2.11 Behavioral bioassays
Behavioral studies were performed in a dark room under red light (2.5-3 lux) at 21±2°C and 70±5% relative humidity. Male moths were tested in a wind tunnel as described in [22]. For odor delivery, a piece of triangular-shaped filter paper (2.5 cm base, 4 cm height) was pipetted with appropriate volume of the test solution and, following solvent evaporation, was hung from the suction hook in the tunnel.
Pheromone standards and BioPhe samples were dissolved in pentane. The major and minor sex pheromone components Z 11-14:OAc andE 11-14:OAc, respectively, were mixed at a 97:3 ratio and were used as a positive control at a 50 μg dose, which, through preliminary testing, was concluded to be the minimum amount eliciting positive courtship behavior, i.e., source-oriented flight pattern, abdominal hair pencils display and abdomen curling upon contact. Additionally, to evaluate potential antagonistic effects of BioPhe blend contaminants on the males’ responses, 14:OAc (most prominent component ~50% of BioPhe blend) and Z 9-11:OAc (~9% of BioPhe blend) have been tested.
A dose of 700μg BioPhe was used in the bioassays (containing 50μgZ 11-14:OAc and 350μg 14:OAc) and a dose of 350μg was added to 50μg pheromone standard (97:3) to emulate abundance and ratio in the BioPhe. Also a dose of 50μg pheromone standard (97:3) with the addition of 4.5μg of Z 9-16:OAc (9% present in the BioPhe blend) was tested, as to our knowledge the role of Z 9-16:OAc has not been investigated as antagonist to male response. Other components of BioPhe, such as Z 9-14:OAc have already been reported in the literature as antagonist to male response [23,24]. Behavioral tests were conducted between the 3rd and 5th hour in the scotophase [24,25]. Two hours before testing, 2-5 days old male moths were transferred in individual 400 mL clear plastic cups covered with perforated lids and left in the conditions of the dark room to acclimate. For each test, the odor source was attached to the tunnel ceiling, and after 30 seconds, an individual male was released in the tunnel, and its behavior was recorded for 10 min.
The following sequences of behavior were recorded: close approach (less than 10 cm) and contact with the source (landing). In addition, the approach and landing steps were further analyzed into four grades, and each grade was assigned its corresponding value. For the landing step, grades were discriminated as follows: grade 1) brief contact with the source (no landing), grade 2) landing on source for 1-2 sec, grade 3) landing on source for more than 2 seconds, and grade 4) landing on source accompanied by hairpencils display and/or abdomen curling (copulation attempt). Similarly, for the approach step were discriminated as: grade 1) close approach to source for 1-2 sec, grade 2) close approach with zig-zag patterned flight for > 2 sec, grade 3) close approach with zig-zag patterned flight and hairpencils display for 1-2 sec, grade 4) close approach with zig-zag patterned flight and hairpencils display for > 2 sec.
Between treatments, the flight tunnel inner walls and suction hook were wiped with acetone and left to aerate for 10 min before the next treatment. For each treatment, 30 males were tested. Males were used once and were discarded after testing.