What's that smell? Hummingbirds avoid foraging on resources with defensive
Ashley Y. Kim1,2 David T. Rankin2,3 and Erin E. Wilson Rankin2*
1Department of Entomology and Nematology, University of California, Davis
2Department of Entomology, University of California, Riverside
3Department of Evolution, Ecology, and Organismal Biology, University of California, Riverside
Supplemental Information
Figure S1. Detection of formic acid up to 30 minutes after application to filter paper. We placed damp litmus paper 2-4 mm above the formic acid droplets (leftmost panel) for 60 seconds every 5 minutes (remaining panels). We observed the damp litmus paper turned pink/red, indicating the presence of an acid. The litmus paper was not allowed to touch the filter paper. A control litmus (dampened only with DI water) is included in photos for comparison. This experiment was replicated 4 times in the lab (T = 21.3-23 °C), with damp litmus turning pink in every instance 5- 30 min after formic acid application.
insect compounds
Behavioral Ecology and Sociobiology
(doi: 10.1007/s00265-021-03067-4)* corresponding author: e.wilson.rankin@gmail.com
Figure S2. Filter papers under ultraviolet light. To rule out any visual cues of our treatments, we took photos of filter paper 1 minute after applying the control and treatment solutions. As
hummingbirds also can see in the UV-spectrum, photos were taken both under white and UV light conditions. We observed no difference between the treatment or control compounds, thus suggesting any behavioral responses observed were not due to visual cues. Hexane control and honey bee cuticular hydrocarbons under white light (A) and UV light (B). Water control and 60% formic acid under white light (C) and UV light (D). Ethanol control and (Z)-9-hexadecenal under white light (E) and UV light (F).
Table S1. Statistical analyses of the effects of temperature, trial, and treatment by temperature interactions. For each analysis, we compared the full model with another model with the factor of interest removed using the anova function. The factor was considered to have a
significant effect if the models were significantly different.
CHC Formic acid (Z)-9-hexadecenal
Hummingbird context Variable Chi sq df p-value Chi sq df p-value Chi sq df p-value Wild
Average duration per feeding visit
(sec)
treatment 0.24 1 0.62 3.31 1 0.07 0.05 1 0.82
temperature 0 1 1 0 1 1 0 1 1
trial 0.011 2 0.99 6.41 2 0.041† 2.02 2 0.36
temperature * treatment 0.096 1 0.76 2.99 1 0.084 5.53 1 0.02
Average number of feeding visits
treatment 0.03 1 0.86 10.9 1 0.001 4.98 1 0.026
temperature 0 1 1 0 1 1 0 1 1
trial 11.312 2 0.0035‡ 2.09 2 0.35 5.65 2 0.59
temperature * treatment 0.63 1 0.43 0.88 1 0.35 0.245 1 0.62
Aviaries
Average duration per feeding visit
(sec)
treatment 0.007 1 0.93 1.05 1 0.31 0.25 1 0.62
temperature 0 1 1 0 1 1 0 1 1
trial 2.9 3 0.41 3.01 3 0.39 1.38 3 0.85
temperature * treatment 1.09 1 0.3 2.61 1 0.11 0.7 1 0.4
Average number of feeding visits
treatment 2.39 1 0.12 4.61 1 0.032 5.79 1 0.016
temperature 0 1 1 0 1 1 0 1 1
trial 2.46 3 0.48 0.87 3 0.83 5.5 4 0.24
temperature * treatment 1.43 1 0.23 0.17 1 0.67 0.99 1 0.32
†: Post-hoc Tukey tests detailed no significant differences among the four trials: trial 1 vs trial 2: p = 1; trial 1 vs trial 3: p = 0.55; trial 1 vs trial 4: p = 1; trial 2 vs trial 3:
p = 1; trial 2 vs trial 4: p = 0.94; trial3 vs trial 4: p = 0.28.
‡: Post-hoc Tukey tests detailed no significant differences among the four trials: trial 1 vs trial 2: p = 0.93; trial 1 vs trial 3: p = 0.27; trial 1 vs trial 4: p = 1; trial 2 vs trial 3: p = 0.058; trial 2 vs trial 4: p = 0.25; trial3 vs trial 4: p = 0.99
Table S2. Statistical results for testing hummingbird response to ethyl butyrate, a food additive that has been used to test olfactory learning and discrimination in a diversity of animal systems (e.g., Goldsmith and Goldsmith 1982, Angely and Coppola 2010, Cunningham et al. 2016, Lee et al. 2017, Stuhl 2020).
5% Ethyl butyrate (EtBu)
Hummingbird context Variable Chi sq df p-value
Wild
Average duration per feeding visit (sec):
13 ± 1 sec (EtBu) vs 16 ± 1 sec (Control) (mean ± SE)
treatment 1.09 1 0.58
temperature 0 1 1
trial 8.1 4 0.09
temperature * treatment 0.001 1 0.99
Average number of feeding visits:
23.6 ± 1.5 visits (EtBu) vs 20.6 ± 3.3 (Control) (mean ± SE)
treatment 1.97 1 0.16
temperature 0.001 1 0.99
trial 9.48 4 0.051
temperature * treatment 1.66 1 0.20
References cited in Electronic Supplementary Material
Angely, C. J., and D. M. Coppola. 2010. How does long-term odor deprivation affect the olfactory capacity of adult mice? Behavioral and Brain Functions 6.
Cunningham, J. P., M. A. Carlsson, T. F. Villa, T. Dekker, and A. R. Clarke. 2016. Do Fruit Ripening Volatiles Enable Resource Specialism in Polyphagous Fruit Flies? Journal of Chemical Ecology 42:931-940.
Goldsmith, K. M., and T. H. Goldsmith. 1982. Sense of smell in the Black-chinned hummingbird.
The Condor 84:237-238.
Lee, S., Y. J. Kim, and W. D. Jones. 2017. Central peptidergic modulation of peripheral olfactory responses. Bmc Biology 15:e35.
Stuhl, C. J. 2020. The development of an attract-and-kill bait for controlling the small hive beetle (Coleoptera: Nitidulidae). Apidologie 51:428-435.
