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Determination of the efficacy
Bioassay procedures
Biological material
Non-blood fed females of Anopheles gambiae s.s., 2 to 5 day old will be used during the study. This strain is a standard susceptible strain (Kisumu, originated from Kenya) colonized for many years in our laboratory and free of any detectable insecticide resistance mechanisms.
WHO cone test method
The WHO cone test measures knock down and mortality of susceptible mosquitoes exposed in a small chamber to a
piece of treated netting (25x25 cm) for a 3 min exposure time (Müller et al. 2002; Ordonez Gonzalez et al. 2002; WHO 1975). In order to stick to this short exposure, batches of only 5 females will be introduced in cones at a time. Four cones will be used on the same net sample. An interval of 10 to 15 seconds will be observed during each batch of 5 mosquitoes. A total of 5 replicates of 10 mosquitoes will be used for each sample tested (10x5 = 50 mosquitoes). After testing, females will be grouped in plastic cups (10 individuals per cup) with honey solution provided and maintained during 24 hours at 25 °C and 80% RH. KD effect will be recorded 60 min post exposure and percentage mortality after 24 hours. Mosquitoes able to fly will be counted as alive. However, it is commonly observed, when testing pyrethroids, surviving mosquitoes able to fly with several legs missing, sometimes up to 5. This phenomenon of leg auto-section related to pyrethroids is likely to occur in the field as well as in the lab. For this reason, we will also record mosquitoes surviving with 3 legs or less, since these mosquitoes are unlikely to survive in the field (not able to bite and/or to lay eggs). We will count them as “functional mortality” (dead+alive with 3 legs or less) by opposition to effective mortality. Difference between effective and functional mortality is sometime important and this double count gives a better estimation of the overall killing effect of the insecticide
Experimental tunnels
The tunnel test measure mortality and blood feeding success of host seeking mosquitoes in an experimental chamber (Corbel et al. 2004). The basic equipment consists of a tunnel (square section 25x25 cm) made of glass, 75 cm length. At one third of the length, a disposable cardboard frame is placed with the treated netting sample. The surface of netting “available” to mosquitoes is 400 sq.cm (20x20 cm) with 9 holes 1 cm diameter precisely positioned. Within the small side, a guinea pig will be placed (unable to move but available for biting). At the end of each side of the tunnel, a 30 cm square cage is fitted, covered with polyester netting. In the cage placed at the end of the long side, 100 females are introduced at 6 PM. Females are free to fly in the cage and to move towards the bait. To reach the bait, they have to fly along the treated net, locate holes and pass through the nets. Then, mosquitoes can easily bite the guinea pig. After blood meal, females usually fly to the cage at the end of this compartment and rest. During exposure, cages are maintained in a tropical glass house at 28°C and 80 % RH with natural day light. The following morning, at 9 AM, females are removed separately from each side of the glass cage and count as alive/dead and blood fed/unfed. Nets washed 20 washes that cause mortality greater than 80% and/or blood feeding inhibition greater than 95% are considered by WHOPES as a Long Lasting Insecticidal Nets.
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