After emerging from the nest during the evening twilight, all the nest-tree foragers followed a stereotypical vertical path up the eucalyptus tree trunk and continued along the same foraging corridor every day. We conducted observations on these experienced foragers for two consecutive nights on their nest trees for control and odour-test purposes. The presence of odours on their foraging corridor resulted in detours for foraging (Fig. 1). Specifically, with Eucalyptus oil, 46.4% of ants walked over the odour, while 53.6% took detours to avoid it. With Olive oil and Flax-seed oil (sources of oleic acid and linoleic acid, respectively), 35% and 40% of foragers walked over the odour, while 65% and 60% took detours. With Tea tree oil and Lavender oil, none of the foragers walked over the odour, with 26% and 22% of foragers taking a U-turn and returning to the nest, while 74% and 78% took detours away from the odour line. These results suggest that foragers exhibit a neophobic response, with nearly half of them avoiding the familiar Eucalyptus oil, more avoiding oils containing oleic and linoleic acid, and complete avoidance of Tea tree and Lavender oils.
In path characteristics, ants exhibited a more curved and meandering trajectory in the odour-change conditions compared to the control conditions. We found differences across conditions in each of our three measures of path meander. Firstly, sinuosity increased significantly in the odour change condition (Olive oil control vs. Olive oil test: t = − 7.47, df = 17.41, P < 0.0001; Flax-seed oil control vs. Flax-seed oil test: t = − 14.12, df = 19.49, P < 0.0001; Tea tree oil control vs. Tea tree oil test: t = − 10.41, df = 18.22, P < 0.0001; Lavender oil control vs. Lavender oil test: t = − 12.2, df = 22.81, P < 0.00001) except for Eucalyptus oil (Eucalyptus oil control vs. Eucalyptus oil test: t = 0.4, df = 23.85, P = 0.68) (Fig. 2A). Secondly, E a max was lower in the odour-change conditions, with the ants having a smaller amount of displacement per unit length travelled compared to the control conditions. The t-tests showed a significant difference in test conditions compared to the control conditions in all comparisions except Eucalyptus oil (Olive oil control vs. Olive oil test: t = 6.18, df = 25.23, P < 0.0001; Flax-seed oil control vs. Flax-seed oil test: t = 7.28, df = 16.86, P < 0.0001; Tea tree oil control vs. Tea tree oil test: t = 9.08, df = 16.98, P < 0.0001; Lavender oil control vs. Lavender oil test: t = 9.64, df = 23.23, P < 0.0001; Eucalyptus oil control vs. Eucalyptus oil test: t = − 0.02, df = 27.59, P = 0.98) (Fig. 2B). With straightness, the odour change led to a significantly higher magnitude of path deviation from a straight-line path compared to paths in the Control conditions, again in all comparisons except Eucalyptus oil (Olive oil control vs. Olive oil test: t = 6.47, df = 14.27, P < 0.001; Flax-seed oil control vs. Flax-seed oil test : t = 10.2, df = 15.52, P < 0.0001; Tea tree oil control vs. Tea tree oil test : t = 8.64, df = 14.32, P < 0.0001; Lavender oil control vs. Lavender oil test: t = 5.03, df = 14.11, P < 0.001; Eucalyptus oil control vs. Eucalyptus oil test: t = 1.75, df = 26.13, P = 0.09) (Fig. 2C).
Odour changes had a noticeable impact on the speed, orientation angular velocity, and duration of the foragers. When the foragers sensed non-visual odour stimuli, they slowed down, frequently shifted their gaze in different directions, and stayed in the recording area longer compared to the respective controls (Fig. 3). In speed, the t-tests revealed significant differences in all comparisons (Fig. 3A), with the ants speeding up with Eucalyptus oil and slowing down with other odours (Eucalyptus oil control vs. Eucalyptus oil test: t = − 3.04, df = 27.63, P < 0.01; Olive oil control vs. Olive oil test: t = 6.57, df = 26.63, P < 0.0001; Flax-seed oil control vs. Flax-seed oil test: t = 5.21, df = 27.01, P < 0.0001; Tea tree oil control vs. Tea tree oil test: t = 8.54, df = 24.25, P < 0.0001; Lavender oil control vs. Lavender oil test: t = 13.94, df = 19.06, P < 0.00001). Changes in odour also increased the magnitude of orientation angular velocity in the foragers (Fig. 3B). The t-tests showed significant differences between all pairs in orientation angular velocity except for Olive oil (Eucalyptus oil control vs. Eucalyptus oil test: t = − 4.64, df = 15.29, P < 0.001; Flax-seed oil control vs. Flax-seed oil test: t = − 20.84, df = 15.16, P < 0.00001; Tea tree oil control vs. Tea tree oil test: t = -8.94, df = 21.12, P < 0.0001; Lavender oil control vs. Lavender oil test: t = − 9.06, df = 25.11, P < 0.0001; Olive oil control vs. Olive oil test: t = 0.33, df = 20.93, P = 0.73). The amount of time spent in the recording area also increased, as foragers stopped just before the odour line and meandered around it (Fig. 3C). Consequently, trips took significantly more time with all odours except for Eucalyptus oil (Olive oil control vs. Olive oil test: t = − 5.3, df = 14.35, P < 0.0001; Flax-seed oil control vs. Flax-seed oil test: t = − 4.8058, df = 16.4, P < 0.0001; Tea tree oil control vs. Tea tree oil test: t = − 6.6, df = 14.66, P < 0.0001; Lavender oil control vs. Lavender oil test: t = − 5.1, df = 14.16, P < 0.0001; Eucalyptus oil control vs. Eucalyptus oil test: t = − 0.70, df = 23.49, P = 0.48).
Odour change increased scanning in foragers. In Control recordings, i.e. those without any odour change, the majority of the foragers performed a single scan across their entire recording area (Fig. 4A), In contrast, on odour trials, where ants experienced a non-visual odour change, foragers scanned at least twice, with a maximum of 28 (Fig. 4A). The t-tests revealed significant differences in the number of scanning bouts in all comparisons (Eucalyptus oil control vs. Eucalyptus oil test: t = − 4.79, df = 18.33, P < 0.001; Olive oil control vs. Olive oil test: t = − 4.8, df = 15.17, P < 0.001; Flax-seed oil control vs. Flax-seed oil test: t = − 4.10, df = 15.94, P < 0.001; Tea tree oil control vs. Tea tree oil test: t = − 5.51, df = 14.93, P < 0.0001; Lavender oil control vs. Lavender oil test: t = − 4.72, df = 15.99, P < 0.001). The duration of scanning bouts also increased significantly with odour changes (Fig. 4B), in all comparisons except for Eucalyptus oil (Olive oil control vs. Olive oil test: t = − 4.29, df = 14.47, P < 0.001; Flax-seed oil control vs. Flax-seed oil test: t = − 2.77, df = 27.87, P < 0.01; Tea tree oil control vs. Tea tree oil test: t = − 4.88, df = 14.56, P < 0.001; Lavender oil control vs. Lavender oil test: t = − 5.42, df = 24.11, P < 0.0001; Eucalyptus oil control vs. Eucalyptus oil test: t = − 1.13, df = 17.60, P = 0.27).