Supplemental 1.Arrangement of odor delivery apparatus (A) and olfactometer arena (B) for bioassays of walking Dendroctonus spp. males exposed either to odors of single female entrances or to synthetic odor blends. Olfactometer is a modification of that in Vet et al. (1983). In single-odor bioassays of entrances, one randomly-selected olfactometer arm was arranged as arm 1 (i.e., receiving odors from a female entrance), whereas the remaining arms were arranged as shown for 4 (i.e., receiving clean air). In odor choice bioassays two opposite arms (i.e., 1-3; 2-4) chosen at random were both arranged as shown for arm 1, but with a different log and species of female for each opposite arm and with the remaining two arms arranged as shown for 4. During assays of synthetic mixtures, a release device (Supplemental2) replaced the aeration funnel.
Fine-mesh screen covered the air outlet of the olfactometer and was the surface on which test beetles were released. From each inlet of the olfactometer a piece of rigid PTFE tubing (8 cm long x 0.8 cm diam.) extended within the interior such that the opening of each was directed toward and positioned 6.8 cm from the center of the opening in the arena floor (air outlet) and ~1 mm above the arena floor. The openings were covered with a piece of PTFE screen to prevent entry of beetles. During trials, the surface of the olfactometer arena floor was covered with a piece of white office paper cut to fit this space. This paper had an opening cut for the air exit and a 1 cm diam. printed circle (the response target) positioned immediately in front of the opening of the tubing for each arm inlet (B). The inlet tubing passed through rectangular strips of PTFE sheeting that were formed into arches traversing the internal corners of the olfactometer arms (B); these prevented beetles from crawling into the crevices formed between the tubing and the interior wall of the olfactometer. Pumped air supplied to each arm was first passed through a rotameter and flow control valve (one for each olfactometer arm), then an activated charcoal filter and a humidifier (a sealed glass container within which the air stream was bubbled through purified water) which were connected to each other and the olfactometer arm inlets by means of PVC tubing or corrugated PTFE tubing (A). Air was drawn out of the olfactometer through the screen-covered opening in the center of the arena floor; PVC tubing connecting to this opening conducted air to a rotameter/flow regulator and the vacuum inlet of the pressure pump. The paper floor covering was replaced at least every 20 trials. All equipment was rinsed at the end of each day with ethanol and air-dried overnight.
During bioassays the olfactometer was covered with a clear acrylic plate to form a semi-airtight enclosure for the arena. Air flow maintained through each of the arms was identical (approximately 50 ml/min), whereas flow from the exit was 1.5 l/min. In pilot trials, we found that the beetles had difficulty orienting toward odorized arms in the olfactometer unless flow out the exit was maintained substantially higher than the sum of flow into the arms. This additional air was apparently being drawn through the space between the top of the olfactometer and the acrylic cover, which did not make a fully airtight seal against the top of the olfactometer during trials. It is possible that the enhanced beetle orientation was the result of this additional flow of non-odorized air into the olfactometer as this could have produced odor streams from the arm inlets with more distinct boundaries.
Vet LEM, van Lenteren JC, Heymans M, Meelis E (1983)An airflow olfactometer for measuring olfactory responses of hymenopterous parasitoids and other small insects. Physiol Entomol 8:97-106