Development of molecular assays to detect target-site mechanisms associated with insecticide resistance in malaria vectors from Latin America.

BACKGROUND: Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America. METHODS: Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed. RESULTS: Primers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids. CONCLUSIONS: New molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

The field evaluation of a push-pull system to control malaria vectors in northern Belize, Central America

BACKGROUND: Campaigns for the continued reduction and eventual elimination of malaria may benefit from new and innovative vector control tools. One novel approach being considered uses a push-pull strategy, whereby spatial repellents are used in combination with outdoor baited traps. The desired effect is the behavioural manipulation of mosquito populations to elicit movement of vectors away from people and into traps. METHODS: Here, a prototype push-pull intervention was evaluated using an experimental hut methodology to test proof-of-principle for the strategy against two natural vector populations, Anopheles albimanus and Anopheles vestitipennis in Belize, Central America. A Latin square study design was used to compare mosquito entry into experimental huts and outdoor traps across four different experimental conditions: 1) control, with no interventions; 2) pull, utilizing only outdoor traps; 3) push, utilizing only an indoor spatial repellent; and 4) push-pull, utilizing both interventions simultaneously. RESULTS: For An. vestitipennis, the combined use of an indoor repellent and outdoor baited traps reduced average nightly mosquito hut entry by 39% (95% CI: [0.37 ­ 0.41]) as compared to control and simultaneously increased the nightly average densities of An. vestitipennis captured in outdoor baited traps by 48% (95% CI: [0.22 ­ 0.74]), compared to when no repellent was used. Against An. albimanus, the combined push-pull treatment similarly reduced hut entry, by 54% (95% CI: [0.40 ­ 0.68]) as compared to control; however, the presence of a repellent indoors did not affect overall outdoor trap catch densities for this species. Against both anopheline species, the combined intervention did not further reduce mosquito hut entry compared to the use of repellent alone. CONCLUSIONS: The prototype intervention evaluated here clearly demonstrated that push-pull strategies have potential to reduce human-vector interactions inside homes by reducing mosquito entry, and highlighted the possibility for the strategy to simultaneously decrease human-vector interactions outside of homes by increasing baited trap collections. However, the variation in effect on different vectors demonstrates the need to characterize the underlying behavioral ecology of target mosquitoes in order to drive local optimization of the intervention…(AU)

A comparison of two commercial mosquito traps for the capture of malaria vectors in northern belize, central america.

To achieve maximum success from any vector control intervention, it is critical to identify the most efficacious tools available. The principal aim of this study was to evaluate the efficacy of 2 commercially available adult mosquito traps for capturing Anopheles albimanus and An. vestitipennis, 2 important malaria vectors in northern Belize, Central America. Additionally, the impact of outdoor baited traps on mosquito entry into experimental huts was assessed. When operated outside of human-occupied experimental huts, the Centers for Disease Control and Prevention (CDC) miniature light trap, baited with human foot odors, captured significantly greater numbers of female An. albimanus per night (5.1 ± 1.9) than the Biogents Sentinel™ trap baited with BG-Lure™ (1.0 ± 0.2). The 2 trap types captured equivalent numbers of female An. vestitipennis per night, 134.3 ± 45.6 in the CDC trap and 129.6 ± 25.4 in the Sentinel trap. When compared to a matched control hut using no intervention, the use of baited CDC light traps outside an experimental hut did not impact the entry of An. vestitipennis into window interception traps, 17.1 ± 1.3 females per hour in experimental huts vs. 17.2 ± 1.4 females per hour in control huts. However, the use of outdoor baited CDC traps did significantly decrease the entry of An. albimanus into window interception traps from 3.5 ± 0.5 females per hour to 1.9 ± 0.2 females per hour. These results support existing knowledge that the underlying ecological and behavioral tendencies of different Anopheles species can influence trap efficacy. Furthermore, these findings will be used to guide trap selection for future push-pull experiments to be conducted at the study site.

A comparison of two commercial mosquito traps for the capture of Malaria vectors in northern Belize, Central America

To achieve maximum success from any vector control intervention, it is critical to identify the most efficacious tools available. The principal aim of this study was to evaluate the efficacy of 2 commercially available adult mosquito traps for capturing Anopheles albimanus and An. vestitipennis, 2 important malaria vectors in northern Belize, Central America. Additionally, the impact of outdoor baited traps on mosquito entry into experimental huts was assessed. When operated outside of human-occupied experimental huts, the Centers for Disease Control and Prevention (CDC) miniature light trap, baited with human foot odors, captured significantly greater numbers of female An. albimanus per night (5.1 6 1.9) than the Biogents SentinelTM trap baited with BG-LureTM (1.0 6 0.2). The 2 trap types captured equivalent numbers of female An. vestitipennis per night, 134.3 6 45.6 in the CDC trap and 129.6 6 25.4 in the Sentinel trap. When compared to a matched control hut using no intervention, the use of baited CDC light traps outside an experimental hut did not impact the entry of An. vestitipennis into window interception traps, 17.1 6 1.3 females per hour in experimental huts vs. 17.2 6 1.4 females per hour in control huts. However, the use of outdoor baited CDC traps did significantly decrease the entry of An. albimanus into window interception traps from 3.5 6 0.5 females per hour to 1.9 6 0.2 females per hour. These results support existing knowledge that the underlying ecological and behavioral tendencies of different Anopheles species can influence trap efficacy. Furthermore, these findings will be used to guide trap selection for future push­ pull experiments to be conducted at the study site...(AU)

A mark-release-recapture study to define the flight behaviors of Anopheles vestitipennis and Anopheles albimanus in Belize, Central America

The recapture rates of wild-caught, unengorged Anopheles vestitipennis and Anopheles albimanus females were determined at 0, 400, and 800 m from a fixed release point in Belize, Central America. Three sampling trials, each consisting of two 12-hour collections, were performed at each distance during September­October 2003. A total of 1,621 An. vestitipennis and 1,326 An. albimanus were marked and released during the course of the study. The recapture rate of An. vestitipennis was greatest at 0 m (7.9%; 44/ 556) and declined from 3.0% (16/531) at 400 m to 0.2% (1/534) at 800 m. Anopheles albimanus females were recaptured only at the 0-m distance and in extremely low numbers (1.1%; 5/446). Biting patterns for the unmarked natural populations were similar to those previously described for Belize, and recaptures for both species occurred during these normal biting times. The overall recapture rates for An. vestitipennis (3.76%; 61/ 1,621) and An. albimanus (0.38%; 5/1,326) indicate that An. vestitipennis has a higher probability of being attracted to a human habitation.