commercial product showed 100 percent mortality up to 10 days, with daily change of water. It was proved that neither esterases nor glutathione transferase played an important role in larval insecticide resistance. Low frequency amplified esterase A4 was present in the studied samples. In adult stage, Boyeros strain was resistant to pyrethroids ciflutrhine and Lambdacyalothrine, in verification to deltamethrine and susceptible to cypermethrine; it was also resistant to organophosphate chlorpiriphos and organochlorate DDT. CONCLUSIONS: these results confirm that although the pyrethroid cipermethrine has been widely used in Boyeros municipality, it continues being effective for Ae. aegypti control(AU)

Determinación in vivo del papel de las enzimas esterasas y glutation transferasa en la resistencia a piretroides en Aedes aegypti (Diptera: Culicidae) / Determination in vivo of the role of esterase and gluthation transferase enzymes in pyrethroid resistance of Aedes aegypti (Diptera: Cullicidae)

Se realizó un estudio in vivo a través del uso de 2 sinergistas, el trifenil fosfato (TFF) inhibidor específico de esterasas y el ácido etacrínico (AE), inhibidor específico de la enzima glutation transferasa (GST), para determinar si estas enzimas eran responsables de la resistencia a piretroides en Aedes aegypti. Para el trabajo se utilizaron 2 cepas de Aedes aegypti resistentes a insecticidas, una cepa que fue seleccionada con temefos por 6 generaciones de selección (SAN-F6) y otra con deltametrina por 12 generaciones de selección con este insecticida (SAN-F12), ambas resultaron ser resistentes a insecticidas piretroides. Se demostró a través del uso de los sinergistas TFF y AE que las enzimas esterasas y GST son responsables de la resistencia a los piretroides en estas cepas. Esos resultados demuestran la existencia de un fenómeno de resistencia cruzada y multirresistencia, lo cual debe tenerse en cuenta en las estrategias de uso de insecticidas para el control de este vector.

An in vivo study of two synergists, that is, Triphenil phosphate -specific esterase inhibitor- and ethacrynic acid specific gluthation transferase inhibitor- was performed to determine if these enzymes were responsible for pyrethroid resistance of Aedes aegypti. To this end, two insecticide resistant Aedes aegypti strains were used, one strain selected with temephos by six selection generations (SAN-F6) and the other strain with delmamethrin by 12 selection generations (SAN-F12), being both strains resistant to pyrethroid insecticices. Through the use of TPP and EA synergists, it was proved that esterase and gluthation-s-transferase (GST) enzymes were responsible for pryrethroid resistance of these strains. These results showed the existence of cross-resistance and multidrug resistance, which should be taken into account for insecticide use strategies aimed at vector control.

Determinación in vivo del papel de las enzimas esterasas y glutation transferasa en la resistencia a piretroides en Aedes aegypti (Diptera: Culicidae) / Determination in vivo of the role of esterase and gluthation transferase enzymes in pyrethroid resistance of Aedes aegypti (Diptera: Cullicidae)

Se realizó un estudio in vivo a través del uso de 2 sinergistas, el trifenil fosfato (TFF) inhibidor específico de esterasas y el ácido etacrínico (AE), inhibidor específico de la enzima glutation transferasa (GST), para determinar si estas enzimas eran responsables de la resistencia a piretroides en Aedes aegypti. Para el trabajo se utilizaron 2 cepas de Aedes aegypti resistentes a insecticidas, una cepa que fue seleccionada con temefos por 6 generaciones de selección (SAN-F6) y otra con deltametrina por 12 generaciones de selección con este insecticida (SAN-F12), ambas resultaron ser resistentes a insecticidas piretroides. Se demostró a través del uso de los sinergistas TFF y AE que las enzimas esterasas y GST son responsables de la resistencia a los piretroides en estas cepas. Esos resultados demuestran la existencia de un fenómeno de resistencia cruzada y multirresistencia, lo cual debe tenerse en cuenta en las estrategias de uso de insecticidas para el control de este vector(AU)

An in vivo study of two synergists, that is, Triphenil phosphate -specific esterase inhibitor- and ethacrynic acid specific gluthation transferase inhibitor- was performed to determine if these enzymes were responsible for pyrethroid resistance of Aedes aegypti. To this end, two insecticide resistant Aedes aegypti strains were used, one strain selected with temephos by six selection generations (SAN-F6) and the other strain with delmamethrin by 12 selection generations (SAN-F12), being both strains resistant to pyrethroid insecticices. Through the use of TPP and EA synergists, it was proved that esterase and gluthation-s-transferase (GST) enzymes were responsible for pryrethroid resistance of these strains. These results showed the existence of cross-resistance and multidrug resistance, which should be taken into account for insecticide use strategies aimed at vector control(AU)

Levels of insecticide resistance and resistance mechanisms in Aedes aegypti from some Latin American countries.

Eight Latin American strains of Aedes aegypti were evaluated for resistance to 6 organophosphates (temephos, malathion, fenthion, pirimiphos-methyl, fenitrothion, and chlorpirifos) and 4 pyrethroids (deltamethrin, lambdacyhalothrin, betacypermethrin, and cyfluthrin) under laboratory conditions. In larval bioassays, temephos resistance was high (resistance ratio [RR50], > or =10X) in the majority of the strains, except for the Nicaragua and Venezuela strains, which showed moderate resistance (RR50, between 5 and 10X). The majority of the strains were susceptible to malathion, fenthion, and fenitrothion. However, resistance to pirimiphos-methyl ranged from moderate to high in most of the strains. Larvae from Havana City were resistant to 3 of the pyrethroids tested and moderately resistant to cyfluthrin. The Santiago de Cuba strain showed high resistance to deltamethrin and moderate resistance to the other pyrethroids (lambdacyhalothrin, betacypermethrin, and cyfluthrin). The rest of the strains were susceptible to pyrethroids, except for the Jamaica and Costa Rica strains, which showed moderate resistance to cyfluthrin, and Peru and Venezuela, which showed resistance to deltamethrin. Adult bioassays showed that all the strains were resistant to dichlorodiphenyl-trichloroethane and to the majority of pyrethroids evaluated. The use of the synergists S,S,S,-tributyl phosphorotrithioate and piperonil butoxide showed that esterase and monooxygenases played an important role in the temephos, pirimiphos-methyl, and chlorpirifos resistance in some strains. Biochemical tests showed high frequencies of esterase and glutathione-S-transferase activity; however, the frequency of altered acetylcholinesterase mechanism was low. The polyacrylamide electrophoresis gel detected the presence of a strong band called Est-A4. Insecticide resistance in Ae. aegypti is a serious problem facing control operations, and integrated control strategies are recommended to help prevent or delay the temephos resistance in larvae and pyrethroids resistance in adults.