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. 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. 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.
Triatomines are hematophagous insects of the order Hemiptera, family Reduviidae, which are widely distributed throughout the Americas. This insect subfamily includes the vectors of Trypanosoma cruzi, the causative agent of Chagas' disease affecting around 8 million people in Central and South America [1]. The most important vector species are Triatoma infestans in the South Cone (especially in Argentina and Bolivia) and Triatoma dimidiata in Northern South America (Colombia, Venezuela, Ecuador and Peru), Central America and Mexico. Rhodnius prolixus (also present in those countries of the North of South America and Central America) and Triatoma pallidipennis in Mexico can also transmit T. cruzi [1]. Because there is still no available vaccine or effective treatment for the chronic stage of the disease, vector control remains the main method to reduce the risk of infection. Vector control efforts have been based mainly on the application of pyrethroid insecticides, achieving an important reduction in the geographic range and level of triatomine infestation of human dwellings [2–4]. However, in the Gran Chaco region, a 1. 1 million km2 ecoregion covering part of Argentina, Bolivia and Paraguay, the elimination of triatomines failed despite intensive use of pyrethroids [5,6]. Resistance to pyrethroids seems to be an important factor for the persistence of vectorial transmission of Chagas' disease in the Gran Chaco [5]. The detection of resistant populations of T. infestans confirmed the potential of triatomines to develop high levels of resistance (with observed resistance ratios from 35 to >1000 when compared with a sensitive strain [6]), contradicting initial assumptions [7,8]. The main cause of the extremely high levels of resistance in T. infestans seems to be the appearance of kdr mutations affecting the target site of pyrethroids, the voltage-gated sodium channel [6,9–11]. We have observed that T. infestans resistant populations having the same kdr mutation at similar frequencies present very different resistance ratios [6]. These data suggest that other mechanisms could be involved in triatomine insecticide resistance. Indeed, metabolic resistance [12,13] and a decreased cuticular penetration [14] have already been reported in particular T. infestans resistant populations, supporting this hypothesis. In insects, detoxification mechanisms transform toxins to non-toxic molecules through Phase I and Phase II reactions. Phase I includes oxidation, reduction and hydrolysis, whereas Phase II occurs through enzyme mediated conjugated reactions to convert the primary products of Phase I into water soluble derivatives, which can be excreted [15]. Genes encoding detoxification enzymes include members of Phase I (Cytochromes P450-CYPs and Carboxyl/Cholinesterases-CCEs), and Phase II (Glutathione Transferases-GSTs) categories, forming broad gene superfamilies. Specific groups of genes within these superfamilies can mediate metabolic resistance to insecticides by means of alterations in their activity [16–18]. Besides their role in detoxification, members of GST, CYP and CCE superfamilies are involved in biosynthesis of endogenous compounds and degradation of odor molecules [19,20]. Here we present a comprehensive study of triatomine detoxification gene superfamilies, discovered in normalized transcriptomes of three epidemiologically relevant triatomine vector species: T. infestans, T. dimidiata and T. pallidipennis. Additionally, we have data-mined the R. prolixus genomic sequences to identify detoxification-related superfamilies and have characterized the expression of these superfamilies in different tissue-specific transcriptomes from R. prolixus. As insect antennae are crucial organs in the detection and processing of olfactory stimuli, we analyzed the expression patterns of GSTs, CYPs and CCEs in three antennal transcriptomes from 5th instar larvae, male and female adults of R. prolixus, with the objective of identifying potential odor/pheromone degrading enzymes. The dramatic physiological alterations triggered by blood feeding in triatomines are mostly regulated by the central nervous system (CNS) [21–23], which is also the target of all the neurotoxic insecticides used. Hence, we studied the changes in expression patterns observed for detoxification gene superfamilies in the CNS of male and female adults of R. prolixus at four different feeding conditions (unfed, 1,4 and 24 hours post blood meal). Finally, we analyzed R. prolixus tissue-specific libraries from anterior and posterior midgut, hindgut [24], Malpighian tubules, fat body, gonads and whole body to obtain transcriptomic information of the detoxification gene superfamilies in those tissues. These libraries were constructed by Oliveira et al. and their sequencing output is publicly available at http: //rhodnius. iq. ufrj. br. The results presented here give a comprehensive panorama of triatomine detoxification genes and their expression in different tissues and under different physiological conditions, constituting an important platform for further physiological hypotheses and experimentation.
Pigeons used in this study were housed, cared, fed and handled in accordance with resolution 1047/2005 (National Council of Scientific and Technical Research, CONICET) regarding the national reference ethical framework for biomedical research with laboratory, farm, and nature collected animals (Marco Ético de Referencia para las Investigaciones Biomédicas en Animales de Laboratorio, de Granja y Obtenidos de la Naturaleza), which is in accordance with the standard procedures of the Office for Laboratory Animal Welfare, Department of Health and Human Services, NIH and the recommendations established by the 2010/63/EU Directive of the European Parliament, related to the protection of animals used for scientific purposes. Biosecurity considerations are in agreement with CONICET resolution 1619/2008, which is in accordance with the WHO Biosecurity Handbook (ISBN 92 4 354 6503). The collection of insects in dwellings was performed in agreement with the Argentinean National Health Ministry ethic requirements. The Roche 454 reads for T. dimidiata, T. infestans and T. pallidipennis were submitted to NCBI Sequence Read Archives (SRA) under the Bioproject ID number PRJNA304741 (SRA numbers: SAMN04317638 for T. infestans; SAMN04317639 for T. dimidiata and SAMN04317640 for T. pallidipennis). For reasons of legibility, we assigned an abbreviated name for each sequence in our manuscript. The correspondence between contig and assigned name is presented in Supplementary S1 File. All Triatoma spp. protein sequences of contigs used to construct the phylogenetic trees are available in Supplementary S2 File. In order to identify genes involved in insecticide resistance we used as queries the PFAM domains [25] of GSTs (PF02798 and PF00043), CYPs (PF00067) and CCEs (PF00135) to perform BLAST searches [26] on the predicted transcriptome of R. prolixus (RproC3. 1 version; available at www. vectorbase. org) and the transcriptomes of T. infestans, T. dimidiata and T. pallidipennis (Martínez-Barnetche et al. , manuscript in preparation). The microsomal GSTs of Triatoma spp. were identified using the orthologues from R. prolixus [27], Drosophila melanogaster, Anopheles gambiae, Apis mellifera and Acyrthosiphon pisum. Briefly, we generated normalized libraries with the kit Mint-2 (Evrogen, Moscow, Russia) using RNA extracted from eggs, unfed and fed insects from larvae (1st to 5th instar) and adults (males and females) of T. infestans, T. dimidiata and T. pallidipennis. Libraries were sequenced on the platform GS FLX+ (454-ROCHE, Basel, Switzerland) and the quality-trimmed reads were subjected to de novo assembly with the GS DeNovo assembler v. 2. 8 software in cDNA mode, using the default parameters. Completeness assessment of the transcriptomes for Triatoma spp. was evaluated searching the Core Eukaryotic Genome Dataset (CEGMA) [28] and the Benchmarking Universal Single Copy Orthologues (BUSCO) [29]. The estimated coverages were between 85. 8% (BUSCO) and 94. 3% (CEGMA) for T. dimidiata; 82. 2% (BUSCO) and 90. 6% (CEGMA) for T. infestans and 86. 9% (CEGMA) and 95. 2% (BUSCO) for T. pallidipennis. Following the same approach, the completeness of the R. prolixus genome was between 97. 0% (BUSCO) and 97. 8% (CEGMA). After BLAST searches, the non-redundant resulting sequences were used to perform an InterProScan search [30] using the Gene3d, PfamA and SuperFamily applications to obtain the protein sequences of interest. After this step, additional tBLASTn searches on the National Center for Biotechnology Information (NCBI) non redundant (nr) database were performed to assess for false positives. In those cases where two or more proteins were obtained from one transcript due to insertions or deletions during sequencing, they were manually reconstructed into a single transcript using as references homologue protein sequences from other insects. Prediction of the subcellular localization for each protein was performed with EuK-mPLoc 2. 0 [31–33] (Supplementary S1 File). The number and location of the transmembrane domains were predicted using the online server TMHMM Server v. 2. 0 [34] (Supplementary S1 File). The protein motif search was performed online using MOTIF (http: //www. genome. jp/tools/motif/) (Supplementary S1 File). We compared protein sequences related to detoxification superfamilies identified in the R. prolixus genome and Triatoma spp. transcriptomes with orthologues previously published for D. melanogaster and other insect species [18,35–41]. Phylogenies for each protein family were based on sequence alignments generated by CLUSTAL Ω [42] using the corresponding Hidden Markov Model for each PFAM superfamily domain as an external profile. Phylogenetic trees were inferred with the software BEAST v1. 8. 3 [43] in the CIPRES Science Gateway [44]. Beauti v1. 8. 3 [43] was used to generate the BEAST input files. Two independent chains were run with the same settings until convergence was achieved (effective sample size values >200), as calculated in the program Tracer v1. 6. 0 (http: //beast. bio. ed. ac. uk/Tracer). To achieve convergence, the number of generations used for each run varied between 5 to 30 million, depending on the tree. Each pair of runs was combined using LogCombiner v1. 8. 3 [43] discarding the first 10% of each chain as a burn-in. The maximum clade credibility tree was generated using TreeAnnotator v1. 8. 3 [43]. The results were visualized with iTol online tool [45]. To maintain the legibility of the figures, the phylogenetic analysis included only D. melanogaster as a reference, and the analysis including more insect species is presented as Supplementary S4 File. Filtered and trimmed Illumina reads for the R. prolixus CNS libraries were obtained from Ons et al. 2016 [46]. Subsequently, these reads and R. prolixus genome sequences (assembly version RproC3. 13) were mapped to the annotated genome assembly by means of TopHat v. 2. 0. 11 [47]. Using mapped read information, a transcriptome assembly and the corresponding gene expression levels (expressed as Fragments Per Kilobase of transcript per Million mapped reads or FPKM) were obtained for each library using Cufflinks v. 2. 1. 0 [47]. Cuffmerge [47] was used for joining the predicted transcriptome assemblies and the annotated genome GTF file. Finally, Cuffdiff [47] allowed comparing FPKM values for each annotated transcript in the different conditions. In the case of R. prolixus antennae, FPKM values were obtained from Latorre-Estivalis et al. 2017 [48]. Heat maps showing gene expression (presented as Log10 FPKM +1) of the target genes in the CNS from pooled male and female R. prolixus adult insects at different times post-blood meal (unfed as basal condition, 1,4 and 24 hours post-feeding) and in the antennae (including 5th instar larvae, female and male adult libraries) were prepared using R package ggplot2 (www. ggplo2. org). For tissue-specific expression analysis, we used public transcriptomes from anterior midgut, fat body, Malpighian tubules, posterior midgut, ovary, rectum, testes and whole body (see [24] for a detailed description on the construction of these transcriptomes). We performed local tBLASTn [26] to correlate the assembled contigs from those transcriptomes to an ID number in v3. 1 of R. prolixus gene set. For this, we used a database containing all the assembled contigs (http: //rhodnius. iq. ufrj. br), and the sequences of detoxification superfamilies of R. prolixus as queries. We analyzed the New-S1-Full. xlsx spreadsheet (available at http: //rhodnius. iq. ufrj. br/), to determine the number of reads for every detoxification-related transcript in each tissue. This analysis allowed the estimation of the tissue-specific expression pattern for each transcript and helped to obtain insights about their relative expression comparing different structures. Insects—Field insects of T. infestans were collected from infested houses in El Juramento, an Argentinean population with a resistance ratio higher than 2,000 and with presence of the L925I kdr mutation. Sensitive laboratory population of T. infestans (without exposure to insecticides for more than 30 generations) from the Reference Center for Vectors of the Ministry of Public Health of Argentina was used as a reference. The two populations analyzed are sensitive to fenitrothion (for a detailed description on sensitive and resistant strains used see [6]). qRT-PCR—Unfed 5th instar larvae of the reference and resistant T. infestans strains were used for transcript abundance comparisons. From each insect, complete CNS, anterior and posterior midgut, rectum, Malpighian tubules and fat body from the abdomen (separated from tracheae and other tissues) were dissected and pooled in a microtube containing Trizol (Ambion, Sao Paulo, Brazil); this reagent was also used for total RNA extraction, according to the manufacturer' s instructions. A total of 1 μg of RNA was treated with DNAseI (Promega, Wisconsin, USA) and used to synthesize cDNA by means of M-MLV Reverse Transcriptase kit (Promega). cDNA amplifications of T. infestans were performed on triplicate in a 25 μL final volume (primers detailed in Supplementary S3 File). α-Tubulin, β-Actin and G6PDH were used as housekeeping genes; they were previously reported as stable genes for qRT-PCR experiments in R. prolixus [49]. The program used in the amplifying reaction was 95°C for 5 min, and 39 cycles of 95°C for 30 sec; 58°C for 30 sec and 72°C for 30 sec in the Mini Opticon Real-Time PCR Detector Separate MJR (Bio-Rad Laboratories, California, USA). Real-time data were collected through the CFX Manager 3. 0 software (Bio-Rad Laboratories). Results were statistically analyzed using One-Way ANOVA.
CYPs are critically involved in metabolic processes such as the biosynthetic pathways of endogenous signaling molecules (e. g. , 20-hydroxyecdysone and juvenile hormone), the degradation of pheromones and the hydroxylation of fatty acids [19]. They are also critically implicated in the detoxification response to xenobiotics, including synthetic insecticides, and are fundamental factors in the adaptation of insects to chemical stress. Insect CYP genes fall into four major clades: CYP2, CYP3, CYP4 and mitochondrial clade. Each one of these clades are further subdivided in several families and subfamilies [19]. New families belonging to clades CYP3 and CYP4 were recently identified in R. prolixus [27]. Our analysis confirmed that, with exception of CYP3096, these new families are also present in other triatomine species (Table 1; Fig 1 and Supplementary S4 File). We detected a total of 87 CYP transcripts in T. dimidiata, 94 in T. infestans, 113 in T. pallidipennis and 119 in R. prolixus, these numbers being higher than those found in A. pisum (64), D. melanogaster (88), and A. mellifera (46) (Table 1). CCEs hydrolyze carboxyl esters to alcohols and acids. They are subdivided in three major classes according to their function: i) the dietary class, containing esterases that metabolize a broad range of substrates including insecticides and xenobiotics from the diet; ii) the hormone and pheromone degrading class, including juvenile hormone esterases and pheromone degrading CCEs; and iii) the neuro/developmental group, containing proteins implicated in neuro/developmental functions as acetylcholinesterases (AChEs), neuroligins, neurotactins, gliotactins and others [18]. AChEs are the only catalytically active component of the neuro/developmental class [18,40]. R. prolixus seems to present an expansion in the number of CCE genes (61) compared with Triatoma spp. (19 in T. infestans, 27 in T. dimidiata and 18 in T. pallidipennis) and the other insect genomes analyzed (35 in D. melanogaster, 51 in An. gambiae, 24 in A. mellifera and 30 in A. pisum) (Table 1; Fig 3 and Supplementary S4 File). The distribution of CCEs across clades was very different among triatomines and the other species analyzed: the dietary class is absent in triatomines, and almost all the CCEs detected are included in clade E from hormone and pheromone degrading esterases (Table 1; Fig 3 and Supplementary S4 File). The GSTs are a family of enzymes widely found in eukaryotic and prokaryotic cells. These enzymes can use glutathione in reactions, contributing to the biodegradation and elimination of xenobiotics and reactive oxygen species generated during aerobic metabolism [16]. Depending on their location in the cell, the insect GSTs are classified in microsomal and cytosolic groups. Within the cytosolic group, Delta, Epsilon, Omega, Sigma, Theta and Zeta classes may be represented in insects. Among these, Delta and Epsilon classes are exclusive for insects, and are the main GST classes that have been implicated in insecticide resistance [16]. Ribeiro et al. [24] found 7 GST transcripts expressed in R. prolixus gut structures. GST genes seem poorly represented in the transcriptomes of T. infestans, T. dimidiata and T. pallidipennis (14,22 and 11 GST encoding genes, respectively), as well as in the genome of R. prolixus (14) when compared to other insect genomes (Table 1). The number of cytosolic GSTs in triatomines did not greatly differ from that found in A. mellifera (Table 1). The Dipterans and A. pisum present the highest numbers of GSTs genes (40 for D. melanogaster, 35 for An. gambiae and 32 in A. pisum) (Table 1). Microsomal GSTs have not been reported to be involved in the metabolism of insecticides, and represent a small subset of the GST complement in triatomines, with 1 representative in R. prolixus, 3 in T. dimidiata, and 2 in T. infestans and T. pallidipennis (Table 1 and Fig 5). The expression levels of three insecticide-resistance related genes were compared between pyrethroid sensitive and resistant populations of T. infestans. For these comparisons we chose three genes belonging to each one of the superfamilies under study. Within each superfamily, we chose to test a gene belonging to a clade related to insecticide resistance (CYP4, Clade E CCE and Delta GST). Interestingly, we found that the CYP4 gene analyzed was significantly overexpressed in insects belonging to the resistant population (p< 0. 05; n = 4) (Fig 7). Overexpression of a CYP4 gene in a resistant T. infestans population was recently reported [13], suggesting that this could be an expanded mechanism of pyrethroid resistance in this species. The expression of the other enzymes studied did not significantly differ between sensitive and resistant populations (Fig 7).
The analysis of three Triatoma spp. transcriptomes and the R. prolixus genome and transcriptomes allowed us to present a functional overview of the three main insecticide detoxification-related enzyme superfamilies found in vectors of Chagas’ disease. Overall, when triatomines were compared with other insect species, the results point to a reduction in some enzyme clades associated to xenobiotic detoxification: absence of Epsilon class and reduced Delta class in GST superfamily; absence of mitochondrial clade CYP12; absence of CYP9 family in CYP3 clade; and the absence of dietary class CCEs. Conversely, other detoxification-related families are expanded in triatomines: CYP3 clade, clade E in the CCE superfamily and Sigma Class GST. From our point of view, those clades present the main detoxification potential of all triatomine species analyzed here. The overexpression of a CYP4 gene found in a T. infestans resistant population suggests that this family would also represent a detoxification mechanism in this insect species. Besides, most of the recently described CYP families within the CYP3 and CYP4 clades in R. prolixus were confirmed in Triatoma spp. The high abundance of some members in these triatomine specific families makes them candidates to perform physiological and genetic studies. Overall, our findings contribute to understanding insecticide resistance in vectors of Chagas' disease, and open up possibilities for functional studies on triatomine detoxification mechanisms.
One of the principal goals of the last decades regarding public health in Latin America was the reduction of the geographic range and infestation rates of triatomines, the insect vectors of Chagas’ disease. However, the elimination of vector transmission has failed in the Gran Chaco ecoregion, even in areas subjected to intense vector control efforts with pyrethroid insecticides. Therefore, detecting the reasons for the persistence of these vectors has been recognized as a priority by public health authorities. Metabolic insecticide resistance due to enhancement of detoxification activity is one of the principal resistance mechanisms observed in insects. In this study, we analyzed the main enzyme superfamilies known to be involved in insecticide resistance in other insects in four important Chagas’ disease vector species from different regions of Latin America. This was made in a comparative manner using well-studied insect genomes as references. We have found gene expansions for several families related to insecticide resistance, while others showed a reduction. Augmented expression of a CYP gene may suggest contribution to pyrethroid resistance in highly resistant populations of Triatoma infestans from the Gran Chaco. Our results increase the knowledge of potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide relevant information to this field.
invertebrates
medicine and health sciences
pathology and laboratory medicine
invertebrate genomics
animals
toxicology
animal models
organisms
drosophila melanogaster
model organisms
animal anatomy
genome analysis
experimental organism systems
antennae (animal physiology)
insect vectors
zoology
drosophila
research and analysis methods
agrochemicals
epidemiology
animal genomics
disease vectors
agriculture
insects
arthropoda
insecticides
animal physiology
detoxification
transcriptome analysis
genetics
biology and life sciences
genomics
computational biology
triatoma
Comparative and functional triatomine genomics reveals reductions and expansions in insecticide resistance-related gene families