Differential spontaneous responses to odorants containing deuterium. The mean relative distribution of flies in the arms of the maze (% excess flies) carrying the indicated odorants ± SEM is shown in all graphs. This metric reflects the prevailing distribution within the arms of the maze of groups of 40 to 60 flies tested each time. The total number of flies tested in each group is shown and the number of groups tested is n ≥ 6 for all groups. (A) Spontaneous responses to 75 μL normal or d₃-, d₅-, or d₈-ACP, each diluted with isopropyl myristate (IPM) to 1 mL. Flies spontaneously preferred h-ACP over the solvent. In contrast, incorporation of five or eight deuterium atoms results in significantly different distribution (P < 0.001) from that toward h-ACP (attraction) to aversion. In contrast, the response to d₃-ACP was not significantly different (P = 0.012) from that of h-ACP. (B) Flies discriminate against d₈-ACP if presented with equal amount (1:1) of h-ACP (75 μL odorant/925 μL IPM). However, a 50% reduction in the amount of deuterated odorant yielded an equal distribution of the flies in the arms (% excess flies not significantly different from zero), defined as a balanced maze. (C) Similarly equal amounts (200 μL) of h-1-octanol (OCT) and d₁₇-1-octanol yielded strong discrimination against the deuterated odorant, which was eliminated upon reducing it by 75% (1:0.25). (D) In contrast, equal amounts of normal and deuterated benzaldehyde (90 μL) did not result in differential discrimination. In accord, decreasing the amount of h-BZA resulted in differential avoidance of d₅-BZA. (E) The preferential discrimination against d₈-ACP was eliminated in Or83b¹ and Or83b² mutants (P < 0.002 and P < 0.001, respectively, Dunnett test). (F) Similarly, discrimination against d₁₇-octanol was eliminated in Or83b¹ and Or83b² mutants (P < 0.001 for both, Dunnett test).

Conditioned avoidance of normal and deuterated chemically identical odorants. The mean relative distribution of flies in the arms of the maze (% excess flies) carrying the indicated odorants ± SEM is shown. The total number of flies tested in each group is shown and mean was derived from at least six repetitions per group. Drosophila was conditioned to selectively avoid the indicated member of an odorant pair by coupling it to electric foot shock (indicated by the lightning symbol). The complementary odor of each pair was used as the unpunished control. Each experiment included a naive group (open bar) to establish balancing of the odors used for testing and was used as control for all statistical comparisons on how conditioning resulted in subsequent selective avoidance of the punished odorants. Asterisks indicate significant such differences that are significant. (A) Flies selectively avoided h-ACP or d₈-ACP as expected based on the conditioning scheme and illustrated by their differential distribution in the arms of the maze. The performance of both groups was significantly different from that of naive animals (P < 0.001, Dunnett test). (B) Selective discrimination (P < 0.001 vs. naïve distribution; open bars) against h-octanol and d₁₇-1-octanol in accord with the punished odorant during training. (C) Conditioned discrimination against h-BZA (P = 0.001 vs. naive) and d₅-BZA (P < 0.001 vs. naive, Dunnett test).

Drosophila can be conditioned to selectively avoid deuterium. The mean relative distribution of flies in the arms of the maze (% excess flies) carrying the indicated odorants ± SEM is shown and the total number of flies in each group is denoted. The mean was derived from at least six repetitions per group. Drosophila was conditioned with the indicated pairs of isotopes, but tested against a different pair as indicated. The test odorants were adjusted such that avoidance of naive animals was as near zero as possible (open bar) and was used to compare the distribution of conditioned animals. (A) Animals conditioned with h-1-octanol, but tested after training with h-BZA versus d₅-BZA exhibited significantly different distribution than that of naive animals (P < 0.001 vs. naive for both, Dunnett test). Animals trained with d₁₇-1-octanol selectively avoided the deuterated test odorant and vice versa. (B) Animals punished to h-ACP discriminated selectively against h-BZA, albeit not as robustly as previously described (P = 0.004 vs. naive, Dunnett test), but ones trained to avoid d₈-ACP avoided the deuterated BZA efficiently (P < 0.001 vs. naive, Dunnett test). (C) Flies conditioned to with h-BZA/d₅-BZA exhibited efficient selective avoidance of d₈-ACP and h-ACP, respectively (P < 0.001 vs. naive for both, Dunnett test).

Drosophila can be conditioned to selectively avoid a vibrational frequency. (A) Computed vibrational spectra (IR intensities) of h-OCT versus d₁₇-OCT. Salient spectral peaks are indicated on the graphs and show that deuteration of octanol shifts the group of C-H stretch vibrations from around 3,000 cm⁻¹ to 2,150 cm⁻¹. Deuteration also shifts downward all of the peaks on the fingerprint region (1,000–1,500 cm⁻¹). (B) Computed IR intensities of citronellal versus citronellyl nitrile with the salient spectral peaks indicated. The spectra of citronellal and citronellyl nitrile are remarkably similar in the fingerprint region. They differ chiefly in the vibrations involving the terminal functional group: in citronellyl nitrile the aldehyde carbonyl stretch around 1,750 cm⁻¹ is absent, replaced by a nitrile stretch around 2,150 cm⁻¹. The low-lying aldehyde C-H stretch vibration is also absent. The vibration band centered at 2,150 cm⁻¹ is the only one common to d₁₇-1-octanol and citronellyl nitrile but not present in h-octanol or citronellal. The spectra were computed using the Amsterdam Density Functional software at DZP/PBE level of theory. (C) Drosophila selectively avoid the molecular vibrations of deuterium. Flies conditioned to selectively avoid d₁₇-1-octanol exhibited strong preferential avoidance of citronellyl nitrile (P < 0.001 vs. naive), but flies punished to h-octanol did not selectively avoid citronellal (P = 0.691 vs. naive). The only common element potentially recognizable in the test odor pair to aid in selective avoidance is the overlap in the vibrational spectrum of the C-D bonds in d₁₇-1-octanol and the C≡N triple bond in citronellyl nitrile as illustrated in A and B. In contrast, they were not selective toward a novel odor without any recognizable molecular features. (D) In the converse experiment, flies conditioned to selectively avoid citronellyl nitrile exhibited highly significant avoidance of d₁₇-1-octanol as a testing odor (P < 0.001 vs. naive), but flies punished to citronellal did not selectively avoid h-octanol (P = 0.999 vs. naive).