The apparatus designed for hygrotactic studies was made of a culture dish (ϕ55 mm; height, 10 mm) covered with a 100 mesh nylon net. 50 flies aged between four and six days were placed inside the apparatus, and were then dehydrated in a chamber with anhydrous calcium chloride (25°C; relative humidity, ~10%) for the times indicated. The apparatus with dehydrated flies was then placed above a water-containing vial (ϕ10 mm). The distance between the nylon net and water surface was 1 mm. The locomotion of dehydrated flies was record by a digital camera. Hygrotactic behavior assays were performed at 25°C with a relative humidity of 40~50%.
To quantify the hygrotactic behavior, we defined a circular region (ϕ25 mm) in the center of the dish and counted the number of flies in the region during the test. The aggregation values at different time points during the test were calculated using the following formula: (NOt—NO0) / NOsum. Here, NOt is the number of flies in the defined region at the moment; NO0 is the number of flies in the region at the beginning of test; the total number of flies (NOsum) in our tests was 50. The hygrotaxis index is defined as the average of all aggregation values measured at five-second intervals within each five-minute test.
To examine the behavioral responses towards other volatile compounds in dehydrated flies, water was replaced by selected volatile compound. Other experimental conditions were the same as described above.
Unless otherwise indicated, flies used in the hygrotaxis assays were female.
To quantify the hygrotactic behavior, we defined a circular region (ϕ25 mm) in the center of the dish and counted the number of flies in the region during the test. The aggregation values at different time points during the test were calculated using the following formula: (NOt—NO0) / NOsum. Here, NOt is the number of flies in the defined region at the moment; NO0 is the number of flies in the region at the beginning of test; the total number of flies (NOsum) in our tests was 50. The hygrotaxis index is defined as the average of all aggregation values measured at five-second intervals within each five-minute test.
To examine the behavioral responses towards other volatile compounds in dehydrated flies, water was replaced by selected volatile compound. Other experimental conditions were the same as described above.
Unless otherwise indicated, flies used in the hygrotaxis assays were female.
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