An in vitro assay for testing mosquito repellents employing a warm body and carbon dioxide as a behavioral activator.

We describe here an in vitro behavioral assay for testing mosquito repellents applied in a dose-based manner to a warm body (34 degrees C) in test cages. The system was used to assess the sensitivity of 4-6-day-old Anopheles gambiae to the insect repellent diethyl methyl benzamide (deet). These tests were made in the absence and presence of additional carbon dioxide (CO2) applied as a pulse to activate mosquitoes in the cages. In the absence of the CO2 pulse the mosquitoes hardly responded to the warm body. Increasing the CO2 level in the cage by 1,000 parts per million caused a 25-fold increase in the number of landings by mosquitoes on the warm body in 2-min tests. This mosquito activation allowed the measurement of a significant reduction in the number of landings to bite on the warm body with increasing doses of deet (0.4 to 3.8 microg/cm2). An asymptotic nonlinear model fitted to the repellency data in the presence of CO2 allowed estimation of the effective dose of deet that reduced landings to bite by 50% (ED50) at 0.95 microg/cm2 (5 nmol/cm2) and the corresponding ED95 at 4.12 microg/cm2 (21.5 nmol/cm2). This in vitro bioassay has the advantage of permitting a fast throughput of test products under standardized conditions and is suitable for screenings designed for the purpose of discovering lead products with as yet unknown human toxicological and dermatological profiles.

Deltamethrin Persistence on Textiles Used to Make Visual Targets for Tsetse Fly Control is Positively Correlated With Their Polyester Content.

Insecticide-impregnated traps and visual targets are used for tsetse (Diptera, Glossinidae) population control. Such devices are made with textiles and deltamethrin is frequently the insecticide of choice. However, persistence of an insecticide on textiles is affected by exposure to weather. Here we examine the effect of weathering on the capacity of four textiles with increasing proportions of polyester (0, 35, 65, and 100%) with cotton and viscose to retain deltamethrin. Textiles tested were those used to make visual targets in a pan-African program to maximize target efficiency for controlling tsetse vectors of African trypanosomiasis. Following impregnation in an aqueous suspension of deltamethrin at 1,000 mg/m2, textiles were weathered for 18 mo at Lambwe Valley, Kenya and sampled every 3 mo to make knockdown tests on the tsetse fly Glossina pallidipes Austen. Deltamethrin content of the textiles was established using gas chromatography mass-spectrometry at impregnation and after 9 mo of weathering. Textiles with higher proportions of polyester retained deltamethrin better: respectively, 100% polyester and 65:35 polyester/viscose textiles retained deltamethrin at 17 and 11 mg/m2 9-mo post-treatment that caused 100% knockdown in G. pallidipes after 1 h, and killed 67 and 47% of flies, respectively, after 24 h. Eighteen-month weathered 100% polyester treated textile still knocked down all tsetse exposed to it within 2 h. The LD50 for deltamethrin on filter paper for G. pallidipes was estimated at 28.8 mg/m2, indicating that deltamethrin is more available on polyester to kill tsetse.

Odorant-binding protein-based identification of natural spatial repellents for the African malaria mosquito Anopheles gambiae.

There is increasing interest in the development of effective mosquito repellents of natural origin to reduce transmission of diseases such as malaria and yellow fever. To achieve this we have employed an in vitro competition assay involving odorant-binding proteins (OBPs) of the malaria mosquito, Anopheles gambiae, with a predominantly female expression bias to identify plant essential oils (EOs) containing bioactive compounds that target mosquito olfactory function. EOs and their fractions capable of binding to such OBPs displayed repellence against female mosquitoes in a laboratory repellent assay. Repellent EOs were subjected to gas chromatographic analysis linked to antennogram (EAG) recordings from female A. gambiae to identify the biologically active constituents. Among these compounds cumin alcohol, carvacrol, ethyl cinnamate and butyl cinnamate proved as effective as DEET at an equivalent dose in the repellent assay, and combinations of carvacrol with either butyl cinnamate or cumin alcohol proved to be significantly more effective than DEET in the assay. When tested as spatial repellents in experimental shelters housing sleeping humans in northern Nigeria a binary mixture of carvacrol plus cumin alcohol caused mosquitoes to leave shelters in significantly higher numbers to those induced by DEET in female Anopheles spp. and in numbers equivalent to that of DEET in Culex spp. mosquitoes. These findings indicate an approach for the identification of biologically active molecules of natural origin serving as repellents for mosquitoes.