Evolutionary genetics of insecticide resistance in Culex quinquefasciatus

Culex quinquefasciatus mosquitoes play an important role in the transmission of vector-borne diseases of public health importance including lymphatic filariasis (LF) as well as many arboviruses. Insecticide-based approaches are one of the most important interventions to mitigate disease burden; nevertheless increased resistance of vectors to insecticides imposes a challenge for the sustainability and effectiveness of both current and future vector control interventions. Hence, understanding the dynamics and likely mechanisms underlying the evolution of resistance will be critical to effective decision-making in insecticide resistance management strategies. The present study was set out to investigate the genetic basis of insecticide resistance in C. quinquefasciatus from Uganda. Two objectives were developed, 1) to investigate patterns of insecticide resistance across the south of the country and how this might reflect local selection and genetic structure and 2) to investigate the basis of the molecular mechanisms underlying resistance to all four classes of insecticides recommended for vector control. The population genetic study compared and contrasted microsatellite markers and two resistance-associated loci (Vgsc-1014F and Ace1-119S). While no significant difference in genetic diversity across populations were detected by microsatellites, higher frequency of Vgsc-1014F compared to the Ace1-119S mutations was observed in all populations suggesting that the Ugandan Eastern – Southwest populations are under a heterogeneous selection pressure, which created a pattern of local adaptation in these populations. Additionally, the copy number (CN) assay developed in this study indicated the presence of CN variation in the voltage gated sodium channel (Vgsc) gene in about 10% of the individuals assayed from these populations. Genotypic/phenotypic association tests conducted on bendiocarb resistant-individuals suggested that this resistant phenotype was not underlying solely by the 119S target-site mutation in the Ace-1 gene. Indeed, synergist bioassays show an increase of mortality of around 25% in mosquitoes pre-exposed to either TTP or PBO, indicating a possible resistance mediated by detoxification enzymes. Using a novel whole-transcriptome microarray we profiled the bendiocarb-resistant phenotype and implicated two P450s (Cyp-Cx1 and Cyp6n23) with the highest up-regulation expression compared to a susceptible strain. Remarkably, the predicted Cyp-Cx1 is closely related to two P450s from the family Cyp6, which were already validated in vitro as insecticide metabolizers in A. gambiae and A. aegypti, which corroborates a likely association of metabolic resistance in the investigated bendiocarb-resistant phenotype. Taken together the results yielded by genomic and transcriptomic experiments provide evidence that Ugandan C. quinquefasciatus mosquitoes are under heterogeneous selection pressure imposed by insecticides from distinct classes, and that the evolution of insecticide resistance is mediated by at least two main genetic mechanisms; target-site mutations (Vgsc-1014F and Ace1-119S) as well as over-expression of detoxification enzymes

Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vector Culex quinquefasciatus

Abstract: Culex quinquefasciatus plays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackle C. quinquefasciatus vectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, including C. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistant C. quinquefasciatus was explored to identify candidate genes associated with insecticide resistance. High levels of insecticide resistance were observed for five out of six insecticides tested, with the lowest mortality (0.97%) reported to permethrin, while for DDT, lambdacyhalothrin, bendiocarb and deltamethrin the mortality rate ranged from 1.63–3.29%. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1 h exposure and 58.02% after 4 h. Genotyping of the G119S Ace-1 target site mutation detected a highly significant association (p 8-fold increase vs unexposed controls). These results provide evidence that bendiocarb resistance in Ugandan C. quinquefasciatus is mediated by both target-site mechanisms and over-expression of detoxification enzymes

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations

Background: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. Methodology/Principal findings: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). Conclusions/Significance: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vector Culex quinquefasciatus

Abstract: Culex quinquefasciatus plays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackle C. quinquefasciatus vectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, including C. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistant C. quinquefasciatus was explored to identify candidate genes associated with insecticide resistance. High levels of insecticide resistance were observed for five out of six insecticides tested, with the lowest mortality (0.97%) reported to permethrin, while for DDT, lambdacyhalothrin, bendiocarb and deltamethrin the mortality rate ranged from 1.63–3.29%. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1 h exposure and 58.02% after 4 h. Genotyping of the G119S Ace-1 target site mutation detected a highly significant association (p 8-fold increase vs unexposed controls). These results provide evidence that bendiocarb resistance in Ugandan C. quinquefasciatus is mediated by both target-site mechanisms and over-expression of detoxification enzymes

Genetic differentiation estimates among the four <i>Cx</i>. <i>quinquefasciatus</i> populations based on allele frequencies at 26 microsatellite markers.

<p>(A) First and second PCs of the Discriminant Analysis of Principal Components (DAPC) to infer population structure. The graph represents the individuals as dots and the groups as coloured ellipses, which model 95% of the corresponding variability plotted. Populations (ellipses) are plotted within the orthogonal space defined by the first two PCA eigenvalues (inserts). (B) Bayesian analysis of population structure performed using STRUCTURE. Diagrammatic representation of population clusters for the most likely <i>K</i> (<i>K</i> = 3) with each coloured segment corresponding to the proportion of individuals assigned to a hypothetical population or cluster. Each vertical bar represents an individual with the height of the column segments showing the probability of assignment of belonging to one of the three clusters. Dark lines correspond to population assignment based on geographic collection-site.</p

<i>F</i>_<i>ST</i> outlier approach for the 26 studied microsatellite loci versus a model of neutral evolution in Ugandan <i>Cx</i>. <i>quinquefasciatus</i>.

<p>Central populations correspond to a cluster of Jinja and Kampala (based on genetic structure analysis, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005917#pntd.0005917.g003" target="_blank">Fig 3</a>). Each dot represents a microsatellite marker. The markers located in the grey middle area are assumed to be neutral. The markers located below the neutral area are candidates for being under balancing selection and the markers located above the neutral area are candidates for being under directional selection. <i>X</i>-axis: estimated heterozygosity values. <i>Y</i>-axis: <i>F</i>_<i>ST</i>-values. Analyses were performed assuming an infinite allele model.</p

Relationship between geographic distance versus genetic distance (<i>F</i>_<i>ST</i>/1- <i>F</i>_<i>ST</i>), analysed in parallel for the microsatellite, <i>Vgsc</i>-<i>L1014F</i> and <i>Ace1-G119S</i> loci.

<p>Significance was tested using a Mantel test. Grey shading corresponds to the 95% confidence interval. Geographic distance corresponding to the great-circle distance, which corresponds to the shortest distance over the earth’s surface between two points.</p

Matrix of inferred gene flow between Ugandan <i>Cx</i>. <i>quinquefasciatus</i> populations.

<p>Matrix of inferred gene flow between Ugandan <i>Cx</i>. <i>quinquefasciatus</i> populations.</p

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.13Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt