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.

Silencing of Two Vitellogenin Genes Inhibits Oviposition in the Chagas Disease Vector Triatoma infestans (Hemiptera: Reduviidae).

RNA interference caused by exogenous double-stranded RNA (dsRNA) is used to downregulate crucial genes to control insects. The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. Adult females of Triatoma infestans were independently injected with two Vg dsRNAs (Vg1 dsRNA or Vg2 dsRNA) or nuclease-free water (control) 24 hours before feeding, and a group of adult females not injected was also analyzed (control). Vg1 and Vg2 messenger RNAs silencing was verified by reverse transcription quantitative polymerase chain reaction. The transcript levels of the Vg1 and Vg2 genes were significantly reduced after dsRNA treatment in fat body and ovary of T. infestans in relation to those detected in individuals injected with nuclease-free water and not injected (controls). Moreover, the present study demonstrated that the silencing of the Vg1 or Vg2 genes inhibits oviposition in the Chagas disease vector T. infestans. These findings may have important implications for the development of novel vector control strategies.

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

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.

cDNA isolation and characterization of two vitellogenin genes in the Chagas' disease vector Triatoma infestans (Hemiptera, Reduviidae).

Two vitellogenin genes (Vg1 and Vg2) were identified in the Chagas' disease vector Triatoma infestans. The putative coding sequence corresponding to Vg2 was found to be 5553bp long, encoding 1851 amino acids in a single open reading frame. The comparative analysis of the deduced amino acid sequences from Vg1 and Vg2 cDNA fragments of T. infestans revealed 58.94% of identity with 76.43% of homology. The phylogenetic tree based on the complete Vg amino acid sequences of hemimetabolous insects unambiguously supported two clusters, one consisting of Vg sequences from dictyopteran and the other containing Vg sequences of hemipteran. The Vg1 and Vg2 mRNAs were detected in fat bodies and ovaries of adult females with the highest levels of both Vg transcripts in the first tissue. Quantitative PCR showed low expression of Vg2 in head and muscle of adult females, while the Vg1 transcript was not present in these organs. Neither Vg1 nor Vg2 was expressed in fifth instar nymph fat bodies or in adult male fat bodies, heads, and muscles.

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.