1. Fecal-Derived Phenol Induces Egg-Laying Aversion in Drosophila
Suzan Mansourian, Jacob Corcoran, Anders Enjin, Christer Löfstedt, Marie Dacke, Marcus C. Stensmyr 
Current Biology 
Volume 26, Issue 20, Pages (October 2016)
DOI: /j.cub
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2. Figure 1 Flies Find the Smell of Carnivore Feces Aversive
(A) Oviposition indices (OI) of wild-type (WT) flies from a binary-choice test between standard cornmeal fly food and fly food mixed with mammalian feces. Error bars represent the SEM. Deviation of the response indices (RI) against zero was analyzed for significance with Student’s t test (p < 0.05).
(B) OI of anosmic flies given a choice between fly food and fly food mixed lion feces. Error bars represent the SEM. Deviation of the RI against zero was analyzed for significance with Student’s t test (p < 0.05).
(C) Gas chromatogram traces from odor collections of mammalian fecal samples.
(D) Heatmap coded matrix showing percent contribution of individual volatile chemicals to the total composition of each examined sample. Samples shown are (1) lager beer, (2) riesling wine, (3) Hanseniaspora uvarum, (4) Torulaspora delbrueckii, (5) Saccharomyces cerevisiae (3–5: [3]), (6) banana, (7) apple, (8) mango, (9) orange, (10) pear, (11) tomato (6–11: [4]), (12) Baringo giraffe, (13) African elephant, (14) eland, (15) white rhinoceros, (16) sable antelope, (17) hippopotamus, (18) impala, (19) bontebok, (20) lion, (21) dog, and (22) painted wolf. Numbers to the right refer to the chemicals shown in (F).
(E) Multidimensional scaling plot from a random forest (RF) analysis of the matrix in (D).
(F) Predictor volatiles separating the different classes. Numbers in parentheses refer to the frequency of their occurrence in RF models.
Current Biology  , DOI: ( /j.cub )
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3. Figure 2
Phenol Is Detected by or46aA and Confers Oviposition Aversion in Flies
(A) Oviposition indices (OI) of WT flies from a binary-choice test between standard cornmeal fly food and fly food mixed with synthetic fecal volatiles (10−2 dilution). Error bars represent the SEM. Deviation of the response indices (RI) against zero was analyzed for significance with Student’s t test (p < 0.05). See also Figures S1A–S1C.
(B) OI of WT flies from a binary-choice test between standard cornmeal fly food and fly food mixed with indole and phenol (both 10−2 dilution). Error bars represent the SEM. Deviation of the RI against zero was analyzed for significance with Student’s t test (p < 0.05).
(C) OI of WT flies from a binary-choice test between standard cornmeal fly food and fly food mixed with giraffe feces and phenol (10−2 dilution). Error bars represent the SEM. Deviation of the RI against zero was treated with a Student’s t test (p < 0.05).
(D) PCA plot showing the distribution of the best ligands for adult olfactory system and phenol in odor space as defined by 32 physiochemical descriptors. Dots are heatmap coded as per Euclidean distance from phenol in odor space. Shown are (1) trans-2-hexen-1-al, (2) acetoin, (3) methyl salicylate, (4) methyl heptenol, (5) valencene, (6) ethyl hexanoate, (7) 2,5-dimethylpyrazine, (8) cyclohexanone, (9) 1-hexanol, (10) 4-hexen-3-one, (11) acetal, (12) ethyl crotonate, (13) p-cresol, (14) pentyl acetate, (15) iridomyrmecine, (16) guaiacol, (17) geosmin, (18) methyl acetate, (19) 3-octanol, (20) phenethyl alcohol, (21) trans-3-hexen-1-ol, (22) ethyl lactate, (23) citral, (24) 4-ethylguaiacol, (25) geranyl acetate, (26) farnesol, (27) ethyl 3-hydroxybutyrate, (28) methyl heptenone, (29) 2-phenylethyl acetate, (30) actinidine, (31) 2,3-butanedione, (32) ethyl benzoate, (33) 2-oxopentanoic acid, (34) 1,4-diaminobutanone, (35) acetic acid, (36) butyric acid, (37) pyrrolidine, (38) phenylacetic acid, (39) phenylethylamine, and (40) ammonia.
(E and F) Or46a-Gal4>UAS-GFP expression pattern in the (E) maxillary palp and (F) antennal lobe (AL). See also Figure S1D.
(G) Prestimulation view of Or46a-Gal4>UAS-GCaMP6m showing intrinsic fluorescence from the VA7l glomerulus.
(H) Pseudocolored image showing phenol-induced fluorescence changes in the AL of a Or46a-Gal4>UAS-GCaMP6m fly.
(I) Averaged traces from VA7l glomerulus of Or46a-Gal4>UAS-GCaMP6m flies stimulated with phenol or solvent (water). Shaded areas represent SEM. Gray bar represents stimulus duration (1 s).
(J) Response of induced and non-induced HEK293/Orco/OR46a cells to vehicle, VUAA1, or 50 μM dose of select phenolic volatiles and the predictive fecal volatiles. Error bars represent the SEM.
(K) Response of induced and non-induced HEK293/Orco/OR46a cells to vehicle, VUAA1, or 50 μM dose of various compounds. Error bars represent the SEM.
(L) Response of induced and non-induced HEK293/Orco/OR46a cells to various doses of agonistic compounds. Error bars represent the SEM. See also Figure S1E.
(M and N) OI of flies expressing TNT from the promoter of Or46a from a binary-choice test between standard cornmeal fly food and fly food mixed with (H) phenol (10−2 dilution) or (I) lion feces. Error bars represent the SEM. Deviation of the response indices was analyzed for significance with Student’s t test (p < 0.05).
(O) Behavioral assay for the optogenetic experiment.
(P) OI of Or46a-Gal4>UAS-CsChrimson flies and parental controls provided a choice between red light illuminated or dark egg-laying substrate. Error bars represent the SEM. Deviation of the RI against zero was analyzed for significance with Student’s t test (p < 0.05).
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4. Figure 3 Bacterial Composition of Mammalian Feces
(A) Animals examined: (1) springbok, (2) Speke’s gazelle, (3) reticulated giraffe, (4) Grevy’s zebra, (5) black rhinoceros, (6) spotted hyena, (7) cheetah, and (8) lion.
(B) Heatmap coded matrix showing percent contribution of individual bacterial families to the total composition of each examined fecal sample.
(C) Multidimensional scaling plot from an RF analysis of the matrix in (B).
(D) Predictor volatiles for carnivore and herbivore feces.
(E) Proportion of bacteria genera from Enterobacteriacae and Clostridiacae:1 sequences from carnivore feces.
(F) Schematic model of phenol production in carnivore feces. Shown are (1) L-tyrosine, (2) bacteria, and (3) L-tyrosine-phenol lyase enzyme.
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5. Figure 4 Dung Beetles Find the Smell of Carnivore Feces Aversive
(A) Drawing of the dung beetle Scarabaeus (Kheper) lamarcki. Scale bar, 1 cm.
(B) Tracks from beetles provided with a choice between lion and giraffe feces in an open olfactory binary-choice arena placed inside a wind tunnel with a laminar airflow.
(C) Tracks from beetles confronted with lion feces and visual control (sand).
(D) Tracks from beetles given a choice between giraffe dung with or without phenol added.
Current Biology  , DOI: ( /j.cub )
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