What happens when the insecticide does not kill? A review of sublethal toxicology and insecticide resistance in triatomines.

Chagas disease is considered one of the most important human parasitosis in the United States. This disease is mainly transmitted by insects of the subfamily Triatominae. The chemical vector control is the main tool for reducing the incidence of the disease. However, the presence of triatomines after pyrethroids spraying has been reported in some regions, as in the case of Triatoma infestans in Argentina and Bolivia. The presence of insects can be explained by the colonization from neighbouring areas, the reduction of insecticide dose to sublethal levels due to environmental factors, and/or by the evolution of insecticide resistance. In the last two scenarios, a proportion of the insects is not killed by insecticide and gives rise to residual populations. This article focuses on the toxicological processes associated with these scenarios in triatomines. Sublethal doses may have different effects on insect biology, that is, sublethal effects, which may contribute to the control. In addition, for insect disease vectors, sublethal doses could have negative effects on disease transmission. The study of sublethal effects in triatomines has focused primarily on the sequence of symptoms associated with nervous intoxication. However, the effects of sublethal doses on excretion, reproduction and morphology have also been studied. Rhodnius prolixus and T. infestans and pyrethroids insecticides were the triatomine species and insecticides, respectively, mainly studied. Insecticide resistance is an evolutionary phenomenon in which the insecticide acts as a selective force, concentrating on the insect population's pre-existing traits that confer resistance. This leads to a reduction in the susceptibility to the insecticide, which was previously effective in controlling this species. The evolution of resistance in triatomines received little attention before the 2000s, but after the detection of the first focus of resistance associated with chemical control failures in T. infestans from Argentina in 2002, the study of resistance increased remarkably. A significant number of works have studied the geographical distribution, the resistance mechanisms, the biological modifications associated with resistance, the environmental influences and the genetic of T. infestans resistant to pyrethroid insecticides. Currently, studies of insecticide resistance are gradually being extended to other areas and other species. The aim of this article was to review the knowledge on both phenomena (sublethal effects and insecticide resistance) in triatomines. For a better understanding of this article, some concepts and processes related to insect-insecticide interactions, individual and population toxicology and evolutionary biology are briefly reviewed. Finally, possible future lines of research in triatomine toxicology are discussed.

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.

Resistance to deltamethrin in Triatoma infestans: microgeographical distribution, validation of a rapid detection bioassay and evaluation of a fumigant canister as control alternative strategy.

Triatoma infestans (Klug) (Hemiptera: Reduviidae) is the main vector of Chagas disease in the Southern Cone of America and resistance to pyrethroid insecticides has been detected in several areas from its geographical distribution. Pyrethroid resistance presents a complex geographical pattern at different spatial scales. However, it is still unknown if the toxicological variability is a common feature within villages of the Gran Chaco were high resistance was descripted. The objectives of this study were to determine: (a) the microgeographical distribution of the deltamethrin-resistance in insects from Pampa Argentina village, (b) the performance of the insecticide impregnated paper bioassay to evaluate deltamethrin-resistance in field collected insects and (c) the lethal activity of the fumigant canister containing DDVP against insects resistant to deltamethrin. High survival of T. infestans exposed to discriminant dose was observed in the samples of all the evaluated dwellings, suggesting that the resistance to deltamethrin is homogeneous at the microgeographical level. Resistance determination by impregnated paper bioassay was similar to traditional topical determination, highlighting the use of this rapid methodology in field large-scale monitoring. The fumigant canister was not effective against resistant insects, remarking the need to develop suitable formulations that ensure minimal toxicological risk and high effectivity.

Genetic structure of deltamethrin-resistant populations of Triatoma infestans (Hemiptera: Reduviidae) in the Gran Chaco.

The genetic structure of natural populations offers insight into the complexities of their dynamics, information that can be relevant to vector control strategies. Microsatellites are useful neutral markers to investigate the genetic structure and gene flow in Triatoma infestans, one of the main vectors of Chagas disease in South America. Recently, a heterogeneous pyrethroid-resistant hotspot was found in the Argentine Gran Chaco, characterized by the highest levels of deltamethrin resistance found at the present time. We applied population genetics analyses to microsatellite and village data and search for associations between the genetic variability and the heterogeneous toxicological pattern previously found. We genotyped 10 microsatellite loci in 67 T. infestans from 6 villages with no, low, and high pyrethroid resistance. The most genetically diverse populations were those susceptible or with low values of resistance. In contrast, high-resistance populations had lower herozygosity and some monomorphic loci. A negative association was found between variability and resistant ratios. Global and pairwise FSTs indicated significant differentiation between populations. The only susceptible population was discriminated in all the performed studies. Low-resistance populations were also differentiated by a discriminant analysis of principal components (DAPC) and were composed mostly by the same two genetic clusters according to STRUCTURE Bayesian algorithm. Individuals from the high-resistance populations were overlapped in the DAPC and shared significant proportions of a genetic cluster. These observations suggest that the resistant populations might have a common origin, although more genetic markers and samples are required to test this hypothesis more rigorously.

Lethal activity of individual and mixed monoterpenoids of geranium essential oil on Musca domestica.

Plant essential oils and its constituent molecules have been suggested as an alternative to control insect. The contribution of the constituents to the effect of the oil is determined by the interactions occurring between them. Synergistic interactions would improve the insecticide efficacy of the compounds due to the utilization of lower doses. We evaluated the insecticidal activity of geranium (Geranium maculatum L.) oil and its major constituents against Musca domestica L. and studied the toxic interactions in artificial mixtures of those constituents in the natural ratio. While synergistic interactions were determined in house fly in this study, these were of low intensity evidencing that the effect of each constituent was slightly modified by the other constituents present in the mixtures. The search for synergism between components is a strategy to improve the insecticide activity of natural compounds. The synergism helps to reduce the environmental and toxicological impact due to the reduction of the dose of use.

Morphometric study of the legs of the main Chagas vector, Triatoma infestans (Hemiptera: Reduviidae).

In triatomines, vectors of Chagas disease, active dispersal takes place by walking and flying. Flight has received more attention than walking although the last is the dispersal modality used by nymphs due to their lack of wings and also used by adults, which would facilitate the colonization and reinfestation of houses after vector control actions. The present work studied the morphometrical variation of Triatoma infestans legs, the main vector of Chagas disease the Southern Cone of South America. We described morphometric traits and the natural variation of each leg segment. Different linear, size and shape variables of each component of the three right legs of fifth instar nymphs of T. infestans were analyzed using morphometric tools. We analyzed differentiation, variation and correlation for each segment across the fore-, mid and hind legs using different statistical approaches such as general linear model, canonical variates analysis, test of equality of coefficient of variation and partial least square analysis. We also analyzed variation and correlation between segments within each leg with partial least square and morphometric disparity analyses. Our results showed that the segments differed between legs, as general trends, the dimensions (length, width and/or size) were greater in the hind legs, smaller in the forelegs and intermediate in the mid ones. The femur and tibia (length and/or width) showed differences in morphometric variation between legs and the femur and tibia showed the highest levels of correlation between legs. On the other hand, in the fore- and mid legs, the femur (length or width) showed similar variation with tibia and tarsus lengths, but in the hind legs, the femur showed similar variation with all segments and not with the tibia length, and there were strong correlations between linear measurement within each leg. Our results suggest that the femur and tibia could play a determining role in the coordination between the legs that determines the walking pattern. Considering that these segments would also be linked to the specific function that each leg has, this study suggests a preponderant role of the femur and tibia in the walking locomotion of T. infestans.

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.

Comparative efficacy of commercial combs in removing head lice (Pediculus humanus capitis) (Phthiraptera: Pediculidae).

The use of a fine comb for removing lice from the head of the human host is a relevant tool both in the diagnosis of infestations and as part of an integrated control strategy of head lice. The effectiveness of a fine comb depends, in part, on the design and material they are built. The aim of this study was to compare in vivo the efficacy of metal and plastic combs that are currently used in the removal of head lice and eggs worldwide. The space between comb teeth and the length was 0.23 and 13 mm in KSL® plastic, 0.3 and 10.7 mm in NOPUCID® plastic, 0.15 and 31 mm in KSL® metal and 0.09 and 37 mm in ASSY® metal. The assays were performed comparing the combs in pairs: (a) KSL® vs. NOPUCID® plastic combs, (b) KSL® vs. ASSY® metal combs and (c) KSL® plastic comb vs. ASSY® metal comb. The most effective plastic comb was KSL®, removing a higher number of individuals of all stages. The most effective metal comb was ASSY®, removing more insects of all stages (except adults). The comparative test between KSL® plastic and ASSY® metal showed that ASSY® was the most effective in removing head lice and their eggs.

Can infection with Trypanosoma cruzi modify the toxicological response of Triatoma infestans susceptible and resistant to deltamethrin?

Chemical control plays a central role in interrupting the vector transmission of Chagas disease. In recent years, high levels of resistance to pyrethroids have been detected in the main vector Triatoma infestans, which were associated with less effectiveness in chemical control campaigns in different regions of Argentina and Bolivia. The presence of the parasite within its vector can modify a wide range of insect physiological processes, including toxicological susceptibility and the expression of resistance to insecticides. This study examined for the first time the possible effects of Trypanosoma cruzi infection on susceptibility and resistance to deltamethrin in T. infestans. Using WHO protocol resistance monitoring assays, we exposed resistant and susceptible strains of T. infestans, uninfected and infected with T. cruzi to different concentrations of deltamethrin in fourth-instar nymphs at days 10-20 post-emergence and monitored survival at 24, 48, and 72 h. Our findings suggest that the infection affected the toxicological susceptibility of the susceptible strain, showing higher mortality than uninfected susceptible insects when exposed to both deltamethrin and acetone. On the other hand, the infection did not affect the toxicological susceptibility of the resistant strain, infected and uninfected showed similar toxic responses and the resistance ratios was not modified. This is the first report of the effect of T. cruzi on the toxicological susceptibility of T. infestans and triatomines in general and, to our knowledge, one of the few on the effect of a parasite on the insecticide susceptibility of its insect vector.

Toxicity of non-pyrethroid insecticides against Triatoma infestans (Hemiptera: Reduviidae).

Triatoma infestans (Klug) is the main vector of Chagas disease, which is a public health concern in most Latin American countries. The prevention of Chagas disease is based on the chemical control of the vector using pyrethroid insecticides. In the last decade, different levels of deltamethrin resistance have been detected in certain areas of Argentina and Bolivia. Because of this, alternative non-pyrethroid insecticides from different chemical groups were evaluated against two T. infestans populations, NFS and El Malá, with the objective of finding new insecticides to control resistant insect populations. Toxicity to different insecticides was evaluated in a deltamethrin-susceptible and a deltamethrin-resistant population. Topical application of the insecticides fenitrothion and imidacloprid to first nymphs had lethal effects on both populations, producing 50% lethal dose (LD50) values that ranged from 5.2-28 ng/insect. However, amitraz, flubendiamide, ivermectin, indoxacarb and spinosad showed no insecticidal activity in first instars at the applied doses (LD50 > 200 ng/insect). Fenitrothion and imidacloprid were effective against both deltamethrin-susceptible and deltamethrin-resistant populations of T. infestans. Therefore, they may be considered alternative non-pyrethroid insecticides for the control of Chagas disease.

Comparative efficacy of new commercial pediculicides against adults and eggs of Pediculus humanus capitis (head lice).

The use of pyrethroids to control head louse infestations have suffered considerable loss of efficacy due to the development of resistance. In the last past years, several new alternative products to synthetic pyrethroids have been developed and are sold in the Argentinean market against head lice. The present study investigated the efficacy of two new Argentinean products Nopucid Qubit® and Nopucid Bio Citrus® and its comparison with two reference products Nyda® and Hedrin®. Nopucid Qubit® is a two-phase lotion containing geraniol and citronellol (phase 1) and ciclopentaxiloxane (phase 2); while Nopucid Bio Citrus® contains dimethicone, ciclopentaxiloxane, and bergamot essential oil. These products are physically acting compounds. The sensitivity of two laboratory assays for testing insecticide activity of new formulations was also compared. Mortality (100%) of motile forms occurred after they were exposed to any product for 1 and 2 min, either by in vitro or ex vivo test. Concerning ovicidal activity, the most effective pediculicides were Nopucid Bio Citrus® and Nyda®, followed by Hedrin® and Nopucid Qubit®. The present study revealed, for the first time, the efficacy of over-the-counter commercial pediculicides available in Argentine (Nopucid Bio Citrus® and Nopucid Qubit®) on either motile stages or eggs against head lice.

Transcriptomic modulation in response to an intoxication with deltamethrin in a population of Triatoma infestans with low resistance to pyrethroids.

BACKGROUND: Triatoma infestans is the main vector of Chagas disease in the Southern Cone. The resistance to pyrethroid insecticides developed by populations of this species impairs the effectiveness of vector control campaigns in wide regions of Argentina. The study of the global transcriptomic response to pyrethroid insecticides is important to deepen the knowledge about detoxification in triatomines. METHODOLOGY AND FINDINGS: We used RNA-Seq to explore the early transcriptomic response after intoxication with deltamethrin in a population of T. infestans which presents low resistance to pyrethroids. We were able to assemble a complete transcriptome of this vector and found evidence of differentially expressed genes belonging to diverse families such as chemosensory and odorant-binding proteins, ABC transporters and heat-shock proteins. Moreover, genes related to transcription and translation, energetic metabolism and cuticle rearrangements were also modulated. Finally, we characterized the repertoire of previously uncharacterized detoxification-related gene families in T. infestans and Rhodnius prolixus. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the detoxification response in vectors of Chagas disease. Given the absence of an annotated genome from T. infestans, the analysis presented here constitutes a resource for molecular and physiological studies in this species. The results increase the knowledge on detoxification processes in vectors of Chagas disease, and provide relevant information to explore undescribed potential insecticide resistance mechanisms in populations of these insects.

Phenotypic plasticity, canalisation and developmental stability of Triatoma infestans wings: effects of a sublethal application of a pyrethroid insecticide.

BACKGROUND: Triatomine control campaigns have traditionally consisted of spraying the inside of houses with pyrethroid insecticides. However, exposure to sublethal insecticide doses after the initial application is a common occurrence and may have phenotypic consequences for survivors. Here, using Triatoma infestans (the main vector of Chagas disease in the Southern Cone of South America) as a model species, we quantified the effects of exposure to a sublethal dose of pyrethroid insecticide on wing morphology. We tested if the treatment (i) induced a plastic effect (change in the character mean); (ii) altered environmental canalisation (higher individual variation within genotypes); (iii) altered genetic canalisation (higher variation among genotypes); and (iv) altered developmental stability (higher fluctuating asymmetry [FA]). METHODS: Each of 25 full-sib families known to be susceptible to pyrethroid insecticides were split in two groups: one to be treated with a sublethal dose of deltamethrin (insecticide-treated group) and the other to be treated with pure acetone (control group). Wings of the emerging adults were used in a landmark-based geometric morphometry analysis to extract size and shape measurements. Average differences among treatments were measured. Levels of variation among families, among individuals within families and among sides within individuals were computed and compared among treatments. RESULTS: Wing size and shape were affected by a sublethal dose of deltamethrin. The treated insects had larger wings and a more variable wing size and shape than control insects. For both wing size and shape, genetic variation was higher in treated individuals. Individual variations and variations in FA were also greater in deltamethrin-treated insects than in control ones for all full-sib families; however, the patterns of shape variation associated with genetic variation, individual variation and FA were different. CONCLUSIONS: Insects exposed to a sublethal dose of deltamethrin presented larger, less symmetrical and less canalised wings. The insecticide treatment jointly impaired developmental stability and genetic and environmental canalisation. The divergent patterns of shape variation suggest that the related developmental buffering processes differed at least partially. The morphological modifications induced by a single sublethal exposure to pyrethroids early in life may impinge on subsequent flight performance and consequently affect the dynamics of house invasion and reinfestation, and the effectiveness of triatomine control operations.

Response of Pediculus humanus humanus (Pediculidae: Phthiraptera) to water or 70% ethanol immersion and determination of optimal times for measuring toxic effects.

Human pediculosis is caused by Pediculus humanus humanus (Linnaeus 1758) and Pediculus humanus capitis (De Geer 1767). We studied the response of body lice to immersion in water and ethanol 70% and determined the optimal times for measuring knockdown and mortality. After immersion in water, all lice remained alive from 5 min to 22 h for both times of exposure. A low proportion of lice were affected after 2 min of immersion in ethanol in the 10-min exposure test, but recovered completely after 5 min. Different proportions of lice were affected between 2 and 7 h after immersion in ethanol, depending on the immersion time. However, a high proportion of lice recovered after 22 h. The results suggest that the optimal times for measuring early knockdown effects of insecticides are the 5-min to 7-h interval for water and 5-min to 1-h interval for ethanol. On the other hand, the best time for measuring mortality is 22 h after immersion. These results should improve the interpretations of the effects of pediculicides in immersion bioassays.

Behavioral response mediated by feces in Triatoma infestans (Hemiptera: Reduviidae: triatominae) susceptible and resistant to deltamethrin.

Chemical cues from feces promote aggregation behavior in Triatoma infestans nymphs and adults. Given the importance of T. infestans resistant to pyrethroids in several areas of Argentina and Bolivia, it would be important to know if there is an association with specific attraction and aggregation behaviors. These behaviors, to and surrounding refuges, play an important role in triatomine population dynamics, an important factor to consider and model for vector control strategies. The aim of the present study was to analyze the behavior of orientation to chemical signals emitted by feces from deltamethrin resistant (R) and susceptible (S) T. infestans. The behavioral assays were performed in a circular glass arena divided in two equal sectors. Fecal signals emitted by both S and R feces are attractants to fifth-instar nymphs of both S and R populations. Both toxicological phenotypes remained significantly longer on R feces, as compared to S feces. This is the first evidence in a triatomine, for the association of an aggregation behavior and insecticide resistance and may be the result of pleiotropic effects surrounding resistance genes.

Insecticide Resistance Mechanisms in Triatoma infestans (Reduviidae: Triatominae): The Putative Role of Enhanced Detoxification and Knockdown Resistance (kdr) Allele in a Resistant Hotspot From the Argentine Chaco.

Chagas disease affects around 6 million people in the world, and in Latin America, it is mainly transmitted by the kissing bug. Chemical control of the vector with pyrethroid insecticides has been the most frequently used tool to reduce the disease incidence. Failures of field control have been detected in areas of the Argentinian Gran Chaco that correlate with high levels of insecticide resistance. Here, we provide evidence of the mechanisms involved in the resistance to insecticides of field populations of T. infestans from General Güemes Department (Chaco Province, Argentina). The biochemical analysis suggests the increase in the activity of the degradative enzymes P450 oxidases and esterases as a minor contributive mechanism in low-resistance populations. The molecular study revealed high frequencies of the kdr L925I mutation at the voltage-gated sodium channel as responsible for the high resistance ratios detected. This knowledge contributes to the generation of comprehensive vector control strategies that reduce the incidence of the disease.

Interaction between two aggregation chemical signals in Triatoma infestans (Hemiptera: Reduviidae).

The nymphs and adults of Triatoma infestans spend much of their time aggregated among themselves within narrow and dark shelters. The search for a suitable shelter depends in part on the recognition of chemical signals coming from the feces and the cuticle of the other individuals who use the refuge. The aim of this study was determine the possible interaction between the chemical signals associated to the feces and to the cuticle of T. infestans. The results showed that the insects remained significantly more time on the feces that had contact with legs and the feces plus footprints than feces or footprints alone, demonstrating the interaction between evaluated signals. These results demonstrates also that feces extracted a chemical stimulus from the legs. Understanding the interaction feces-legs as an interaction feces-cuticle of legs, the results suggest that the feces could extract some cuticular compound with activity on the behavior of the insects. This is the first report of the interaction between the two aggregation signals recognized in T. infestans and of the increase in the behavioral response of insects exposed to feces that had contact with a cuticular structure.

Fluctuating asymmetry and exposure to pyrethroid insecticides in Triatoma infestans populations in northeastern Argentina

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.

Transcriptomic modulation in response to an intoxication with deltamethrin in a population of Triatoma infestans with low resistance to pyrethroids

Background Triatoma infestans is the main vector of Chagas disease in the Southern Cone. The resistance to pyrethroid insecticides developed by populations of this species impairs the effectiveness of vector control campaigns in wide regions of Argentina. The study of the global transcriptomic response to pyrethroid insecticides is important to deepen the knowledge about detoxification in triatomines. Methodology and findings We used RNA-Seq to explore the early transcriptomic response after intoxication with deltamethrin in a population of T. infestans which presents low resistance to pyrethroids. We were able to assemble a complete transcriptome of this vector and found evidence of differentially expressed genes belonging to diverse families such as chemosensory and odorantbinding proteins, ABC transporters and heat-shock proteins. Moreover, genes related to transcription and translation, energetic metabolism and cuticle rearrangements were also modulated. Finally, we characterized the repertoire of previously uncharacterized detoxification-related gene families in T. infestans and Rhodnius prolixus. Conclusions and significance Our work contributes to the understanding of the detoxification response in vectors of Chagas disease. Given the absence of an annotated genome from T. infestans, the analysis presented here constitutes a resource for molecular and physiological studies in this species. The results increase the knowledge on detoxification processes in vectors of Chagas disease, and provide relevant information to explore undescribed potential insecticide resistance mechanisms in populations of these insects.

Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families.

BACKGROUND: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas' disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas' disease. METHODS AND FINDINGS: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas' disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. CONCLUSIONS AND SIGNIFICANCE: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas' disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms.