Determining Airborne Concentrations of Spatial Repellent Chemicals in Mosquito Behavior Assay Systems

Background

Mosquito behavior assays have been used to evaluate the efficacy of vector control interventions to include spatial repellents (SR). Current analytical methods are not optimized to determine short duration concentrations of SR active ingredients (AI) in air spaces during entomological evaluations. The aim of this study was to expand on our previous research to further validate a novel air sampling method to detect and quantitate airborne concentrations of a SR under laboratory and field conditions.

Methodology/Principal Findings

A thermal desorption (TD) gas chromatography-mass spectrometry (GC-MS) method was used to determine the amount of dichlorodiphenyltrichloroethane (DDT) in samples of air. During laboratory experiments, 1 L volumes of air were collected over 10 min intervals from a three-chamber mosquito behavior assay system. Significantly higher levels of airborne DDT were measured in the chamber containing textiles treated with DDT compared to chambers free of AI. In the field, 57 samples of air were collected from experimental huts with and without DDT for onsite analysis. Airborne DDT was detected in samples collected from treated huts. The mean DDT air concentrations in these two huts over a period of four days with variable ambient temperature were 0.74 µg/m³ (n = 17; SD = 0.45) and 1.42 µg/m³ (n = 30; SD = 0.96).

Conclusions/Significance

The results from laboratory experiments confirmed that significantly different DDT exposure conditions existed in the three-chamber system establishing a chemical gradient to evaluate mosquito deterrency. The TD GC-MS method addresses a need to measure short-term (<1 h) SR concentrations in small volume (<100 L) samples of air and should be considered for standard evaluation of airborne AI levels in mosquito behavior assay systems. Future studies include the use of TD GC-MS to measure other semi-volatile vector control compounds.     

Evaluation of a peridomestic mosquito trap for integration into an Aedes aegypti (Diptera: Culicidae) push-pull control strategy

We determined the feasibility of using the BG‐Sentinel? mosquito trap (BGS) as the pull component in a push‐pull strategy to reduce indoor biting by Aedes aegypti. This included evaluating varying numbers of traps (1–4) and mosquito release numbers (10, 25, 50, 100, 150, 200, and 250) on recapture rates under screen house conditions. Based on these variations in trap and mosquito numbers, release intervals were rotated through a completely randomized design with environmental factors (temperature, relative humidity, and light intensity) and monitored throughout each experiment. Data from four sampling time points (05:30, 09:30, 13:30, and 17:30) indicate a recapture range among treatments of 66–98%. Furthermore, 2–3 traps were as effective in recapturing mosquitoes as 4 traps for all mosquito release numbers. Time trends indicate Day 1 (the day the mosquitoes were released) as the “impact period” for recapture with peak numbers of marked mosquitoes collected at 09:30 or 4 h post‐release. Information from this study will be used to guide the configuration of the BGS trap component of a push‐pull vector control strategy currently in the proof‐of‐concept stage of development in Thailand and Peru.     

Contact Irritant Responses of Aedes aegypti Using Sublethal Concentration and Focal Application of Pyrethroid Chemicals

Background

Previous studies have demonstrated contact irritant and spatial repellent behaviors in Aedes aegypti following exposure to sublethal concentrations of chemicals. These sublethal actions are currently being evaluated in the development of a push-pull strategy for Ae. aegypti control. This study reports on mosquito escape responses after exposure to candidate chemicals for a contact irritant focused push-pull strategy using varying concentrations and focal application.

Methods

Contact irritancy (escape) behavior, knockdown and 24 hour mortality rates were quantified in populations of female Ae. aegypti under laboratory conditions and validated in the field (Thailand and Peru) using experimental huts. Evaluations were conducted using varying concentrations and treatment surface area coverage (SAC) of three pyrethroid insecticides: alphacypermethrin, lambacyhalothrin and deltamethrin.

Results

Under laboratory conditions, exposure of Ae. aegypti to alphacypermethrin using the standard field application rate (FAR) resulted in escape responses at 25% and 50% SAC that were comparable with escape responses at 100% SAC. Significant escape responses were also observed at <100% SAC using ½FAR of all test compounds. In most trials, KD and 24 hour mortality rates were higher in mosquitoes that did not escape than in those that escaped. In Thailand, field validation studies indicated an early time of exit (by four hours) and 40% increase in escape using ½FAR of alphacypermethrin at 75% SAC compared to a matched chemical-free control. In Peru, however, the maximum increase in Ae. aegypti escape from alphacypermethrin-treated huts was 11%.

Conclusions/Significance

Results presented here suggest a potential role for sublethal and focal application of contact irritant chemicals in an Ae. aegypti push-pull strategy to reduce human–vector contact inside treated homes. However, the impact of an increase in escape response on dengue virus transmission is currently unknown and will depend on rate of biting on human hosts prior to house exiting.     

Spatial repellency and other effects of transfluthrin and linalool on Aedes aegypti and Aedes albopictus

In the present study, the effects of two spatial repellents (SR) were determined for Aedes aegypti and Ae albopictus, the main vectors of dengue, Chikungunya, and Zika fever. The modular high‐throughput screening system (HITSS ) was used to evaluate the response of both species to transfluthrin and linalool SR at different concentrations. The highest spatial repellency results for Ae. aegypti were obtained by transfluthrin to 0.001% with 37.50 ± 4.33%, and for linalool to 10% with 77.50 ± 3.90%. For Ae. albopictus, the highest spatial repellency percentages for transfluthrin 0.01% were 45.00 ± 3.78%, and linalool at 1% and 10% were 56.25 ± 7.06% and 56.25 ± 6.46%, respectively. Transfluthrin caused high levels of mortality with 71.25 ± 6.66%, 79.75 ± 8.65%, and 100% to Ae. aegypti and 70.00 ± 5.98% and 98.75 ± 0.82% to Aedes albopcitus. With the results of this study, we concluded that both the transfluthrin and linalool could be used as protection measures against the bite of Ae. aegypti and Ae. albopictus in the integral strategies for the control of vectors in Mexico.     

A new classification system for the actions of IRS chemicals traditionally used for malaria control

Knowledge of how mosquitoes respond to insecticides is of paramount importance in understanding how an insecticide functions to prevent disease transmission. A suite of laboratory assays was used to quantitatively characterize mosquito responses to toxic, contact irritant, and non-contact spatial repellent actions of standard insecticides. Highly replicated tests of these compounds over a range of concentrations proved that all were toxic, some were contact irritants, and even fewer were non-contact repellents. Of many chemicals tested, three were selected for testing in experimental huts to confirm that chemical actions documented in laboratory tests are also expressed in the field. The laboratory tests showed the primary action of DDT is repellent, alphacypermethrin is irritant, and dieldrin is only toxic. These tests were followed with hut studies in Thailand against marked-released populations. DDT exhibited a highly protective level of repellency that kept mosquitoes outside of huts. Alphacypermethrin did not keep mosquitoes out, but its strong irritant action caused them to prematurely exit the treated house. Dieldrin was highly toxic but showed no irritant or repellent action. Based on the combination of laboratory and confirmatory field data, we propose a new paradigm for classifying chemicals used for vector control according to how the chemicals actually function to prevent disease transmission inside houses. The new classification scheme will characterize chemicals on the basis of spatial repellent, contact irritant and toxic actions.     

Effects of environmental conditions on the movement patterns of Aedes aegypti (Diptera: Culicidae) into and out of experimental huts in Thailand

Mark‐release‐recapture experiments with Aedes aegypti were performed using experimental huts equipped with entrance and exit traps to evaluate their movement patterns during a two‐year period in Thailand. Results indicate no significant differences in the patterns of movement between the two years of observation. Movement into the huts occurred during the early morning period (06:00‐11:00) with a peak at 07:00 in the summer and rainy season and 09:00 in the winter. In contrast, the exit pattern was observed during the late morning (09:00‐12:00) and early afternoon (12:00‐16:00), with a peak at 16:00 in the winter, 11:00 in the summer, and 14:00 in the rainy season. Multiple regression analysis indicated that movements of Ae. aegypti females into and out of the huts were impacted by humidity and temperature during the day.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Evaluation of habitat management strategies for the reduction of malaria vectors in northern Belize.

Mowing and burning of emergent vegetation were evaluated as potential management strategies for the control of the malaria vector, Anopheles vestitipennis, in northern Belize, Central America. The primary aim was reduction of tall dense macrophytes (dominated by Typha domingensis) as preferred larval habitat for An. vestitipennis. Nine experimental plots were established in a Typha marsh in Orange Walk District, Belize. Three plots were burned, three were treated by subaquatic mowing, and three were unaltered controls. After treatment, Typha height was most dramatically affected by the mow treatment. Plant heights at 21 and 95 days post-treatment reflected an 89% and 48% decrease, respectively, compared to pretreatment conditions. The Typha height in the burn plots was not as severely affected. Heights at 21 days post-treatment were 39% lower than those of pre-treatment vegetation, with a return to near pre-test heights by 95 days post-treatment. Both treatments resulted in a significant reduction in the number of An. vestitipennis larvae collected as compared to control plots. Conversely, the treatments resulted in increased larval densities of several other vector and pest mosquito species. Larval population densities ofAn. albimanus, Ochlerotatus taeniorhynchus, and Culex coronator were significantly higher in burn plots. In mow plots, there were significant increases in An. albimanus and Oc. taeniorhynchus larval populations. Non-target invertebrate species affected by the treatments were adult Tropisternus collaris, larval Corythrella, and adult Parapleapuella.