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. 2020 Jun 29;21(13):4601. doi: 10.3390/ijms21134601

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Nlg3^Def1 but not nlg3^L04 or nlg3^GS32 have altered social space. Mean number of flies within four body lengths (4BL) ± s.e.m. (A) Males nlg3^Def1 have significantly less flies within 4BL than male Cs, regardless of their age (two-way ANOVA: F_1,49=5.538, p = 0.0227; 3–4 days old: n = 15 trials for Cs, n = 13 for nlg3^Def1; 7–10 days old: n = 14 for Cs and n = 11 for nlg3^Def1). (B) No difference was observed between young Cs and nlg3^Def1 females; however old nlg3^Def1 females had less flies within 4BL than Cs (Welch’s t-test: T_13.62=2.097, p = 0.0276; 3–4 days old: n = 12 trials for Cs and nlg3^Def1; 7–10 days old: n = 14 for Cs and n = 7 for nlg3^Def1). The 7–10 day-old nlg3^Def1 and Cs females were tested on different days than 3–4 day-old flies, they were therefore plotted on separate axis and Welch’s t-tests were performed on the difference between nlg3^Def1 and Cs at each age. There was no effect of age, genotype, or any interaction for nlg3^L04 male (C) or female (D) and nlg3^GS32 male (E) or female (F). Color coding of bars corresponds to type of mutation: grey represents Cs, red represents nlg3^Def1, blue represents nlg3^L04, and orange represents nlg3^GS32. The p-values were determined using a two-way ANOVA. Error bars represent s.e.m. (C,D): Males: 3–4 days old: n = 9 trials for Cs and n = 8 for nlg3^GS32; 7–10 days old: n = 9 for Cs and nlg3^GS32. Females: 3–4 days old: n = 9 for Cs and n = 8 for nlg3^GS32; 7–10 days old: n = 9 for Cs and nlg3^GS32. E-F: Males: 3–4 days old: n = 9 for Cs and n = 8 for nlg3^L04; 7–10 days old: n = 9 trials for Cs and nlg3^L04. Females: 3–4 days old: n = 9 for Cs and n = 7 for nlg3^L04; 7–10 days old: n = 7 for Cs and nlg3^L04. (G) The 3–4 day-old elav > nlg3 males were not different from UAS-nlg3/+ control flies but elav-Gal4/+ flies had a higher number of flies within 4BL. elav > nlg3 7–10 day-old males were however more social than the two genotype controls (two-way ANOVA—effect of genotype: F_2,30 = 7.440, p = 0.0024; effect of age: F_2,30 = 0.02485, p = 0.8758; interaction of age and genotype: F_1,30 = 6.496, p = 0.0024; 3–4 days old: n = 6 trials for elav-Gal4/+, n = 6 UAS-nlg3/+ and n = 5 for elav > nlg3; 7–10 days old: n = 6 for elav-Gal4/+, n = 8 for UAS-nlg3/+ and n = 6 for elav > nlg3). (H) Female elav > nlg3 3–4 days old had an increased number of flies within 4BL compared to elav-Gal4/+ and UAS-nlg3/+ controls. The 7–10 day-old elav > nlg3 females had an intermediate phenotype with higher number of flies within 4BL than elav-Gal4/+ but lower number of flies within 4BL than UAS-nlg3/+ 7–10 days old (two-way ANOVA—effect of genotype: F_2,32 = 2.993, p = 0.0644; effect of age: F_1,32 = 9.240, p = 0.0047; interaction of age and genotype: F_2,32 = 3.633, p = 0.0379; 3–4 days old: n = 6 trials for elav-Gal4/+ and UAS-nlg3/+, n = 5 for elav > nlg3; 7–10 days old: n = 5 for elav-Gal4/+, n = 8 UAS-nlg3/+ and elav > nlg3). For (A–H): n = 5–15 trials–as detailed above–with 12–17 flies–always some escapees–per trial. Lighter colors represent 3–4 day-old flies. Darker colors represent 7–10 day-old flies.

Nlg3^Def1 but not nlg3^L04 or nlg3^GS32 have altered social space. Mean number of flies within four body lengths (4BL) ± s.e.m. (A) Males nlg3^Def1 have significantly less flies within 4BL than male Cs, regardless of their age (two-way ANOVA: F_1,49=5.538, p = 0.0227; 3–4 days old: n = 15 trials for Cs, n = 13 for nlg3^Def1; 7–10 days old: n = 14 for Cs and n = 11 for nlg3^Def1). (B) No difference was observed between young Cs and nlg3^Def1 females; however old nlg3^Def1 females had less flies within 4BL than Cs (Welch’s t-test: T_13.62=2.097, p = 0.0276; 3–4 days old: n = 12 trials for Cs and nlg3^Def1; 7–10 days old: n = 14 for Cs and n = 7 for nlg3^Def1). The 7–10 day-old nlg3^Def1 and Cs females were tested on different days than 3–4 day-old flies, they were therefore plotted on separate axis and Welch’s t-tests were performed on the difference between nlg3^Def1 and Cs at each age. There was no effect of age, genotype, or any interaction for nlg3^L04 male (C) or female (D) and nlg3^GS32 male (E) or female (F). Color coding of bars corresponds to type of mutation: grey represents Cs, red represents nlg3^Def1, blue represents nlg3^L04, and orange represents nlg3^GS32. The p-values were determined using a two-way ANOVA. Error bars represent s.e.m. (C,D): Males: 3–4 days old: n = 9 trials for Cs and n = 8 for nlg3^GS32; 7–10 days old: n = 9 for Cs and nlg3^GS32. Females: 3–4 days old: n = 9 for Cs and n = 8 for nlg3^GS32; 7–10 days old: n = 9 for Cs and nlg3^GS32. E-F: Males: 3–4 days old: n = 9 for Cs and n = 8 for nlg3^L04; 7–10 days old: n = 9 trials for Cs and nlg3^L04. Females: 3–4 days old: n = 9 for Cs and n = 7 for nlg3^L04; 7–10 days old: n = 7 for Cs and nlg3^L04. (G) The 3–4 day-old elav > nlg3 males were not different from UAS-nlg3/+ control flies but elav-Gal4/+ flies had a higher number of flies within 4BL. elav > nlg3 7–10 day-old males were however more social than the two genotype controls (two-way ANOVA—effect of genotype: F_2,30 = 7.440, p = 0.0024; effect of age: F_2,30 = 0.02485, p = 0.8758; interaction of age and genotype: F_1,30 = 6.496, p = 0.0024; 3–4 days old: n = 6 trials for elav-Gal4/+, n = 6 UAS-nlg3/+ and n = 5 for elav > nlg3; 7–10 days old: n = 6 for elav-Gal4/+, n = 8 for UAS-nlg3/+ and n = 6 for elav > nlg3). (H) Female elav > nlg3 3–4 days old had an increased number of flies within 4BL compared to elav-Gal4/+ and UAS-nlg3/+ controls. The 7–10 day-old elav > nlg3 females had an intermediate phenotype with higher number of flies within 4BL than elav-Gal4/+ but lower number of flies within 4BL than UAS-nlg3/+ 7–10 days old (two-way ANOVA—effect of genotype: F_2,32 = 2.993, p = 0.0644; effect of age: F_1,32 = 9.240, p = 0.0047; interaction of age and genotype: F_2,32 = 3.633, p = 0.0379; 3–4 days old: n = 6 trials for elav-Gal4/+ and UAS-nlg3/+, n = 5 for elav > nlg3; 7–10 days old: n = 5 for elav-Gal4/+, n = 8 UAS-nlg3/+ and elav > nlg3). For (A–H): n = 5–15 trials–as detailed above–with 12–17 flies–always some escapees–per trial. Lighter colors represent 3–4 day-old flies. Darker colors represent 7–10 day-old flies.