A common genetic target for environmental and heritable influences on aggressiveness in Drosophila

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   Fig. 1.

   Social experience influences Drosophila aggressiveness. (Upper) Experimental manipulations. (Lower) (A) Mean fighting frequencies in 3-day-old, single-housed and group-housed flies (mean ± SEM; n = 6 experiments each containing five fly pairs; ∗∗, P < 0.01). (B) Mean fighting frequencies in 6-day-old, S → S, G → S, G → G, and S → G flies (mean ± SEM. n = 6 experiments each containing five pairs. Significant differences (P < 0.05) are indicated by letters above bars). (C) Median lunging intensities of 3-day-old, single-housed flies, and 6-day-old, S → S, G → S, G → G, and S → G flies [n = 16, 25, 19, 2, and 1 pairs, respectively; N.S., not significantly different (P > 0.05)]. (D) Median lunging latencies of 3-day-old, single-housed flies and 6-day-old, S → S, G → S, G → G, and S → G flies [n = 16, 25, 19, 2, and 1 pairs, respectively; N.S. (P > 0.05)]. Comparisons between groups were made by using the Mann–Whitney U test (A), ANOVA followed by post hoc test (B) and Kruskal–Wallis ANOVA (C and D).

   
   
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   Fig. 2.

   Cyp6a20 expression is correlated with social experience. (Upper) Experimental manipulations. (Lower) (A) Relative levels of Cyp6a20 mRNA (normalized to Ddc mRNA levels) in 3-day-old, single-housed and group-housed flies (mean ± SEM; n = 4; ∗∗, P < 0.01). (B) Relative levels of Cyp6a20 mRNA in 6-day-old, S → S, G → S, G → G, and G → S flies (mean ± SEM; n = 4; significant differences are indicated by letters above each bar). (C) Negative correlation between relative levels of Cyp6a20 mRNA and fighting frequency. The linear regression plot (R ² = 0.922) is compiled by using the data in A and B and Fig. 1 Lower A and B. Comparisons between groups were made by using student's t test (A), or ANOVA followed by a post hoc test (B).

   
   
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   Fig. 3.

   Cyp6a20 mutants exhibit increased aggressiveness only under group-housing conditions. (A) Mean fighting frequencies of 6-day-old, single-housed and group-housed flies, of the indicated genotypes [mean ± SEM; n = 6 experiments each containing five pairs; significant differences (P < 0.05) are indicated by letters above the bars]. Comparison between groups was made by using ANOVA followed by a post hoc test. (B) Median lunging intensities of 6-day-old, single- or group-housed flies of the indicated genotypes (n = 10, 2, 9, 11, 15, 1, 17, 13 pairs, respectively; N.S., P > 0.05). (C) Median lunging latencies of 6-day-old, single- or group-housed flies of different genotypes (n = 10, 2, 9, 11, 15, 1, 17, 13 pairs, respectively; N.S., P > 0.05). Comparisons between groups were made by using Kruskal-Wallis ANOVA. (D) Two classes of negative genetic regulators of aggressiveness and their interaction with social experience. Class I genes, when mutated, increase aggressiveness under single-housing conditions, where their expression levels are normally relatively lower, but this phenotype is not observed in group-housing. Class II genes, when mutated, increase aggressiveness under group- but not single-housing conditions, overriding the effect of social experience to suppress aggressiveness. Cyp6a20 is a class II gene.

   
   
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   Fig. 4.

   Group-housed Cyp6a20 mutants exhibit normal locomotor, olfactory and courtship behavior. (A) Mean walking distances of 6-day-old, single-housed and group-housed flies of the indicated genotypes (n = 20; N.S., P > 0.05). Comparisons between groups were made by using ANOVA followed by post hoc test. (B) Median number of flies of the indicated genotypes (n = 15) trapped in food-containing (food+) vs. empty (food−) traps. Significant differences are indicated by letters above the boxes). Comparisons between groups were made by using Kruskal–Wallis ANOVA followed by a post hoc test. (C–F) Cyp6a20 mutants have normal courtship behavior. (C) Percentage of fly pairs of the indicated genotypes that copulated in 30 min. (D) Mean courtship latency of flies of the indicated genotypes (n = 21 and 20, respectively; N.S., P > 0.05). (E) Mean copulation latency of flies of the indicated genotypes (n = 21 and 20, respectively; N.S., P > 0.05). (F) Mean courtship index of flies of the indicated genotypes (n = 21 and 20, respectively; N.S., P > 0.05). For D–F, comparisons between groups were made by using Student's t test.

   
   
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   Fig. 5.

   Cyp6a20 expression in olfactory sensory organs. (A and B) GFP expression in the antennae (A) and palps (B). Whole-mount antennae and palps were stained with rabbit anti-GFP (green). (C and D) GFP⁺ cells are nonneuronal. Frozen sections were stained with rabbit anti-GFP (green) and rat anti-ELAV (red). (E and F) a subset of GFP⁺ cells in the antennae (E), but not in the palps (F), coexpressed LUSH. Frozen sections were stained with chick anti-GFP (green) and rabbit anti-LUSH (red). Confirmation of colabeling of one cell (white arrow) by z-series analysis is shown below and to the right of E. Inset in E is a higher magnification view of the boxed region (arrowhead), illustrating GFP⁺ LUSH⁻ and GFP⁺ LUSH⁺ cells. In all images, TOPRO-3 (blue) was used for nuclear staining. (Scale bars, 50 μm.)

   
   
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   Fig. 6.

   Cyp6a20 is a common genetic target of environmental and heritable influences on aggressive behavior. (A) Social experience influences aggressiveness in a reversible manner (bidirectional arrows), mediated by differential expression of Cyp6a20. (B) Genetic selection over multiple generations establishes neutral and aggressive populations with differential levels of aggressiveness, which correlate with differential Cyp6a20 expression (4). (C) Cyp6a20 regulation constitutes a common molecular target of environmental and genetic influences on aggressiveness. Circular arrowheads indicate that both positive and negative influences are possible. Environmental influences act on a time scale of the lifespan of the organism (Left), whereas genetic influences act over multiple generations as a consequence of selection (natural or artificial) (Right).