Development of a unified method for the determination of legacy and metabolites of current pesticides in serum for exposure assessment.

The use of pesticides is often regarded as a fundamental aspect of conventional agriculture. However, these compounds have gained recognition as some of the oldest and most widely employed xenobiotic contaminants, necessitating effective strategies for human biomonitoring. In this context, a method was developed for the determination of 16 legacy organochlorine pesticides, 6 metabolites of current pesticides (2,4-D, malathion, parathion, fipronil, pyraclostrobin, cypermethrin, permethrin, cyfluthrin), and 1 triazine herbicide (atrazine) in serum. Samples were prepared with water, formic acid, acetonitrile, and ultrasound irradiation, followed by solid-phase extraction with Oasis Prime HLB. Subsequently, metabolites from current pesticides underwent derivatization using MTBSTFA with 1% TBDMSCl for analysis via gas chromatography-tandem mass spectrometry (GC-MS/MS), employing an SLB-5MS fused silica capillary column. Analytical curves were generated with limits of quantification from 0.3 to 4.0 ng.mL-1. Accuracy ranged from 69 to 124%, and the coefficient of variation from 2 to 28%. Moreover, determining 1-(4-chlorophenyl)-1H-pyrazol-3-ol was suggested as a biomarker for pyraclostrobin biomonitoring. This analytical approach facilitated the determination of both legacy and metabolites of current pesticides in the same serum sample, presenting an interesting and cost-effective option for large cohorts, and multi-omics studies that evaluate time-dependent biomarkers in blood samples, thereby enabling biomonitoring within the same matrix. Furthermore, a proof-of-concept involving 10 volunteers demonstrated exposure to 9 pesticides at mean concentrations measured in ng mL-1, consistent with findings from various biomonitoring initiatives.

Slow recovery rates and spatial aggregation of Triatoma infestans populations in an area with high pyrethroid resistance in the Argentine Chaco.

BACKGROUND: The emergence of pyrethroid resistance has threatened the elimination of Triatoma infestans from the Gran Chaco ecoregion. We investigated the status and spatial distribution of house infestation with T. infestans and its main determinants in Castelli, a municipality of the Argentine Chaco with record levels of triatomine pyrethroid resistance, persistent infestation over 2005-2014, and limited or no control actions over 2015-2020. METHODS: We conducted a 2-year longitudinal survey to assess triatomine infestation by timed manual searches in a well-defined rural section of Castelli including 14 villages and 234 inhabited houses in 2018 (baseline) and 2020, collected housing and sociodemographic data by on-site inspection and a tailored questionnaire, and synthetized these data into three indices generated by multiple correspondence analysis. RESULTS: The overall prevalence of house infestation in 2018 (33.8%) and 2020 (31.6%) virtually matched the historical estimates for the period 2005-2014 (33.7%) under recurrent pyrethroid sprays. While mean peridomestic infestation remained the same (26.4-26.7%) between 2018 and 2020, domestic infestation slightly decreased from 12.2 to 8.3%. Key triatomine habitats were storerooms, domiciles, kitchens, and structures occupied by chickens. Local spatial analysis showed significant aggregation of infestation and bug abundance in five villages, four of which had very high pyrethroid resistance approximately over 2010-2013, suggesting persistent infestations over space-time. House bug abundance within the hotspots consistently exceeded the estimates recorded in other villages. Multiple regression analysis revealed that the presence and relative abundance of T. infestans in domiciles were strongly and negatively associated with indices for household preventive practices (pesticide use) and housing quality. Questionnaire-derived information showed extensive use of pyrethroids associated with livestock raising and concomitant spillover treatment of dogs and (peri) domestic premises. CONCLUSIONS: Triatoma infestans populations in an area with high pyrethroid resistance showed slow recovery and propagation rates despite limited or marginal control actions over a 5-year period. Consistent with these patterns, independent experiments confirmed the lower fitness of pyrethroid-resistant triatomines in Castelli compared with susceptible conspecifics. Targeting hotspots and pyrethroid-resistant foci with appropriate house modification measures and judicious application of alternative insecticides with adequate toxicity profiles are needed to suppress resistant triatomine populations and prevent their eventual regional spread.

Pyrethroid resistance distribution in Triatoma infestans and environmental association along the Argentine endemic zone.

Insecticide resistance is considered a barrier to chemical control of Triatoma infestans, the main vector of Chagas disease in the Southern Cone of South America. Although initiatives to reduce the incidence of the disease in the region have integrated different strategies, they have mainly relied on vector elimination using pyrethroid insecticides such as deltamethrin. Reports of pyrethroid resistance in connection with T. infestans control failures first emerged in northern Argentina and southern Bolivia. Recently, a mosaic pyrethroid-resistant focus has been described in the center of the Argentine Gran Chaco (Department of General Güemes, Chaco Province), characterized by the presence of susceptible and very highly resistant populations in the same area. The involvement of different resistance mechanisms has been proposed, together with the contribution of environmental variables that promote the toxicological heterogeneity described. In the endemic zone of Argentina, however, new questions arise: Are there any other clusters of resistance? Is there a relationship between the distribution of resistance and environmental variables (as has been observed at smaller scale)? We studied toxicological data from insects collected and analyzed at 224 localities between 2010 and 2020 as part of the resistance monitoring conducted by the Chagas National Program. The sites were classified according to the survival rate of insects exposed to a discriminant dose of deltamethrin: 0-0.19 were considered susceptible, 0.2-0.79 low-resistance, and 0.8-1 high-resistance. Localities were georeferenced to describe the spatial distribution of resistance and to identify environmental variables (demographics, land use, urbanization, connectivity, and climate) potentially associated with resistance. We used Generalized Linear Models (GLMs) to examine the association between resistance and environmental predictors, selecting error distributions based on the response variable definition. For the entire period, 197 susceptible localities were distributed across the endemic zone. Localities with different survival rates were found throughout the area; 9 high-resistance localities circled the two previously identified resistant foci, and 18 low-resistance in 6 provinces, highlighting their relevance for control planning. Precipitation variables were linked to resistance in all the GLMs evaluated. Presence/absence models were the most accurate, with precipitation, distance from the capital city, and land use contributing to the distribution of resistance. This information could be valuable for improving T. infestans control strategies in future scenarios characterized by unpredictable changes in land use and precipitation.

RNA interference of the clock gene period disrupts circadian rhythms in the expression of genes related to insecticide resistance in the chagas disease vector Triatoma infestans (Hemiptera: Reduviidae).

In Triatoma infestans it was observed pyrethroid resistance attributed in part to an elevated oxidative metabolism mediated by cytochromes P450. The nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) plays a crucial role in catalysing the electron transfer from NADPH to all cytochrome P450s. The daily variations in the expression of CPR gene and a P450 gene (CYP4EM7), both associated with insecticide resistance, suggested that their expressions would be under the endogenous clock control. To clarify the involvement of the clock in orchestration of the daily fluctuations in CPR and CYP4M7 genes expression, it was proposed to investigate the effect of silencing the clock gene period (per) by RNA interference (RNAi). The results obtained allowed to establish that the silencing of per gene was influenced by intake schemes used in the interference protocols. The silencing of per gene in T. infestans reduced its expression at all the time points analysed and abolished the characteristic rhythm in the transcriptional expression of per mRNA. The effect of the per gene silencing in the expression profiles at the transcriptional level of CPR and CYP4EM7 genes showed the loss of rhythmicity and demonstrated the biological clock involvement in the regulation of t heir expression.

High Frequencies of kdr Mutation and Chikungunya Infection in Aedes aegypti Population from Minas Gerais, Brazil.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

Risk assessment of a Brazilian urban population due to the exposure to pyrethroid insecticides during the COVID-19 pandemic using wastewater-based epidemiology.

Pyrethroids are synthetic insecticides commonly used in agriculture and homes due to their low toxicity to mammals and effectiveness at low doses. However, exposure to pyrethroids can cause various symptoms, depending on the route of exposure. To measure human exposure to pyrethroids, researchers used wastewater-based epidemiology (WBE) with polar organic chemical integrative samplers (POCIS) sampling. This approach is a cost-effective and efficient way to assess exposure to pyrethroids. The study aimed to evaluate the exposure of an urban population in Brazil to pyrethroids during the COVID-19 pandemic using WBE with POCIS sampling. Researchers analyzed 3-phenoxybenzoic acid (3-PBA) in wastewater using passive sampling with POCIS, which was extracted with methanol and analyzed using UPLC-MS/MS. The range of CTWA concentrations of 3-PBA in wastewater was 24.3-298.2 ng L-1, with a mean value of 134 ± 76.5 ng L-1. The values were used to estimate the exposure of the population to pyrethroid insecticides. Three different conversion factors were applied to determine the range of exposure to at least 20 different pyrethroid insecticides. The exposure values ranged from 18.08 to 1441.49 mg day-1 per 1000 inhabitants. The toxicological risk posed to the exposed population was evaluated by calculating the WBE toxicological level (WBE-TL). Lambda-cyhalothrin was used as a reference for risk assessment, and the WBE-TL values for lambda-cyhalothrin ranged from 0.5 to 8.29 (considering the high CF). We compared mobility trends to 3-PBA exposure during the COVID-19 pandemic. The study highlighted the effectiveness of POCIS sampling in WBE and provided useful information for policymakers and regulatory agencies. POCIS sampling has practical advantages, including analyte pre-concentration, low operational cost, and ease of use. Overall, the study shows the importance of monitoring and understanding the exposure of the population to pyrethroid insecticides, especially during the pandemic when people may be spending more time at home.

Extended stage duration and diminished fecundity in deltamethrin-resistant Triatoma infestans (Klug, 1834) of the Argentinean Chaco.

Pyrethroid-resistance is an emergent trait in populations of various insect species. For Triatoma infestans (Klug, 1834) (Heteroptera: Reduviidae), the major vector of Chagas disease in the southern part of South America, hotspot areas of pyrethroid-resistance have recently been found in the Chaco Province of Argentina. Resistant condition can reduce fitness of individuals in the absence of insecticide exposure, that is, fitness costs. We evaluated the existence of developmental and/or reproductive costs in T. infestans collected from two areas of pyrethroid-resistance in Chaco Province, Argentina. Three toxicological groups were defined from field-collected insects: susceptible (survival <20%), moderately resistant (survival between 20% and 80%) and highly resistant (survival >80%). Cohorts of the three toxicological groups were followed-up to study life cycle and reproductive parameters. Additionally, we parameterized matrix population growth models. First and IV nymphal stages of the resistant groups exhibited a longer stage duration than susceptible ones. The reproductive days and hatching success showed significant lower values revealing reproductive costs for the resistant groups. Matrix analysis showed lower population growth rates for the resistant groups. Our results support developmental and reproductive costs for pyrethroid-resistant individuals. This trait could be interpreted as lower population recovery ability for pyrethroid-resistant individuals compared to susceptible insects after alternative vector control actions.

The Pampa del Indio project: sustainable vector control and long-term declines in the prevalence and abundance of Triatoma infestans infected with Trypanosoma cruzi in the Argentine Chaco.

BACKGROUND: The Gran Chaco region is a major hotspot of Chagas disease. We implemented a 9-year program aimed at suppressing house infestation with Triatoma infestans and stopping vector-borne transmission to creole and indigenous (Qom) residents across Pampa del Indio municipality (Argentine Chaco). The aim of the present study was to assess the intervention effects on parasite-based transmission indices and the spatial distribution of the parasite, and test whether house-level variations in triatomine infection with Trypanosoma cruzi declined postintervention and were influenced by household ethnicity, persistent infestation linked to pyrethroid resistance and other determinants of bug infection. METHODS: This longitudinal study assessed house infestation and bug infection with T. cruzi before and after spraying houses with pyrethroids and implemented systematic surveillance-and-response measures across four operational areas over the period 2007-2016. Live triatomines were individually examined for infection by optical microscopy or kinetoplast DNA (kDNA)-PCR and declared to be infected with T. cruzi when assessed positive by either method. RESULTS: The prevalence of infection with T. cruzi was 19.4% among 6397 T. infestans examined. Infection ranged widely among the study areas (12.5-26.0%), household ethnicity (15.3-26.9%), bug ecotopes (1.8-27.2%) and developmental stages (5.9-27.6%), and decreased from 24.1% (baseline) to 0.9% (endpoint). Using random-intercept multiple logistic regression, the relative odds of bug infection strongly decreased as the intervention period progressed, and increased with baseline domestic infestation and bug stage and in Qom households. The abundance of infected bugs and the proportion of houses with ≥ 1 infected bug remained depressed postintervention and were more informative of area-wide risk status than the prevalence of bug infection. Global spatial analysis revealed sharp changes in the aggregation of bug infection after the attack phase. Baseline domestic infestation and baseline bug infection strongly predicted the future occurrence of bug infection, as did persistent domestic infestation in the area with multiple pyrethroid-resistant foci. Only 19% of houses had a baseline domestic infestation and 56% had ever had ≥ 1 infected bug. CONCLUSIONS: Persistent bug infection postintervention was closely associated with persistent foci generated by pyrethroid resistance. Postintervention parasite-based indices closely agreed with human serosurveys at the study endpoint, suggesting transmission blockage. The program identified households and population subgroups for targeted interventions and opened new opportunities for risk prioritization and sustainable vector control and disease prevention.

Comparative Transcriptomic Analysis of Insecticide-Resistant Aedes aegypti from Puerto Rico Reveals Insecticide-Specific Patterns of Gene Expression.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

First report on knockdown resistance mutations in wild populations of Aedes aegypti from Argentina determined by a novel multiplex high-resolution melting polymerase chain reaction method.

BACKGROUND: The mosquito Aedes aegypti is an urban vector of dengue and other arboviruses. During epidemics of these viruses, pyrethroid insecticides are used for the control of adult mosquitoes. The worldwide resistance of Ae. aegypti to these insecticides is a cause of failure of vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel. Point mutations in the gene coding for this channel, called knockdown resistance (kdr) mutations, are associated with pyrethroid resistance. Two kdr mutations, V1016I and F1534C, have increased in frequency in natural populations of Ae. aegypti in the Americas during the last decade. Their association with pyrethroid resistance has been largely demonstrated in field populations throughout the Americas, and in in vitro assays. Diagnostics for kdr polymorphism allow early detection of the spread of insecticide resistance, which is critical for timely decisions on vector management. Given the importance of resistance management, high-throughput methods for kdr genotyping are valuable tools as they can be used for resistance monitoring programs. These methods should be cost-effective, to allow regional-scale surveys. Despite the extensive presence of Ae. aegypti and incidence of dengue in Argentina, the presence, abundance, and distribution of kdr mutations in populations of this mosquito have yet to be reported for the country. METHODS: Aedes aegypti samples were collected as immature stages or adults from Buenos Aires Metropolitan Area and northern localities of Tartagal (Salta Province) and Calilegua (Jujuy Province). Immature stages were maintained in the laboratory until they developed into adults. A high-resolution melting assay, based on an analysis of melting temperatures, was developed for the simultaneous genotyping of V1016I and F1534C kdr mutations. We used this method to infer the presence and frequencies of kdr alleles in 11 wild populations from Argentina. RESULTS: We demonstrated the presence of kdr mutations in Ae. aegypti in Argentina in regions where this species is under different selection pressures due to the use of pyrethroids. The populations under analysis are located in geographically distant regions of the species' distribution in Argentina: the northern provinces of Salta and Jujuy and the Buenos Aires Metropolitan Area. Higher frequencies of resistant-associated alleles were detected in the northern region. We report a multiplex high-throughput assay based on a high-resolution melting polymerase chain reaction method for the simultaneous genotyping of V1016I and F1534C kdr mutations. This assay was shown to be cost-effective, and thus provides an interesting molecular tool for kdr genotyping in A. aegypti control campaigns. CONCLUSIONS: We report, to the best of our knowledge for the first time, the presence of kdr mutations in populations of Ae. aegypti from geographically distant locations of Argentina that differ with respect to their epidemiological situation and history of mosquito control. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the Americas. Given its affordability and short running time, this method can be used in control campaigns to monitor the presence and spread of kdr alleles. The information provided here is relevant for the rational design of control strategies in the context of integrated vector management.

Pyrethroid genetic resistance in the dengue vector (Aedes aegypti) in Posadas, Argentina.

Pyrethroids are extensively used to control adult populations of the arboviral vector Aedes aegypti, raising concerns regarding the increasing frequency and distribution of insecticide resistance mutations (kdr: knock-down resistance) in the voltage-gated sodium channel gene (Nav). The widespread use of pyrethroids imposes a threat to the success of mosquito control and the environment. In this study, we investigated the presence of two kdr mutations (V1016I and F1534C) in the Nav gene and their distribution across four neighborhoods in Posadas, Argentina, with different Ae. aegypti abundance and contrasting socioeconomic status (SES). Alleles at each locus were interrogated using TaqMan SNP genotyping assays in DNA extracted from adult females collected in a longitudinal study. We report the presence of both pyrethroid resistance alleles (kdr 1016I = 29.08%; kdr 1534C = 70.70%) among adult females. The frequency of combined kdr genotypes reveals that approximately 70% of local adult females have enhanced resistance to pyrethroids. Both, the proportion of resistant adult females (with at least one kdr allele in each locus) and Ae. aegypti abundance showed an uneven distribution between neighborhoods with different SES (p < 0.001). In high-SES neighborhoods, we found more mosquitoes and a higher frequency of pyrethroid resistance, possibly as a consequence of different public health interventions, social habits, and insecticide use. This is the first report of kdr mutations in Ae. Aegypti in the northeast region of Argentina. Our results focus on the need for within-population (city) distribution analyses of kdr mutations and highlight the relevance of incorporating insecticide resistance monitoring within the Integrated Vector Management initiative.

Study of the Use of Permethrin 5% Cream in Galicia (Spain) between 2018 and 2021.

Drug utilization studies can provide direct insights into how a drug is used in real-world conditions and can give a rough estimate of the proportion of the study population treated with it. In the present work, we examined the consumption of permethrin 5% cream in the four provinces of Galicia (a Spanish autonomous community) and described the seasonal variability and the annual evolution of its consumption between 2018 and 2021. A descriptive, cross-sectional, and retrospective study of the consumption of this drug, expressed in defined daily dose per 1000 inhabitants per day (DID), was carried out. The results obtained revealed differences between the amounts consumed in the four Galician provinces (p < 0.001). No specific geographical pattern was observed; however, the results suggested a marked seasonality and a slightly increasing global trend in the consumption of permethrin 5% cream throughout the study period. Since the only authorized indication of this drug in the study area is the treatment of scabies, this work may give an idea of the epidemiological situation of the disease in Galicia and serve to establish public health strategies against this parasitosis.

Marginal risk of domestic vector-borne Trypanosoma cruzi transmission after improved vector control of Triatoma infestans across a rural-to-urban gradient in the Argentine Chaco.

The interruption of domestic vector-borne transmission of Trypanosoma cruzi in the Americas remains one of the main goals of the World Health Organization 2021-2030 road map for neglected tropical diseases. We implemented a longitudinal intervention program over 2015-2022 to suppress (peri)domestic Triatoma infestans in the municipality of Avia Terai, Chaco Province, Argentina and found that house infestation (3851 houses inspected) and triatomine abundance decreased over the first 2 years post-intervention (YPI), and remained stable thereafter associated to moderate pyrethroid resistant foci. Here we assessed selected components of transmission risk after interventions across the rural-to-urban gradient. We used multistage random sampling to select a municipality-wide sample of T. infestans. We examined 356 insects collected in 87 houses for T. cruzi infection using kDNA-PCR and identified their bloodmeal sources using an indirect ELISA. The overall prevalence of T. cruzi infection post-intervention was 1.7% (95% CI 0.7-3.6). Few houses (5.7%) (95% CI 2.5-12.8) harbored infected triatomines across the gradient. Infected triatomines were found in 5 peri­urban or rural dwellings over 1-4 years post-intervention. No infected insect was found in the urban area. The human blood index decreased from 66.2 at baseline to 42.8 at 1YPI and then increased to 92.9 at 4-5 YPI in the few infested domiciles detected. The percentage of houses with human-fed bugs displayed a similar temporal trend. Our results indicate marginal risks of domestic vector-borne transmission across the district after implementation of the intervention program. Ensuring sustainable vector surveillance coupled with human etiological diagnosis and treatment in hiperendemic areas like the Gran Chaco region, is urgently needed. 252 words.

Global Pattern of kdr-Type Alleles in Musca domestica (L.).

Purpose of Review: Houseflies, Musca domestica L., are an important sanitary pest that affects human and domesticated animals. They are mechanical carriers of more than 100 human and animal diseases including protozoan, bacterial, helminthic, and viral infections. Recently, it was demonstrated that houseflies acquired, harbored, and transmitted SARS-CoV-2 (COVID-19) for up to 1 day post-exposure. The most widely used control strategy relays on the application of pyrethroid insecticides due to their effectiveness, low mammalian toxicity, low cost, and environmental safety. The main mechanism of action of pyrethroids is to exert their toxic effects through affecting the voltage-sensitive sodium channel (VSSC) modifying the transmission of the nerve impulse and leading to the death of the insects. Target site insensitivity of the VSSC is due to the presence of single nuclear polymorphisms (SNPs) named knockdown mutations (kdr). In this review, we synthetize recent data on the type and distribution of these mutations globally. Recent Findings: Housefly resistance is reported in several countries. Increased applications of pyrethroids to control housefly populations led to the emergence of multiple evolutionary origins of resistance determined by five amino acid substitutions or specific mutations in the VSSC: kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L1014F), type N (D600N + M918T + L1014F), and 1B (T929I + L1014F). According to the global map obtained, high levels of resistance to pyrethroids are associated with the L1014F mutation found mostly in North America, Europe, and Asia, while the super-kdr mutation was mostly found in the American continent. The level of protection conferred by these alleles against pyrethroids was generally kdr-his < kdr < Type N ≤ super-kdr ≤ 1B. The relative fitness of the alleles under laboratory conditions was susceptible ≅ kdr-his > kdr > super-kdr suggesting that the fitness cost of an allele was relative to the presence of other alleles in a population and that the reversion of resistance in a free insecticide environment might be quite variable from one region to another. Summary: An adequate integrated pest management program should consider monitoring susceptibility to pyrethroids to detect early levels of resistance and predict the spread and evolution of resistant phenotypes and genotypes. From this review, the pyrethroid resistance status of housefly population was determined in very few countries and has evolved independently in different areas of the world affecting chemical control programs.

Esfenvalerate biodegradation by marine fungi is affected by seawater and emulsifier formulation.

Pesticides already were detected in the oceans, and their fates require evaluation in these environmental conditions. Therefore, marine-derived fungi were assessed for Esfenvalerate biodegradation, approaching the effects of seawater and use of commercial emulsifiable formulation. Residual pesticide and four metabolites were quantified. Furthermore, kinetics were determined for the three tested strains (Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, and Westerdykella sp. CBMAI 1679). These facultative marine fungi biodegraded up to 87 ± 2% of 100 mg L-1 Esfenvalerate in liquid media. However, Esfenvalerate biodegradation was faster in low salinity conditions than in artificial seawater. Moreover, rates of consumption were higher for Esfenvalerate in the pure form than for the commercial emulsifiable formulation. These results suggest that half-life of Esfenvalerate formulated with inert ingredients in seawater can have a double prolongation effect that can contribute to health and environmental issues.

Biodegradation of the Pesticides Bifenthrin and Fipronil by Bacillus Isolated from Orange Leaves.

The pyrethroid bifenthrin and the phenylpyrazole fipronil are widely employed insecticides, and their extensive use became an environmental issue. Therefore, this study evaluated their biodegradation employing bacterial strains of Bacillus species isolated from leaves of orange trees, aiming at new biocatalysts with high efficiency for use singly and in consortium. Experiments were performed in liquid culture medium at controlled temperature and stirring (32 °C, 130 rpm). After 5 days, residual quantification by HPLC-UV/Vis showed that Bacillus amyloliquefaciens RFD1C presented 93% biodegradation of fipronil (10.0 mg.L-1 initial concentration) and UPLC-HRMS analyses identified the metabolite fipronil sulfone. Moreover, Bacillus pseudomycoides 3RF2C showed a biodegradation of 88% bifenthrin (30.0 mg.L-1 initial concentration). A consortium composed of the 8 isolated strains biodegraded 81% fipronil and 51% bifenthrin, showing that this approach did not promote better results than the most efficient strains employed singly, although high rates of biodegradation were observed. In conclusion, bacteria of the Bacillus genus isolated from leaves of citrus biodegraded these pesticides widely applied to crops, showing the importance of the plant microbiome for degradation of toxic xenobiotics.

Genetic diversity and Kdr mutations of natural Aedes (Stegomyia) aegypti (Diptera: Culicidae) populations of Brazil

ABSTRACT Aedes (Stegomyia) aegypti is an important vector of dengue, yellow fever, chikungunya and Zika virus. It is well known that resistance monitoring and genetic diversity data help designing the vector control programs. This study aimed to evaluate resistance to pyrethroids (PYs) through the frequency of kdr mutations Val1016IIe and F1534C, and the genetic variation of the mitochondrial gene ND4 in six natural populations of A. aegypti from Paraná - Brazil. Adults were obtained from eggs collected from Alvorada do Sul, Marilena, Maringá, Nova Londrina, Paranavaí and São Carlos do Ivaí. From these adults, 345 were used to identify the 1016 and 1534 sites, and 120 were used to perform the ND4 gene analysis. The studied populations from Paraná showed PYs resistance, low gene flow and genetic diversity. Additionally, a relationship was observed among the haplotypes of populations from the Amazon and Southeastern Brazil, Peru, Mexico, and North America.

Anonaine from Annona crassiflora inhibits glutathione S-transferase and improves cypermethrin activity on Rhipicephalus (Boophilus) microplus (Canestrini, 1887).

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) is one of the most important ectoparasites of cattle, causing severe economic losses in tropical and subtropical regions of the world. The selection of resistance to the most commonly used commercial acaricides has stimulated the search for new products for tick control. The identification and development of drugs that inhibit key tick enzymes, such as glutathione S-transferase (GST), is a rational approach that has already been applied to other parasites than ticks. In this context, alkaloids such as anonaine display several biological activities, including an acaricidal effect. This study aimed to assess the specific inhibition of the R. microplus GST by anonaine, and analyze the effect on ticks when anonaine is combined with cypermethrin. For this purpose, a molecular docking analysis was performed using an R. microplus GST three-dimensional structure model with anonaine and compared with a human GST-anonaine complex. The absorption, distribution, metabolism, excretion, and toxicity properties of anonaine were also predicted. Then, for in vitro analyses, anonaine was isolated from Annona crassiflora (Martius, 1841) leaves. The inhibition of purified recombinant R. microplus GST (rRmGST) by anonaine and the effect of this alkaloid on cypermethrin efficacy towards R. microplus were assessed. Anonaine has a higher affinity to the tick enzyme than to the human enzyme in silico and has moderate toxicity, being able to inhibit, in vitro, rRmGST up to 37.5% in a dose-dependent manner. Although anonaine alone has no activity against R. microplus, it increased the cypermethrin effect on larvae, reducing the LC50 from 44 to 22 µg/mL. In conclusion, anonaine is a natural compound that can increase the effect of cypermethrin against R. microplus.

Morphophysiological alterations in fruit-eating bats after oral exposure to deltamethrin.

Deltamethrin (DTM) is a synthetic pyrethroid widely used in the cultivation and management of several crops due to its insecticidal action. Application to crops of pyrethroids such as DTM can result in the exposure of water and fruit consumed by fruit bats having a high pyrethroid content which may be harmful. Therefore the objective of this study was to evaluate the effects of short-term oral exposure of the fruit-eating bats (Artibeus lituratus) to two concentrations of DTM (0.02 and 0.04 mg/kg of papaya) on histopathology of the intestine, liver and kidney. The intestine of the animals exposed to both concentrations showed inflammatory infiltrate, degeneration, necrosis and goblet cell hyperplasia as the most frequent pathologies. Besides, the acid mucins showed an increase in the frequency of non-viable cells. The liver showed hepatocyte vacuolizatio and nuclear enlargement, as well as inflammatory infiltrate and steatosis. The kidneys of the exposed animals showed and inflammatory infiltrate, benign nephrosclerosis, vacuolization and necrosis. Also, DTM reduced nitric oxide synthesis, decreased glomerular diameter and increased glycogen percentage in the proximal tubules. Our results suggest that acute exposure to DTM at low concentrations has the potential to induce pronounced histopathological changes in vital organs, such as intestine, liver and kidney of fruit-eating bats.

Sublethal effects of a pyrethroid insecticide on cuticle thickness, wing size and shape in the main vector Triatoma infestans.

Exposure to sublethal doses of insecticide may affect biological traits in triatomines. We investigated the effects of toxicological phenotype (pyrethroid resistance status) and exposure to sublethal doses of deltamethrin on two traits of Triatoma infestans Klug (Heteroptera: Reduviidae) using a phenotypic plasticity experimental design. First-instar nymphs from 14 and 10 full-sib families from pyrethroid-susceptible and pyrethroid-resistant populations, respectively, were used. For the susceptible population, we treated first instars topically with acetone (control) or deltamethrin (treatment) once. For the resistant population, instars were treated once, twice and three times as first, third or fifth-instar nymphs, respectively. We measured cuticle thickness, wing size and wing shape of 484 emerging adults, and tested for treatment effects using mixed ANOVA and MANOVA models. Toxicological phenotype, exposure to deltamethrin and full-sib family exerted significant effects on cuticle thickness, wing size and wing shape. Adult triatomines previously treated with deltamethrin developed significantly thicker cuticles than control triatomines only in the resistant population and significantly bigger wings in both populations. Mean cuticle thickness and wing size increased with increasing exposures to deltamethrin. Exposure to sublethal doses of deltamethrin generated morphological modifications that may affect insect survival and flight dispersal, and hence may have evolutionary and epidemiological consequences.