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.

MS4A2-rs573790 Is Associated With Aspirin-Exacerbated Respiratory Disease: Replicative Study Using a Candidate Gene Strategy.

Aspirin exacerbated respiratory disease (AERD) is a set of diseases of the unified airway, and its physiopathology is related to disruption of the metabolism of arachidonic acid (AA). Genetic association studies in AERD had explored single nucleotide polymorphism (SNPs) in several genes related to many mechanisms (AA metabolism, inflammation, drug metabolism, etc.) but most lack validation stages in second populations. Our aim is to evaluated whether contribution to susceptibility of SNPs reported in other populations are associated with AERD in Mexican Mestizo patients. We developed a replicative study in two stages. In the first, 381 SNPs selected by fine mapping of associated genes, (previously reported in the literature), were integrated into a microarray and tested in three groups (AERD, asthma and healthy controls -HC-) using the GoldenGate array. Results associated to risk based on genetic models [comparing: AERD vs. HC (comparison 1, C1), AERD vs. asthma (C2), and asthma vs. HC (C3)] were validated in the second stage in other population groups using qPCR. In the first stage, we identified 11 SNPs associated with risk in C1.The top SNPs were ACE-rs4309C (p = 0.0001) and MS4A2-rs573790C (p = 0.0002). In C2, we detected 14 SNPs, including ACE-rs4309C (p = 0.0001). In C3, we found MS4A2-rs573790C (p = 0.001). Using genetic models, C1 MS4A2-rs57370 CC (p = 0.001), and ACE-rs4309 CC (p = 0.002) had associations. In C2 ACE-rs4309 CC (p = 0.0001) and C3 MS4A2-rs573790 CC (p = 0.001) were also associate with risk. In the second stage, only MS4A2-rs573790 CC had significance in C1 and C3 (p = 0.008 and p = 0.03). We concluded that rs573790 in the MS4A2 gene is the only SNP that supports an association with AERD in Mexican Mestizo patients in both stages of the study.

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.

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.

Application of Inter-Simple Sequence Repeat Markers in the Analysis of Populations of the Chagas Disease Vector Triatoma infestans (Hemiptera, Reduviidae).

Here we apply inter-simple sequence repeat (ISSR) markers to explore the fine-scale genetic structure and dispersal in populations of Triatoma infestans. Five selected primers from 30 primers were used to amplify ISSRs by polymerase chain reaction. A total of 90 polymorphic bands were detected across 134 individuals captured from 11 peridomestic sites from the locality of San Martín (Capayán Department, Catamarca Province, Argentina). Significant levels of genetic differentiation suggest limited gene flow among sampling sites. Spatial autocorrelation analysis confirms that dispersal occurs on the scale of ∼469 m, suggesting that insecticide spraying should be extended at least within a radius of ∼500 m around the infested area. Moreover, Bayesian clustering algorithms indicated genetic exchange among different sites analyzed, supporting the hypothesis of an important role of peridomestic structures in the process of reinfestation.

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.

Dynamics of expression of two vitellogenin genes in the Chagas' disease vector Triatoma infestans: Analysis throughout pre-vitellogenesis and vitellogenesis.

The reproductive success of all oviparous species depends on vitellogenin (Vg) biosynthesis and its accumulation in the developing oocytes. The expression levels of two Vg genes (Vg1 and Vg2) were analyzed by qPCR and western blot in fat body and ovaries of adult females, at different times after ecdysis (pre-vitellogenic phase) and after blood feeding of females (vitellogenic phase). Vg genes were also evaluated in fat bodies of adult males as well as in female fifth instar nymphs. No trace of Vg mRNA was detected in adult males or in nymphs. Vg1 and Vg2 were expressed in the fat bodies and ovaries of adult females. The Vg genes start to be expressed slightly in both tissues of adult females during pre-vitellogenesis. After blood feeding, Vg1 and Vg2 were up regulated and significant levels of Vg transcripts as well as protein expression were observed in fat bodies sampled throughout vitellogenesis. During this period however, the distribution patterns of Vg1 and Vg2 transcripts showed two peaks around early and advanced vitellogenesis (days 4 and 12 post-feeding, respectively). In the ovaries, levels of mRNAs increased from the day 10 post-blood feeding onwards. In addition, the immunofluorescence assays showed a strong signal for vitellin in the yolk bodies of terminal follicles of vitellogenic females. The involvement of fat body and ovary in the synthesis of Vg suggests different roles of Vgs in supporting the growth of oocytes.

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.

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.

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.

Fine-scale genetic structure in populations of the Chagas' disease vector Triatoma infestans (Hemiptera, Reduvidae).

Fine scale patterns of genetic structure and dispersal in Triatoma infestans populations from Argentina was analysed. A total of 314 insects from 22 domestic and peridomestic sites from the locality of San Martín (Capayán department, Catamarca province) were typed for 10 polymorphic microsatellite loci. The results confirm subdivision of T. infestans populations with restricted dispersal among sampling sites and suggest inbreeding and/or stratification within the different domestic and peridomestic structures. Spatial correlation analysis showed that the scale of structuring is approximately of 400 m, indicating that active dispersal would occur within this distance range. It was detected difference in scale of structuring among sexes, with females dispersing over greater distances than males. This study suggests that insecticide treatment and surveillance should be extended within a radius of 400 m around the infested area, which would help to reduce the probability of reinfestation by covering an area of active dispersal. The inferences made from fine-scale spatial genetic structure analyses of T. infestans populations has demonstrated to be important for community-wide control programs, providing a complementary approach to help improve vector control strategies.

Variation in mitochondrial NADH dehydrogenase subunit 5 and NADH dehydrogenase subunit 4 genes in the Chagas disease vector Triatoma infestans (Hemiptera: Reduviidae).

Variation in mitochondrial NADH dehydrogenase subunit 5 (ND5) and NADH dehydrogenase subunit 4 (ND4) genes was surveyed in Triatoma infestans from 24 localities of Argentina. The DNA sequence comparisons of 2,183 basepairs of the mitochondrial genome, which include the complete sequence of ND5 (1,712 basepairs) and 401 basepairs of ND4 genes, showed 19 haplotypes determined by 48 variable sites and a nucleotide diversity value of 0.292%. Twenty-six (65%) substitutions were synonymous, and there were 14 (35%) predicted amino acid replacements in ND5. In ND4, 5 (62.5%) substitutions were synonymous and 3 (37.5%) were replacement sites. Samples from six localities studied shared one haplotype and the rest of the localities had different haplotypes. The amplified regions should be useful for population genetic studies.

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.

Microsatellite analysis of genetic structure in natural Triatoma infestans (Hemiptera: Reduviidae) populations from Argentina: its implication in assessing the effectiveness of Chagas' disease vector control programmes.

The genetic structure in populations of the Chagas' disease vector Triatoma infestans was examined. Comparisons of the levels of genetic variability in populations of this species from areas with different periods since last insecticide treatment and from areas that never received treatment were also carried out. A total of 598 insects from 19 populations were typed for 10 polymorphic microsatellite loci. The average observed and expected heterozygosities ranged from 0.186 to 0.625 and from 0.173 to 0.787, respectively. Genetic drift and limited gene flow appear to have generated a substantial degree of genetic differentiation among the populations of T. infestans. Departures from Hardy-Weinberg expectations due to an excess of homozygotes suggested the presence of null alleles and population subdivision. Microgeographical analysis supports the existence of subdivision in T. infestans populations. Levels of genetic diversity in the majority of the populations of T. infestans from insecticide-treated localities were similar or higher than those detected in populations from areas without treatment. Since the populations of T. infestans are subdivided, a population bottleneck would result in independent genetic drift effects that could randomly preserve different combinations of alleles in each subpopulation. These events followed by a rapid population growth could have preserved high levels of genetic diversity. This study supports the hypothesis of vector population recovery from survivors of the insecticide-treated areas and therefore highlights the value of population genetic analyses in assessing the effectiveness of Chagas' disease vector control programmes.

Phylogeny of triatomine vectors of Trypanosoma cruzi suggested by mitochondrial DNA sequences.

The subfamily Triatominae (Hemiptera: Reduviidae) comprises hematophagous insects, most of which are actual or potential vectors of Trypanosoma cruzi, the protozoan agent of Chagas' disease (American trypanosomiasis). DNA sequence comparisons of mitochondrial DNA (mtDNA) genes were used to infer phylogenetic relationships among 32 species of the subfamily Triatominae, 26 belonging to the genus Triatoma and six species of different genera. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes (totaling 848-851 bp) from each of the 32 species, as well as of the cytochrome oxidase I (COI, 1447 bp) gene from nine. The phylogenetic analyses unambiguously supported several clusters within the genus Triatoma. In the morphological classification, T. costalimai was placed tentatively within the infestans complex while T. guazu was not included in any Triatoma complex. The placement of these species in the molecular phylogeny indicated that both belong to the infestans complex. We confirmed with a strong support the inclusion of T. circummaculata, a member of a different complex based on morphology, within the infestans complex. On the other hand, the present phylogenetics analysis did not support the monophyly of the infestans complex species as it was suggested in our previous studies. While no strong inference of polyphyly of the genus Triatoma was provided by the bootstrap analyses, the other species belonging to Triatomini analyzed could not be distinguished from the species of Triatoma.

Short report: variation in mitochondrial 12S and 16S ribosomal DNA sequences in natural populations of Triatoma infestans (Hemiptera: Reduviidae).

Mitochondrial DNA sequences of the 12S and 16S ribosomal RNA genes were analyzed in five natural populations of the Chagas' disease vector Triatoma infestans from Argentina. DNA sequence comparisons of 878 basepairs (12S plus 16S) revealed 13 haplotypes. A total of 10 private haplotypes were found in four of the populations analyzed, suggesting low current levels of genetic exchange. The levels of genetic differentiation between the population of Chancaní (Córdoba) and other two of the populations analyzed indicated significant deviation from a pattern of unrestricted gene flow. The haplotypic diversity and the private haplotypes found in the geographically closest localities of Chancaní and El Jardín (La Rioja) suggest that the reduction in the population size by insecticide treatment did not avoid the recovery of the populations apparently from survivors of the same area.