In old flies, speed decays gradually whereas courtship performances collapses, but a weak-to-moderate correlation is found between speed and STM.

(A) Climbing speed measurements during the last days of the cohort (>10 weeks). Not the final drop of speed past the 75th day. (B) Courtship and Short Term Memory (STM) Indexes remain stable during the last days. (C) Patterns of change in courtship and climbing speed during the last three days prior to courtship collapse. Note that courtship against the tester (CI test) and the trainer (CI initial) abruptly drop to very low levels whereas the climbing speed declines gradually. (One-Way ANOVA, Kruskal-Wallis multiple comparison test, * p(D) No correlation between speed and both courtship (CI trainer, CI tester) and STM indexes. Note that a slow individual can out-perform in courtship engagement and vice versa.</p

Courtship rituals latencies, that is the time it takes for an individual to initiate any of the behavior, increase in late life.

Courtship rituals latencies, that is the time it takes for an individual to initiate any of the behavior, increase in late life.</p

Comparison of latencies for male courtship parameters depicted in Fig 4B.

p -values from One-way ANOVA, Kruskal-Wallis test, Tukey’s multiple comparisons test. Values in bold indicate significance.</p

Courtship LTM is absent during mid and late life.

(A) When presented with a virgin female, courtship indices of the Tester (males presented with a mated female on the previous day) and Sham males (naïve males) differ in young flies (5d), thus indicating courtship LTM. However, Tester and Sham courtship indices are not different during mid and late life. Courtship index was estimated as the percentage of male courtship within a 10 minutes time window. Tested males first courted towards a mated female for 8 hours, were then isolated for 24 hours, and finally courted against a virgin tester female. (B) LTM (24 hours) courtship memory index in mid and late life. Note that the LTM index approaches 1 at 4 weeks and all older ages tested, indicating absence of LTM from 4 weeks on. (T-test for A, One-Way Anova, Kruskal-Wallis multiple comparison test for B. ** p<0.01, *** p<0.001, **** p<0.0001).</p

Climbing performance remains stable in mid life but decreases during late life.

(A) Male flies of different ages were individually tested for their performance in climbing a 6 cm vertical distance after having been startled with different stimulation regimes. The strongest stimuli caused the fastest responses at all ages. (B) Successful responses (completion of the 6 cm climbing task within 10 s) remain unaltered between 4 and 8 weeks of age (mid life) but decline after the 8th week (late life). (One-Way ANOVA, Kruskal-Wallis multiple comparison test, * p<0.05, ** p<0.01, *** p<0.001).</p

4–10 week old males.

R and p values from Spearman rank correlation test. Values in bold indicate significance.</p

Supplemental Material for Tran et al., 2018

Table S1. <i>dCORL</i> mutant virgin and mated adult longevity defects compared to seven control lines.<div><br></div><div>Table S2. Mean & median longevity of <i>dCORL</i> virgin and mated adults with seven controls.<br></div><div><br></div><div>Table S3. Significant brain size reduction is present in <i>Df(4)dCORL</i> larvae but not adults.<br></div

>10 week old males.

R and p values from Spearman rank correlation test. Values in bold indicate significance.</p

Male courtship index decays in old flies, but courtship learning and (STM) remain stable.

(A). Courtship Index is estimated as the percentage of male courtship within a 10 min time window. Courtship conditioning assays were performed with the males of different ages (4, 6, 8, and 10 weeks of age). First the trained male courts against a mated female trainer for one hour [recorded:10 first min with trainer (CI initial), 10 last mins with trainer (CI final)]. Next, after 5 min isolation the trained male is presented to a young virgin female tester [recorded:10 first mins with the virgin tester (CI test)]. As a control, age-matched, non-trained males (Sham) are presented with a virgin female. (B) The learning index is the ratio of the courtship indices during the final 10 min of the training (CI final) and that of the initial 10 min (CI initial). (C) The memory index is calculated by dividing CI test by the mean of the sham control courtship levels (CI sham).</p

Late life pathophysiology of locomotor behavior.

(A) Life history chart for 53 male Oregon-R flies tested daily and individually in the startle assay from the age of 60 days until death. Gray bars indicate health-span and colored bars disabilities of different categories as defined in ([4]; see also D). 39.6% (21 out of 53) of the flies show no sign of impairment until the last day of life. All flies, with or without disabilities during the last day of life, undergo a dramatic functional collapse (terminal stage) characterized by immobility, unresponsiveness and finally death within few hours [4]. Since terminal stage lasts only few hours and is followed by death it was not detected in the majority of flies by testing once a day. (B) Time enlargement of the last 10 days for all flies. Note that days are separated by vertical lines and combinations of colored bars show co-morbidities. (C) Survival curves for the tested population in A and non-tested control flies are not significantly different [Log-rank (Mantel-Cox) test; X2 = 0.001431, p = 0.9698]. (D) Color-code for behavioral impairments as defined in [4]. (E) Maximum climbing speed drops significantly between the 8th and 9th week. (F) Severe reduction (>90%) in male courtship occurs on average 4 days prior to death, 2.5 days earlier than the onset of motor impairments. (One-Way ANOVA, Kruskal-Wallis multiple comparison test for E; T-test for F. ** p<0.01, *** p<0.001).</p