Experiment 3—Olfactometry Bioassays

Olfactometry bioassays were conducted to verify the odor-mediated preference of male B. germanica for the TSP extract or control food. Experiments comprised three distinct phases.

Ten males were placed in a 1-liter airtight container fitted with a lid containing drilled holes for internal ventilation, provided with a water dispenser and a cardboard strip as harborage. Food was offered in solid form inside a 1.5-ml Eppendorf tube continuously for 5 d. Three treatment groups were prepared: a control group (BLA) where individuals were fed on control food, a TSP group where the males fed on TSP-extract-treated food, and a starved group where no food was provided.

All the treatment groups from phase 1 were starved for 48 h.

Individual males were allowed to make a choice between two olfactory stimuli presented in a Y-tube olfactometer, which was assembled such that the insects were unable to come into contact with the odor sources kept in airtight glass vials placed upstream to the Y-shaped arena. The olfactometer comprised a one-piece Y-shaped glass tube with an internal diameter of 2 cm. The two arms and the stem of the Y-tube were equal in length (22 cm). A 5-cm-long glass tube expansion located on the stem of the Y-tube served as the entry point for an individual cockroach and functioned as an acclimatizing chamber. Male cockroaches were anesthetized by keeping at −18°C for 2 min and then placed individually in the acclimatizing chamber. Air entering the system was purified by means of a carbon filter and then humidified by passing through distilled water. The flow rate was set at 150 ml/min. Bioassays were carried out in the dark, under a dim red light to avoid light interference. Variables recorded were 1) the time spent by each male cockroach in either arm of the Y-tube olfactometer and 2) the first choice and the total number of entries per minute. Each experiment lasted 5 min, and experiments were replicated four times. After testing ten individuals, the connections of the odor sources to the olfactometer arms were exchanged to remove any asymmetrical bias in the set up, and the odor sources were replenished with fresh material. At the end of each day, the Y-tube and glass vials were thoroughly washed with soap, rinsed with ethanol (90%) and dried overnight in an oven at 200°C.