First, to determine how an invertebrate blood-seeking animal responds to the odor of E2D, we examined blood chemotaxis of the stable fly, Stomoxys calcitrans, a livestock pest of which both sexes require blood meals for survival and reproduction26. We assayed flies (n = 35) in a Y-maze setup (Fig. 1B) in which the flies were first provided with a choice between E2D and a near-odorless organic solvent (diethyl phthalate) within the context of a host odor (horse or cattle). Given this choice, the flies significantly preferred E2D (χ 2(1) = 25.7195, p < 0.001; see Fig. 1C, left panel). Next, we contrasted E2D against real blood (cattle), again within the context of a host odor, and the flies now displayed the same level of approach response to E2D as to the odor of blood, i.e., equating the two odor sources (χ 2(1) = 1.847, p = 0.17; see Fig. 1C, right panel).

We then sought to determine whether E2D would be sufficient to elicit approach responses typical to those elicited by real blood in a large mammalian predator. To this end, we tested the Eurasian wolf (Canis lupus), a gregarious carnivore that relies heavily on olfactory information when tracking and hunting prey27. We placed odor-impregnated wooden logs into the wolf packs’ (n = 7) outdoor enclosure to allow the animals to freely interact with the logs. Over a twenty-day experimental period, the wolf pack was presented with logs impregnated with either E2D, real blood (horse), or two control conditions – a fruity odor (iso-pentyl acetate) or the near-odorless organic solvent (diethyl phthalate), while individual interactions with the logs were recorded (see Fig. 1D). There were significant differences in the number of interactions between odorized logs (χ 2(3) = 17.597, p < 0.001). As hypothesized, the wolves displayed a higher number of interactions when presented with E2D compared to the fruity odor (p < 0.05) and the near-odorless solvent (p < 0.05). There was, however, no significant difference between E2D and the full blood odor mixture (p = 0.997). The wolves also displayed a significantly higher number of interactions when presented with horse blood compared to fruity odor (p < 0.05) and the near-odorless solvent (p < 0.05). The number of interactions did not differ significantly between the fruity odor and the solvent (p = 1.0; see Fig. 1E).

These findings suggest that for blood-seeking invertebrates and carnivorous mammals alike, E2D serves as a conserved chemical cue indicating the presence of blood. In short, we conclude that for both vertebrate and invertebrate blood-seeking animals, the monomolecular odor E2D is sufficient as a chemical cue to initiate an approach behavior that is indistinguishable from that elicited by the complex odor mixture of natural mammalian blood.

Having concluded that predators and blood-feeding animals use E2D as a chemical cue to locate and approach blood, we next asked whether E2D would induce avoidance responses in prey species. First, we tested the behavioral response of mice (Mus musculus), a known prey species for which the odor of blood might indicate the presence of injured conspecifics. We tested predator-naïve CD-1 mice, an outbred strain which has a behavioral phenotype more similar to wild mice compared to inbred strains28. We measured time spent on either side of a 2-compartment test arena, with the near-odorless organic solvent (diethyl phthalate) on one side and either E2D, real blood, or a fruity odor (n-pentyl acetate) on the other (see Fig. 2A). Both the odor of E2D and the odor of the full blood mixture generated aversive behavior in mice. The mice (n = 60) spent significantly more time in the near-odorless compartment compared to both E2D (V = 2792.5, p < 0.05) and blood (V = 58893.5, p < 0.0001), but not compared to the fruity odor (V = 31595, p = 0.65). As with flies and wolves, the response to E2D and blood did not significantly differ from each other (χ 2(1) = 2.3726, p = 0.12).

Figure 2 E2D induces avoidance behavior in the prey species mouse and in human participants. (A) Upper panel: A mouse displaying aversion towards E2D. Lower panel: mean aversion index of the time spent in the non-odorized compartments. (B) A human participant standing on the force platform measuring approach (anterior)-avoidance (posterior). (C) Mean total movement (cm) in the anterior-posterior direction as a function of E2D, and the control odors trans-2-decenal (T2D) and butanol (BUT). (D) Mean GSR signal as a function of E2D, T2D and BUT. (E) Mean visual reaction time to affective faces in human participants as function of E2D, BUT, and odorless control. Error bars indicate s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

Taken together, these experiments confirmed that the mammalian blood odor component E2D elicits approach-avoidance responses in mammalian predator and prey species as well as in blood-feeding invertebrates, in accordance with their ecological niches. Our findings also show that E2D initiates a behavioral response as efficiently as a natural, complex blood odor.

Next, we sought to determine whether E2D is conserved as a chemical alarm cue in humans (Homo sapiens) as well. We hypothesized that E2D would serve as a contextual threat and alarm cue that warns the individual of impending danger. According to the well-validated threat-imminence and defense-cascade model, behavioral responses to stimuli that warn the individual of a potential threat in the vicinity are manifested by active avoidance responses, increased arousal, vigilance, and attention29,30. We first assessed whether participants would demonstrate an automatic avoidance response towards the E2D odor using a strain gauge force plate (see Fig. 2B). This is a method for measuring approach-avoidance responses in humans by the use of balance-dependent sway in the anterior–posterior (AP) direction31. Moreover, we measured physical arousal by means of galvanic skin responses (GSR). Specifically, we hypothesized that participants would demonstrate an avoidance response by initiating a backward leaning motion and elevated physical arousal response towards the E2D odor. To control for odor specificity of the behavioral and autonomic response, we used two control odors, n-butanol, frequently used in human olfactory research, and trans-2-decenal, a molecule that is structurally similar to E2D, differing only by the presence vs. absence of a functional epoxy group. We matched all three odors on intensity, using weak but detectable iso-intense concentrations. Notably, there were no significant differences between odors in either pleasantness or familiarity. All three odors were rated as neutral in valence (iso-pleasant) and low in familiarity (see Materials and Methods section for ratings and analysis). Participants (n = 40) were standing on the force plate blindfolded and E2D, n-butanol, and trans-2-decenal were birhinally presented using a computer-controlled olfactometer32 (see Fig. 2B). Importantly, participants could not anticipate the stimuli onsets, and no mention of blood or blood odor was given either before, during, or after the experiment. Post-experiment control questions showed that no participant had any association with the odor of blood (see Materials and Methods section for more information). In response to 2 s long odor puffs, we observed that E2D increased movement in the AP axis significantly more than both odorant controls, n-butanol (estimate = −0.010851, t(2242) = 2.395, p < 0.05), and trans-2-decenal (estimate = −0.013613, t(2242) = 3.158, p < 0.01), whereas the latter two did not significantly differ from each other in their total AP axis movement (estimate = −0.002762, t(2242) = 0.610, p = 0.81; see Fig. 2C). Subsequent post-hoc tests against zero (no movement) demonstrated that E2D increased movement towards the posterior, a leaning back motion (p < 0.05) whereas both control odors did not differ from baseline (n-butanol: p = 0.15, and trans-2-decenal(p = 0.31). Similarly, E2D increased the participants’ physical arousal by inducing a significantly higher GSR signal than both control odors (n-butanol, estimate = 4.2615, t(2242) = 4.515, p < 0.001, and trans-2-decenal (estimate = −4.0195, t (2242) = 4.478, p < 0.001), which in turn did not significantly differ from each other (estimate = 0.2421, t(2242) = 0.256, p = 0.96). Both of these responses, the leaning back motion and the increased physical arousal, are viewed as hallmarks of avoidance behavior in humans31,33.

Having established that E2D induced an avoidance response in humans, we next asked if this alarm cue has broader cognitive implications. We tested this by studying if E2D modifies the emotional saliency of visual stimuli by increasing vigilance and attention. Earlier studies have shown that ecologically relevant chemosensory cues, such as body odors, can modify behavioral responses to ambiguous signals of visual threat by making them more similar to unambiguous warning signals34. Accordingly, we used a well-validated visual search task35 that contained affective facial stimuli while measuring reaction time (RT) and GSR, as the autonomic measure of arousal. We used schematic rather than real faces for three principal reasons: (i) their stereotypical expressions of affective valence, (ii) to limit the influence of previous associations and gender/racial biases that real faces would evoke, and (iii) ensuring low perceptual expertise with the images across participants32. These parameters mean schematic faces produce lower variability in evoked arousal than real faces do32. In three separate blocks, participants (n = 33) were instructed to, as quickly as possible, determine whether all faces in a 3 × 3 array of schematic faces were identical or if one was different while being exposed to either E2D, n-butanol, or clean air (same concentrations as in the previous experiment). An angry or a happy face was present as a target among neutral distractor faces; previous studies have demonstrated an angry/threat superiority within this task32. As hypothesized, there was a main effect of chemosensory cue on both RT (F(2, 64) = 6.168, p < 0.01 (see Fig. 2E), and GSR (F(2, 64) = 3.241, p < 0.05; see Fig. 2D). Pairwise comparisons revealed lower RTs for E2D than for both the odorant control n-butanol (p < 0.01), and the odorless control, p < 0.05, but the latter two did not significantly differ from each other (p = 0.32). Exposure to E2D, but not n-butanol or clean air, speeded RTs to happy faces bringing them to the same level of response time as to that of angry faces. Replicating our previous GSR finding, E2D increased GSR significantly more than n-butanol (p < 0.05), and odorless control (p < 0.05), whereas the latter two did not significantly differ (p = 0.625).