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.