RNA interference of NADPHcytochrome P450 increased deltamethrin susceptibility in a resistant strain of the Chagas disease vector Triatoma infestans.

The enzyme NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochromes P450 activity. Gene expression analysis of cytochromes P450 and CPR in deltamethrin-resistant and susceptible populations revealed that P450s genes are involved in the development of insecticide resistance in Triatoma infestans. To clarify the role of cytochromes P450 in insecticide resistance, it was proposed to investigate the effect of CPR gene silencing by RNA interference (RNAi) in a pyrethroid resistant population of T. infestans. Silencing of the CPR gene showed a significant increase in susceptibility to deltamethrin in the population analysed. This result support the hypothesis that the metabolic process of detoxification mediated by cytochromes P450 contributes to the decreased deltamethrin susceptibility observed in the resistant strain of T. infestans.

Clock Gene Timeless in the Chagas Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

To contribute to a better understanding of the molecular basis of the circadian biological rhythms in Chagas disease vectors, in this work we identified functional domains in the sequences of the clock protein TIMELESS (TIM) in Rhodnius prolixus and analyzed the expression of the timeless (tim) gene at the mRNA level in Triatoma infestans. The tim gene expression in nervous tissue of adult T. infestans revealed clear oscillations in the abundance of the transcript in both sexes in the group maintained under photoperiod with a daily canonical rhythm, showing a significant increase in expression at sunset. As expected, in the group maintained in constant light, no daily increase was detected in the tim transcript level.

Daily Variations in the Expression of Genes Related to Insecticide Resistance in the Chagas Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

Pyrethroid resistance has been detected in Triatoma infestans, which in part has been attributed to increased oxidative metabolism of the insecticide by cytochrome P450s. Nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) catalyzes electron transfer from NADPH to all known cytochrome P450s. In this study, the expression of the CPR gene at transcriptional level was determined in different tissues and two life stages. The expression patterns showed differences in the tissues and stages studied, suggesting differential metabolic requirements. On the other hand, to investigate the presence of rhythms in the expression of genes related with insecticide resistance, we explored the daily expression profile of the CPR gene and a P450 gene (CYP4EM7) in fat body from adults of T. infestans under different dark/light regimes. The results suggest that CPR gene expression is under endogenous clock regulation and show a rhythmic profile in the expression of the CYP4EM7 gene.

cDNA Isolation and Expression of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Cytochrome P450 Reductase Gene in the Chagas Disease Vector Triatoma infestans.

Pyrethroid resistance has been detected in Triatoma infestans (Hemiptera: Reduviidae), which was atributed to target site insensitivity and increased oxidative metabolism of the insecticide by cytochrome P450s. Nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome P450 reductase (CPR) plays an essential role in transferring electrons from NADPH to the P450-substrate complex. In this study, the full length CPR cDNA of T. infestans was isolated and gene expression was determined by quantitative polymerase chain reaction. The open reading frame is 2,046 bp long, encoding a protein of 682 amino acids. Amino acid sequence analysis indicates that the T. infestans CPR and the putative Rhodnius prolixus and Triatoma dimidiata CPRs present conserved ligand-binding domains. Congruent with a previous study of our laboratory, in which the expression of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6 genes) was induced by deltamethrin, the levels of T. infestans CPR mRNA were upregulated in the fat body of fifth instar nymphs after topical application of deltamethrin. Besides, as it was observed in the CYP4EM7 gene, it was detected overexpression of the CPR gene in the most resistant strain of T. infestans included in the study. These results suggest that CPR plays an essential role in P450-mediated resistance of T. infestans to insecticides.

Clock Gene Period in the Chagas Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

To contribute to a better understanding of the molecular bases of the circadian biological rhythms in Chagas disease vectors, in this work we identified functional domains in the sequences of the clock protein PERIOD (PER) in Rhodnius prolixus and Triatoma infestans and analyzed the expression of the PER gene at mRNA level in T. infestans. The PER protein sequences comparison among these species and those from other insects revealed that the most similar regions are the PAS domains and the most variable is the COOH-terminal. On the other hand, the per gene expression in nervous tissue of adult T. infestans varies with a daily canonical rhythm in groups of individuals maintained under photoperiod (light/dark, LD) and constant dark (DD), showing a significant peak of expression at sunset. The pattern of expression detected in LD persists under the DD condition. As expected, in the group maintained in constant light (LL), no daily increase was detected in per transcript level. Besides, the presence of per transcript in different tissues of adult individuals and in nervous tissue of nymphs evidenced activity of peripheral clocks in adults and activity of the central clock in nymphs of T. infestans.

Daily variations in the glycerol-3-phosphate dehydrogenase isoforms expression in Triatoma infestans flight muscles.

Triatoma infestans, the main vector of Chagas disease, is a blood-sucking insect. Flight dispersal of adults is the most important mechanism for reinfestation of houses after insecticide spraying. Flight muscles have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. In this study, we explored the profile of GPDH expression in females and males adult flight muscles under light/dark cycle, constant light, and constant dark conditions. Under constant dark conditions, GPDH-1 flight muscles of T. infestans showed a rhythmic pattern of transcription synchronous with a rhythmic profile of activity suggesting regulation by the endogenous circadian clock. Otherwise, the GPDH-2 expression analysis showed no regulation by the endogenous clock, but showed that an external factor, such as the dark/light period, was necessary for synchronization of GPDH-2 transcription and activity.

Differential expression of glycerol-3-phosphate dehydrogenase isoforms in flight muscles of the Chagas disease vector Triatoma infestans (Hemiptera, Reduviidae).

Flight muscles of Triatoma infestans have two glycerol-3-phosphate dehydrogenase (GPDH) isoforms: GPDH-1 is involved in flight metabolism and GPDH-2 provides lipid precursors. Total GPDH activity was greater in the natural population and almost only due to GPDH-1. Different expression and activity observed between GPDH isoforms in the natural population and the first laboratory generation was not detected in the second laboratory generation. This pattern may be caused by gradual adaptation to laboratory nutritional conditions. During development, the expression of GPDH-2 increased with a longer time of intake, which would imply an increment in lipid biosynthesis. The GPDH-1 transcript predominated with respect to that of GPDH-2 in the lower nutritional condition, suggesting the necessity of insects to fly during this nutritional status. The transcriptional pattern showed a delay at 22°C. The isoforms activities and transcript patterns in flight muscles suggest transcriptional adaptation to metabolic requirements originated by alternative splicing.

Molecular Population Genetics and Evolution of the Chagas' Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

Triatoma infestans (Klug) is the main vector of Chagas' disease in the Southern Cone of Latin America between the latitudes 10° S and 46° S. The long-term effectiveness of the control campaigns is greatly dependent upon the vector population structure. Mitochondrial DNA (mtDNA) genes have been used in a number of T. infestans population genetic analyses. However, the maternally inherited markers as well as nuclear ribosomal DNA analyzed until the present exhibited low or limited levels of variation. Analyses based on microsatellite markers strongly supported the existence of some type of stratification in T. infestans populations and supported the hypothesis of vector population recovery from survivors of the insecticide-treated areas, highlighting the value of population genetic analyses in assessing the effectiveness of Chagas' disease vector control programmes. Although phylogeographic studies have generally suggested a Bolivian Andean origin of T. infestans, they recovered two reciprocal monophyletic groups of T. infestans and Bolivian populations who were not basal as expected for an ancestral group. In addition, a non-Andean origin could not be excluded by mtDNA genealogies that included sylvatic bugs from Gran Chaco. On the other side, mitochondrial and microsatellite markers supported the hypothesis of two independent migration events of colonization and secondary contacts in southern South America. Since the phylogenetic analyses remain inconclusive, more sequences, not only from mitochondrial genes but also from nuclear genes, need to be examined.

Gene discovery in Triatoma infestans.

BACKGROUND: Triatoma infestans is the most relevant vector of Chagas disease in the southern cone of South America. Since its genome has not yet been studied, sequencing of Expressed Sequence Tags (ESTs) is one of the most powerful tools for efficiently identifying large numbers of expressed genes in this insect vector. RESULTS: In this work, we generated 826 ESTs, resulting in an increase of 47% in the number of ESTs available for T. infestans. These ESTs were assembled in 471 unique sequences, 151 of which represent 136 new genes for the Reduviidae family. CONCLUSIONS: Among the putative new genes for the Reduviidae family, we identified and described an interesting subset of genes involved in development and reproduction, which constitute potential targets for insecticide development.