Patent Application: US-90855805-A

Abstract:
this invention provides an apparatus and a method for testing insect repellents that is derived from the behavior of mosquitoes in nature . the method is based on the displacement of mosquitoes from a place on a created habitat using a repellent substance . it does not require attractants , or blood - hungry mosquitoes , and it is appropriate for high throughput testing of different materials at the same time and in few hours .

Description:
the natural tendency of the most routine testing mosquito model , aedes aegypti , is to rest on the walls and ceiling of the common laboratory wire mesh cage rather than to stand on the bottom . this tendency is observed in many mosquito species . when the surfaces of walls and ceiling of the cage are changed to smooth and polished — such as acrylic plastic or glass —, the insects are forced to rest on the bottom . if a rough surface is provided on the ceiling of that smooth box , that rough surface becomes the preferred resting place for the mosquitoes . it is on the above observations that the present method is based . the following description of the bioassay uses definite dimensions and materials for the corked - box ; however , other dimensions and materials can be used according to the needs . to perform the bioassay , a 2 ft × 1 ft × 1 ft box , such as the one depicted in fig1 , is built of plexiglass with minute aeration holes on the side panels , 8 holes ( 1 . 5 inches diameter each , 6 inches apart from each other ) on the top to accommodate as many # 21 cork stoppers . two larger holes ( 4 inches diameter ) on the front panel fitted with a cotton sleeve for easy hand access , and a 4 inch square mosquito access opening with door at the bottom completes the box design . food ( 10 % sucrose ) and water are provided inside the box in two small erlenmeyer flasks with cotton wicks . under normal conditions , when the box is filled through the mosquito access opening , with about 400 mixed - sex mosquitoes , 20 to 40 will rest on each cork . thus , the size of the corks and the number of mosquitoes per cubic feet are important for the test to be statistically significant . the cork material is not an attractant ; a cork sitting at the bottom of the cage does not attract mosquitoes . the design of this box and testing conditions permits the evaluation of 8 substances at the same time with no crossover effect from one cork to its neighbors . when a cork impregnated with a repellent is replaced with a new cork , mosquito landing on the new cork is immediate . thus , testing can be performed continuously on different compounds . repellent testing is performed with mosquitoes 3 days to 3 weeks old that are maintained at 70 - 80 % humidity and a temperature of 27 - 29 ° c . with an artificial 16 - 8 day - night cycle . on average 200 - 300 mosquitoes per cubic foot provide optimum results . substances to be tested are prepared at a concentration 0 . 015m in ethanol . this concentration is important because 1 ) the activity of repellents is directly proportional to concentration and 2 ) for relative comparison to the standard deet . a cork impregnated with a solution of deet at that concentration ( 0 . 015m ) produces a repellence time ( rpt ) of about 4 h . repellency time ( rpt ) is defined as the time between the introduction of an impregnated cork into the testing chamber and the time one mosquito lands and remains on that cork for 1 minute . in a typical experiment , a cork is immersed for 30 seconds in an ethanol solution of the compound under study at a concentration of 0 . 015m . the alcohol is allowed to evaporate ( about 10 min .) and the cork is inserted into one of the top holes of the testing chamber that contains between 200 and 300 mosquitoes per cubic foot . the time is recorded and the cork is observed directly by a technician or monitored by a video camera connected to recorder for later evaluation . the presence of one mosquito on the cork for 1 minute determines the end of the test , and the repellency time ( rpt ) is then noted . the area of substance coverage on the cork is about 30 cm 2 , and the amount of substance on the cork after evaporation of the alcohol is about 7 . 7 × 10 − 4 mmol / cm 2 . blank corks are immersed in pure ethanol for the same amount of exposure and evaporation times . however , blank corks are not required except to verify that the alcohol does not contain repellent substances as contaminants . the following comparison of repellencies was made between reported in - vivo values from different sources and those obtained using the in - vitro method presented here . the list shows that a correlation exists between the two methods and that the in - vitro method can be used to predict the repellency time of a given repellent in humans . compounds o - deet , m - deet , and p - deet correspond to the three isomers of the same material ; the m - deet is the most active and it is the one used as the standard . rpt ( min ) rpt ( min ) compound in - vivo in - vitro o - deet 90 20 ± 5 m - deet 300 240 ± 20 p - deet 240 210 ± 20 camphor 0 - 60 15 ± 2 geraniol 120 - 180 315 ± 20 nerolidol 120 - 180 300 ± 25 citronellal 0 - 60 0 ± 5 2 - tridecanone 0 - 60 20 ± 5 amyl alcohol 0 - 60 5 ± 1 cyclohexanol 0 - 60 10 ± 1 cyclohexanone 0 - 60 0 oleic acid 0 - 60 0 abdel - rahman , a ., shetty , a . k ., abou - donnia , m . b . 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