Olfactometer Bioassay

In order to validate the olfactory orientation and behavioral response of M. separata to volatiles based on binding assays, olfactometer bioassay was performed ³⁹. A glass Y-tube olfactometer (arms: 3.0cm diameter by 26cm long, base: 4.0cm diameter by 25cm long) was used in this bioassay. The incoming air to the tube was allowed to pass via activated charcoal filter, then humidified with ultra-pure water. The humidified air coming from activated charcoal was divided into two tiny chambers; one holding tested volatile and the other holding liquid paraffin (control). Both tiny chambers were attached with arms of Y-tube. An inline flow-meter (Gilmont Instruments, USA) was used to keep the airflow constant (6.0 L min^-1). Considering the nocturnal behavior of M. separata, the whole experiment was conducted after 1 to 2 h of sunset in the dark room; orange light was used, the temperature was 25±1°C and the relative humidity was 70%±5%. A 10µl of tested volatile was applied to 10×10mm filter paper (Whatman No.1) and was placed in one tiny chamber attached with one arm of olfactometer. A separate olfactometer was used for female and male moth. A three-day-old moth was released in the base of Y-tube and was given 10 min to respond to the volatile or control. To make sure that insects have no exposure to the tested volatiles, female and male moths were kept into isolated test tubes (50ml) and closed with sterilized cotton plug for approximately 30 min before the test. Thirty female and thirty male moths were used for each tested volatile in this behavioral bioassay.