Research Paper Volume 15, Issue 4 pp 947—981

Figure 2. Male flies with either elevated or downregulated neuronal dLdh expression exhibit decreased long-term courtship memory and survival with age. (A) Western blot analysis of head extracts from neuronal transgenic male flies aged 21 days at 29°C showing elevated ectopic HA tagged dLdh expression (upper panel) and decreased endogenous dLdh levels (lower panel) in flies using a Elavts-Gal4 driver to drive UAS-dLDH (with 3xHA tag) and UAS-dLdh-RNAi expression respectively (n = 3), expression was normalized to total protein content (based on Ponceau-S staining). Each sample consisted of protein extracts from 20 heads. Comparison of endogenous dLdh level were made using an unpaired t test, ^*p < 0.05. (B) Courtship indexes for neuronal transgenic male flies aged 7, 14, or 21 days at 29°C post-eclosion showed a decrease in courtship conditioning rejection trained vs. naïve for all genotypes at all ages, except for 21 day upregulation. N = 59–125. Naïve and trained flies were compared within each genotype at each age using one-sided Mann-Whitney U tests, ^****p < 0.0001, ^***p < 0.001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (C) Long-term courtship memory indexes for neuronal transgenic trained flies represented in B. Memory in neuronal transgenic flies only differed from control at 21 days of age (H = 21.33, p < 0.0001), with both flies with dLdh upregulation and downregulation showing reduced memory at 21 day of age compared to control. Genotypes at each age were compared using Kruskal-Wallis tests with Dunn’s multiple comparisons to control, ^****p < 0.0001, ^*p < 0.05. Data presented as violin plot of frequency distribution. (D) In male flies, both neuronal upregulation and downregulation of dLdh resulted in reduced survival compared to control. Shaded area represents the 95% confidence interval. Curve comparisons were made using a Log-rank (Mantel-Cox) test, ^****p < 0.0001. Ages chosen for memory testing are highlighted with vertical dotted lines. (E) Upregulation of neuronal dLdh in male flies decreased climbing ability during negative geotaxis compared to control and dLdh downregulation (genotype effect F(2, 33) = 4.355, p = 0.0210). Comparisons across age and genotype were made using a mixed-effects model with Geisser-Greenhouse correction and Šídák’s multiple comparisons within each age group, ^*p < 0.05, ^****p < 0.0001. Effects of age, genotype, and age by genotype interaction are denoted on the bottom left.