Elsevier

Infection, Genetics and Evolution

Volume 44, October 2016, Pages 459-470
Infection, Genetics and Evolution

Research paper
Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations

https://doi.org/10.1016/j.meegid.2016.07.027Get rights and content

Highlights

  • Isolation and expression of three cytochrome P450 genes in Triatoma infestans

  • Gene expression was induced by deltamethrin in susceptible and resistant populations.

  • Overexpression of a P450 gene was demonstrated in the most resistant strain.

  • P450 genes would be involved in the detoxification of deltamethrin.

Abstract

Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance.

Introduction

Chagas' disease (American trypanosomiasis) is produced by infection with Trypanosoma cruzi, which is transmitted by hematophagous insects of the subfamily Triatominae (Hemiptera: Reduviidae). The disease is a serious public health problem in Latin America, where about 8 million people are estimated to be infected with T. cruzi and > 25 million people are at risk of contracting the infection (Rassi et al., 2010, World Health Organization, 2014). From an epidemiological standpoint, the most important species of triatomines involved in the transmission of Chagas' disease are those that combine a high degree of adaptation to the domestic environments, have a wide geographical distribution, possess a high vectorial capacity, and are anthropophilic (Lent and Wygodzinsky, 1979). Among them, Triatoma infestans is the main vector of T. cruzi in the Southern Cone of Latin America between latitudes 10° S and 46° S, where it is primarily restricted to domestic and peridomestic environments.

The interruption of transmission of this parasitic and infectious disease consists of vector control by insecticide treatment of infested dwellings. T. infestans has been the target of control programs as part of the Southern Cone Initiative (Moncayo and Silveira, 2009). Pyrethroid insecticides remain the first choice of chemical for indoor residual spraying due to their low mammalian toxicity, rapid breakdown in the environment and efficacy. However, vector control has proven to be difficult, in part as a consequence of the variability and extension of endemic areas, and because of the difficulties to implement sustained entomological vigilance to prevent the recovery of treated bug populations (Tarleton et al., 2014). In this regard, high levels of T. infestans reinfestation after spraying were observed in Argentina, Bolivia, and Paraguay (Gürtler et al., 2007). Coincidently, pyrethroid resistance in the vector insect has been reported in some of those countries and emerges as one of the main explanations of the unsatisfactory control observed (Vassena et al., 2000, Picollo et al., 2005, Santo Orihuela et al., 2008, Germano et al., 2010, Lardeux et al., 2010, Gurevitz et al., 2012, Mougabure-Cueto and Picollo, 2015). Analyses of genes involved in insecticide resistance in insect vectors of diseases as T. infestans are of considerable importance. Particularly, the importance of studying the expression patterns of genes involved in their resistance lies in its potential for exploitation in novel insect control strategies.

In insects, cytochrome P450 monooxygenases (cytochrome P450s) play a predominant role in the metabolism of insecticides, which often results in the development of insecticide resistance in insect populations (Zhou et al., 2010). Most insect cytochrome P450 genes belong to microsomal CYP4, CYP6, CYP9, CYP28, CYP321 and mitochondrial CYP12 families and many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides (Feyereisen, 2005, Li et al., 2007). An important characteristic of insect P450s that is associated with enhanced metabolic detoxification of insecticides is the constitutively increased levels of P450 proteins and P450 activity that result from constitutively transcriptional overexpression of cytochrome P450 genes in insecticide resistant insects (Carino et al., 1994, Liu and Scott, 1997, Liu and Scott, 1998, Kasai et al., 2000, Feyereisen, 2005, Daborn et al., 2002, Daborn et al., 2007). In addition, the expression of some cytochrome P450 genes can be increased by exogenous and endogenous compounds (Feyereisen, 2005), a phenomenon known as induction. In insects, the induction of P450s and their activities would be involved in the adaptation of insects to their environment and the development of insecticide resistance (Terriere, 1983, Terriere, 1984).

Resistance to deltamethrin was associated with oxidative metabolism in specimens of T. infestans from Brazil and Argentina and Rhodnius prolixus from Venezuela. Deltamethrin resistance in both species was decreased by piperonyl butoxide, an inhibitor of cytochrome P450 enzymes (Vassena et al., 2000, Picollo et al., 2005). Gonzáles Audino et al. (2004) and Picollo et al. (2005) measured cytochrome P450 activity on individual T. infestans through ethoxycoumarin-deethylase (ECOD) activity. The ECOD activity was significantly higher in a deltamethrin-resistant colony than in a susceptible colony. These results suggested that cytochrome P450s play an important role in the resistance to deltamethrin. On the other hand, other mechanism that has been associated to pyrethroid resistance in insects involves changes in the voltage-gated sodium channel which is the target site of these insecticides. To this respect, Fabro et al. (2012) identified the presence of a resistant-conferring mutation (L1014F) in a pyrethroid-resistant population of T. infestans from Argentina. Subsequently, Capriotti et al. (2014) identified a new pyrethroid resistant-conferring mutation (L925l) in T. infestans that was associated with inefficiency in the control campaigns.

Because the increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects (Carino et al., 1992, Carino et al., 1994, Liu and Scott, 1997, Liu and Scott, 1998, Li et al., 2007), it was of interest to analyze in T. infestans genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, in this study initially the cDNA sequences of three cytochrome P450 genes were identified. Comparative analysis of transcriptional expression of these P450 genes in susceptible and resistant strains of T. infestans was performed. Besides, the expression patterns of the P450 genes isolated were determined at different interval times after the application of deltamethrin in insects from the different strains analyzed.

Section snippets

Insects

Four different laboratory strains of T. infestans were studied. Two strains were provided by the Centro de Referencia de Vectores of the Servicio Nacional de Chagas de Córdoba (Córdoba province, Argentina). One belonging to a colony susceptible to deltamethrin (CRV-susceptible strain) that was originated in 2006 from insects collected in the locality of Chuña (Department of Ischilín, Córdoba province, Argentina) (30° 28′ S, 64° 40′ W) and other belonging to a 2nd generation of a colony

Sequence and phylogenetic analyses of three cytochrome P450 genes in Triatoma infestans

In this study, three cytochrome P450 cDNAs were isolated using the 3′-RACE methodology. Subsequent reverse transcription PCR (RT-PCR) amplifications using specific primers designed from these 3′ fragments and unspecific or degenerate primers designed from conserved regions of cytochrome P450 sequences of other insects, allowed to obtain longer cDNA partial sequences of three cytochrome P450 genes for T. infestans. These genes were named CYP4EM7, CYP3085B1, and CYP3092A6 (GenBank accession

Discussion

Increased metabolic detoxification of insecticides, primarily by activities of cytochrome P450 monooxygenases (P450s), is the most frequent type of resistance mechanism occurring in insects (Li et al., 2007). It has been proposed that induction and/or constitutive overexpression of P450s is linked to the adaptation of insects to their environment (Terriere, 1983, Terriere, 1984). As part of this adaptation, it has been hypothesized that both constitutively increased expression (overexpression)

Acknowledgments

We thank the Centro de Referencia de Vectores, Servicio Nacional de Chagas de Córdoba, Argentina, for providing insects used in our studies. This research was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT-0293, FONCyT-Argentina), Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 11220120100361, CONICET-Argentina), and Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (203/14-103/15, SECyT-UNC-Argentina). The experiments of

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