Supplemental Information. Octopamine Drives Endurance. Exercise Adaptations in Drosophila

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1 Cell Reports, Volume 21 Supplemental Information Octopamine Drives Endurance Exercise Adaptations in Drosophila Alyson Sujkowski, Divya Ramesh, Axel Brockmann, and Robert Wessells

2 Supplemental Figures Figure S1

3 Supplemental Figure S1: Confirmation of post-developmental sex-specific transcript expression by qrt- PCR, Related to Figures 2 and 7. Elav Gene Switch Gal4 was used to express (A) UAS tra F in neurons of adult male flies and (B) UAS tra F RNAi in neurons of adult female flies as described in Supplemental Experimental Procedures. (C-H) Confirmation of knockdown from experiments using daughterless gal4 to drive RNAi in specified tissues. qrt-pcr was performed using SYBR green after RNA isolation from fly heads (A,B) or whole flies (C-H). Bars represent triplicate biological replicates of 20 individuals each. p< for all sex-specific and/or RNAi transcripts, analyzed using single ANOVA with Bonferroni post-hoc comparison. See supplemental Experimental Procedures for primer sequences.

4 Table S1: Octopaminergic neurons mediate sex-specific endurance in D. melanogaster, Related to Figure 2. Targeted Gal4/UAS screen based on tissue-specific masculinization and feminization indicates that octopaminergic neurons are sufficient to control sexually dimorphic endurance. Gal4 Target tissue tra F feminization Time to fatigue (minutes) tra F RNAi masculinization Time to fatigue (minutes) Mef2 Muscle ElavGS Adult nervous system D42 Motor neurons Akh Adipose tissue Ddc Dopaminergic & Serotonergic neurons MT14 Olfactory, gustatory, mechanosensory neurons OK371 Glutamatergic neurons Tdc2 Octopaminergic neurons Pdf CNS circadian control centers Tim Pacemaker neurons TrH Serotonergic neurons TH Dopaminergic neurons To Midgut, feeding centers Npf Feeding, flight, sensorimotor centers

5 Figure S2

6 Supplemental Figure S2: Octopaminergic neurons mediate sex differences in exercise response, Related to Figures 2 and 4. (A) Tdc2>tra F females and background control females do not increase runspan after exercise training (log-rank, p=0.2585, n=160). (B) Tdc2>tra F females and background control females do not increase climbing speed after exercise training (2-way ANOVA with Tukey multiple comparisons, training effect, p=0.9483, n 100). (C) Lysotracker staining in abdominal fat is low in female Tdc2>tra F and background controls whether exercised or not (ANOVA with Tukey multiple comparisons, p=0.1250, n=10). (D) Tdc2>tra F male flies and background control males display increased post-training runspan at day 25 (log-rank, p=0.0121, n=160). (E) Trained Tdc2>tra F male flies and trained background control males display increased climbing speed relative to untrained groups (2-way ANOVA with Tukey multiple comparisons, training effect, p<0.0001, n 100). (F) Lysotracker staining is increased in abdominal adipose tissue of exercised Tdc2>tra F males and exercised background controls (t-test between trained and untrained groups, p<0.0001, n=10). (G) Female Tdc2>VMAT RNAi flies and background control females do not increase climbing speed with training (2-way ANOVA with Tukey multiple comparisons, training effect, p=0.8879, n 100). (H) Tdc2>VMAT RNAi females and background control females do not show increased day-25 flight performance, and in fact display lower landing height than untrained controls (ANOVA with Bonferroni post hoc comparison, p= (control UN vs control EX) and p= Tdc2>VMAT RNAi UN vs Tdc2>VMAT RNAi EX), n 59). (I) Lysosomal activity in abdominal adipose tissue is low regardless of training status in Tdc2>VMAT RNAi and control females (ANOVA with Tukey post hoc comparison, p=0.5711, n=10). (J) Neither ElavGS>OAMB RNAi RU+ females nor RU- control females increase climbing speed with exercise (2-way ANOVA with Tukey multiple comparisons, training effect, p=0.9607, n 100). (K) ElavGS>OAMB RNAi RU+ females and RU- control females do not increase landing height after exercise, and display lower landing height than untrained females at day 25 (ANOVA with Bonferroni post hoc comparison, p< (RU- control UN vs RU- control EX) and p= (ElavGS>OAMB RNAi RU+ UN vs ElavGS>OAMB RNAi RU+ EX), n 76). (L) Abdominal adipose tissue Lysotracker staining is low in ElavGS>OAMB RNAi RU+ females and RU- control females regardless of training status (ANOVA with Tukey post hoc comparison, p=0.2518, n=10). (M) Female Tβh mt flies and female background controls do not increase climbing speed with exercise (2-way ANOVA with Tukey multiple comparisons, training effect, p=0.9721, n 100). (N) Female Tβh mt flies and female background controls do not increase flight performance with exercise (ANOVA with Bonferroni post hoc comparison, training effect: p=0.5182, n n 119). (O) Female Tβh mt flies have higher lysosomal activity in abdominal adipose tissue than female background controls (ANOVA with Tukey post-test, p<0.0001, n=10). Exercised female Tβh mt flies do not display increased adipose tissue Lysotracker staining relative to untrained female Tβh mt flies (t-test, p=0.1598, n=10).

7 Figure S3

8 Supplemental Figure S3: Octopamine feeding rescues exercise response in T h mt flies, Related to Figure 3. (A) T h mt flies have reduced day-5 endurance in comparison to background controls (log-rank, p<0.0001), but 3 days of octopamine feeding improves both male and female T h mt endurance to that of wild-type females (p=0.9751). (B) Following endurance training, background control Canton S (CS) females do not improve runspan, as previously observed. Octopamine feeding, however, enhances female CS endurance in trained and untrained groups (log-rank, p<0.0001). Octopamine feeding improves runspan of T h mt females to the level of wild-type females (log-rank, p=0.7379). (C) Male CS flies improve endurance with exercise training, and octopamine feeding increases runspan in both trained and untrained groups (log-rank, p=0.0078). T h mt males have reduced runspan independent of training, and octopamine feeding improves endurance to resemble wild-type, untrained male CS flies (log rank, p<0.0001, , respectively). (D) T h mt female flies have reduced climbing speed across ages, but octopamine feeding increases climbing speed to equal the speed of octopamine-fed CS female flies by weeks 3 and 4 (Two-way ANOVA, p=0.3033). (E) Male T h mt flies improve climbing performance across ages after octopamine feeding, equaling speed of exercise-trained or octopamine-fed CS male flies (n 100 for all climbing experiments, Two-way ANOVA, p< for genotype effect, exercise effect, drug effect for all significantly different groups). Octopamine feeding also enhanced flight performance and cardiac stress resistance in T h mt female (F,H) and T h mt male (G,I) flies whether exercised or not. (p values listed, n 100 for all experiments). Lysosomal activity was high in both T h mt female (J) and T h mt male (K) flies independent of training status. Octopamine feeding reduced lysosomal activity in the fat body back to wild-type, pre-training levels in both sexes.

9 Table S2: Smaller subsets of octopaminergic neurons were not sufficient to feminize endurance in male flies, Related to Figure 2. Targeted Gal-4/UAS screen based on tissue-specific feminization yields wild-type runspan in day-5 adult male flies. Each line expresses in a subset of the tdc2 expression pattern. Images of the specific patterns of each driver are available at the Janelia Farms FlyLight Website: ( along with more information about their derivation. Gal4 tra F feminization Time to fatigue (minutes) Citation R16B Pfeiffer et al. (2008) R57F Pfeiffer et al. (2008) MB Jenett et al. (2012) MB022B Jenett et al. (2012) MB113C Jenett et al. (2012)

10 Figure S4 Supplemental Figure S4: Octopamine affects cardiac performance in Drosophila, Related to Figure 4. (A) Female flies with RNAi against synaptic release of octopamine (Tdc2>VMAT RNAi) and background control groups have normal day-25 pacing-induced cardiac failure whether exercised or not. (B) Tdc2>VMAT RNAi males do not exhibit enhanced cardiac pacing resistance after training, while background controls display reductions in cardiac failure typical of exercised male flies (2-way ANOVA with Tukey multiple comparisons, training effect, p=0.0042, n 104 for all groups).

11 Figure S5

12 Supplemental Figure S5: Intermittent octopaminergic activation mimics exercise training and has no additive effect with machine-training, Related to Figure 6. (A) After 25 days of temperature training, Tdc2>TrpA1 females had much greater endurance than background controls under the same conditions (log rank, p<0.0001, n=160). TT EX indicates flies under identical intermittent temperature training conditions to those in figure 7, but simultaneously placed on the Power Tower for exercise training. TT UN indicates flies treated as TT EX but mobility-restricted by placing a foam stopper low in the vial (B) Temperature trained exercised and unexercised Tdc2>TrpA1 females displayed increased climbing speed (2-way ANOVA, genotype effect: p<0.0001, n=100), (C) flight performance (p<0.0001, n 160) and (D) enhanced cardiac pacing resistance (p=0.0268, n 100). (E) TT EX and TT UN Tdc2>TrpA1 males displayed similar enhancements to endurance (p<0.0001, n=160), (F) climbing speed (p<0.0001, n=100), (G) flight performance (p<0.0001, n 160) and (H) decreased cardiac failure in response to electrical pacing (p=0.0148, n 100).

13 Figure S6 Supplemental Figure S6. Female flies expressing ubiquitous, adult-specific RNAi against octopamine receptors have wild-type, low exercise-training response, Related to Figure 7. (A-D) Female day 25 runspan is not significantly different between RU- controls nor RU+ experimental groups expressing RNAi against Octβ1R (A), Octβ2R (B), Octβ3R (C) or Octβ3/1R (D) whether exercised or not. (E-H) Neither female RU- controls nor RU+ experimental flies expressing RNAi against Octβ1R (E), Octβ2R (F), Octβ3R (G) or Octβ3/1R (H) respond to endurance exercise with increased climbing speed. Similar adaptive deficiencies are observed for flight performance (I-L), cardiac pacing (M-P) and lysosomal activity (Q-T) in the same female cohorts.

14 Figure S7

15 Supplemental Figure S7: Spontaneous activity does not correlate with exercise adaptations in Drosophila, Related to Figures 1, 3, and 5. (A) Naïve 25-day-old Tdc2>TrpA1 male flies never exposed to the exercise machine have increased spontaneous activity in comparison to male background controls (ANOVA with Bonferroni post hoc comparison, p=0.0003, n 16). (B) 5-day old Tdc2>TrpA1 flies of either sex have increased activity in comparison to background controls (ANOVA with Bonferroni post hoc comparison, p=0.0317, p= n 16). 5-day old background control females are more active than background control males (ANOVA with Bonferroni post hoc comparison, p=0.0234, n 16), but Tdc2>TrpA1 females and males display indistinguishable spontaneous activity (t-test, p=0.0945, n 16). (C) Day-5 Tβh mt flies of either sex have lower spontaneous activity than background controls of the corresponding sex (ANOVA with Bonferroni post hoc comparison, p=0.0039, p= n 16). (D) Machine-exposed day 25 wild-type females have higher spontaneous activity than males independent of training status controls (ANOVA with Bonferroni post hoc comparison, p<0.0001, n 16) Exercise training does not change the spontaneous activity of female (t-test, p=0.0751, n 16) or male (t-test, p=0.3279, n 16) wild-type flies. (E) Tdc2>TrpA1 females have higher day-25 spontaneous activity than both w 1118 ;Tdc2 (ANOVA with Bonferroni post hoc comparison, p=0.0008, n 16) and w 1118 ;TrpA1 (ANOVA with Bonferroni post hoc comparison, p<0.0001, n 16) control females, but do not differ with training (t-test, p=0.6708, n 16). (F) Tdc2>TrpA1 males have higher day-25 spontaneous activity than both w 1118 ;Tdc2 (ANOVA with Bonferroni post hoc comparison, p<0.0001, n 16) and w 1118 ;TrpA1 (ANOVA with Bonferroni post hoc comparison, p<0.0001, n 16) control females, but do not differ with training (t-test, p=0.8480, n 16). (G) Neither Tβh mt females nor (H) Tβh mt males display spontaneous activity that is different from day-25 background controls, regardless of training status (ANOVA with Bonferroni post hoc comparison, p=0.7278, p= n 16).

16 Supplemental Experimental Procedures Fly Stocks and Maintenance RNAi lines were validated by crossing to GS-tub-Gal4 and comparing expression in RU+ and RU- flies. All Drosophila lines were from the Bloomington Drosophila Stock Center with the following exceptions: ElavGS- Gal4 was provided by Scott Pletcher (University of Michigan), and the Gal4 insert was mobilized to the third chromosome using standard cross schemes. Tβh nm18 flies were provided by Chun-Fang Wu (University of Iowa) and Sarah Certel (University of Montana). UAS-VMAT RNAi was obtained from the Vienna Drosophila RNAi center. All UAS and Gal4 containing lines not in the w 1118 background were backcrossed ten generations before analysis. The presence of the insertions was verified by the presence of a w + marker associated with the insertion. Control flies for all non-gene-switch UAS Gal4 experiments consisted of a single outcrossed population of both the UAS and Gal4 lines into w For experiments in which both the w 1118 ;Gal4 control and w 1118 ;UAS control flies were found to be statistically identical, results were pooled and graphed as a single datum. In gene-switch experiments, possible genetic background effects were further controlled for by defining RU- flies of the same w 1118 background as the negative control. Temperature Training On adult day 5, flies were transferred to fresh food vials and shifted to the activating temperature (25 ºC), considered day-1 of temperature training. Temperature training was accomplished by placing TT and TT + restraint flies in the 25ºC incubator and TT EX and TT UN flies on the Power-Tower in a temperature controlled, 25ºC room. In week 1, flies were trained for 2 hours, in week 2 for 2.5 hours, and in week 3, 3 hours. After each day s training period, all cohorts were returned to the restrictive temperature. All cohorts were transferred to fresh food vials every day and assessed for climbing speed immediately prior to the temperature training period. Upon completion of the 3 week training program, all groups were tested for post-training endurance, flight performance, fat body lysotracker and cardiac pacing within a 72 hour window. qrt-pcr For confirmation of adult-specific activation of sex-specific transcripts dependent on transformer, cdna was prepared from 20 adult fly heads after 7 days of exposure to either 70% ethanol vehicle or 100 µm mifepristone. For confirmation of RNAi constructs, cdna was prepared from whole flies. Three independent RNA extractions were prepared for each sample. Differences between genotypes were assessed by t-test or nested ANOVA. Primer sequences are listed below. 5 tra F AACCCAGCATCGAGATTCCC 3 tra F ACCTCGTCTGCAAAGTACGG 5 dsx F GAATCGAAGAGGGCCAATACG 3 dsx F AAATTATCATCCACATTGCCGC 5 dsx M GCCACGTAGCAGTATCGCAA 3 dsx M CTCCAGTGAAAATGGTGCTGC 5 fru M CCCGCATCCCCTAGGTACAA 3 fru M GACTGTTTCGCCCTCGCAGG 5 fru C CAAATTTGACCGGCGTGCTAACCT 3 fru C AGTCGGAGCGGTAGTTCAGATTGT 5 Octβ1R GGCAACGAGTAACGGTTTGG 3 Octβ1R TCATGGTAATGGTCACGGGC 5 Octβ2R TTAGTGTGCAAGTAACTGGGC 3 Octβ2R TGAGAAGTAGACATCGAGGCTG 5 Octβ3R TGTGGTCAACAAGGCCTACG 3 Octβ3R GTGTTCGGCGCTGTTAAGGA 5 VMAT- AAAATTGGACGATGGTTTGC 3 VMAT- ATTCGGGATGATCAGGTGAG 5 OAMB- CGGTTAACGCCAGCAAGTG

17 3 OAMB- AAGCTGCACGAAATAGCTGC 5 rp49 GCCAAACTGATGCTAGGC 3 rp49 CCACCTCCACTTCAGGATAC 5 act5c GGCGCAGAGCAAGCGTGGTA 3 act5c GGGTGCCACACGCAGCTCAT Quantification of OA from fly heads Fly heads were homogenized in 190ul of acidified acetone, 10ul of 10mM ascorbic acid and 10ul of 1.25ug/ml DOPA-D3 (CDN Isotopes, Quebec, Canada) as the internal standard. The supernatants were collected, dried and derivatized using in-house synthesized 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Sample cleanup was done using solid phase extraction (SPE) cartridges (Phenomenex, Inc. Hyderabad, India). The eluted samples were completely dried before reconstitution (2% acetonitrile with 0.5% formic acid) and injection into the HPLC- ESI-MS. The liquid chromatography (LC) gradient and electro-spray ionization (ESI) conditions were as described in (Natarajan et al., 2015). The calibration curves were made in neat solvents, after testing for matrix effects, and found to be linear over a 64-fold concentration range with the highest concentration being 0.8ug/ml. Quantitation was done using the Xcalibur software (version 2.2 SP1.48). Endurance Climbing endurance was measured using the fatigue assay described previously (Tinkerhess et al. 2012a, Tinkerhess et al. 2012b). Eight vials of flies from each cohort were subjected to the fatigue assay at two time points: once on day 5 and once on day 25 of adulthood. For each assessment, the flies were placed on the Power Tower exercise machine and made to climb until they were fatigued, or no longer responded to the negative geotaxis stimulus. Monitored at 15 min intervals, a vial of flies was visually determined to be fatigued when five or fewer flies could climb higher than 1 cm after four consecutive drops. A minimum of 8 vials containing 20 flies each was used for each fatigue assessment with each vial plotted as a single datum. Runspan graphs with fewer than 8 data points indicate that 2 or more vials were scored as fatigued at the same time. Each experiment was performed in duplicate or triplicate, and runspans were scored blindly when possible. The time from the start of the assay to the time of fatigue was recorded for each vial, and the data analyzed using log-rank analysis in GraphPad Prism (San Diego, CA, USA). Negative Geotaxis Speed Adult flies were collected with light CO 2 anesthesia within 2 hours of eclosion and housed in appropriate fresh food vials. Negative geotaxis was assessed in Rapid Negative Geotaxis (RING) assays in groups of 100 flies as described (Piazza et al., 2009a, Piazza et al. 2009b). Flies were transferred to individual polypropylene vials in a RING apparatus and allowed to equilibrate for 1 minute. Negative geotaxis was elicited by sharply rapping the RING apparatus four times in rapid succession. The positions of the flies were captured in digital images taken 2s after eliciting the behavior. Images were analyzed using ImageJ (Bethesda, MD). The relative distance climbed by each fly was converted into quadrants using Microsoft Excel. The performance of 20 flies was calculated as the average of four consecutive trials to generate a single datum. Flies were longitudinally tested 5 times per week for 5 weeks to assess decline in negative geotaxis speed with age. For exercise experiments, data were further consolidated into weekly performance and normalized to the starting climbing index of each individual cohort. Between assessments, flies were returned to food vials and housed until the following RING test. Negative geotaxis results were analyzed using two-way ANOVA analysis with post hoc Tukey multiple comparison tests in GraphPad Prism (San Diego, CA, USA). All negative geotaxis experiments were performed in duplicate or triplicate. Flight performance Flight was analyzed as in Sujkowski et al. (2015). Duplicate or triplicate cohorts of at least 100 flies were aged and/or exercise trained in narrow vials housing groups of 20 age-matched siblings. Acrylic sheeting with paintable adhesive was placed in the flight tube, and fly cohorts were ejected into the apparatus to record flight performance and subsequent landing height after release. Fly cohorts were introduced to the flight tester one vial at a time using a gravity-dependent drop tube in order to reduce variability. After a full cohort of flies was captured on the adhesive, the sheeting was removed to a white surface in order to digitally record the landing height of each fly. Flies with damaged wings were censored from final analysis to control for mechanical stress not related to training

18 performance. Images were analyzed using ImageJ. Landing height was averaged and compared in Prism using a student t-test. Cardiac Pacing 5- or 25-day old flies were removed from appropriate experimental cohorts and subjected to electrical pacing as in Wessells et al. (2004). The percentage of fly hearts that responded to pacing with either fibrillation or arrest were recorded as % failure. Percent failure is a marker for stress sensitivity and characteristically declines with age (Piazza et al., 2009b, Wessells et al. 2004). Endurance exercise reduces cardiac failure rate across ages in trained male Drosophila (Piazza et al. 2009a, Sujkowski et al. 2015). Lysotracker Lysotracker staining of adult fat bodies was performed as in Sujkowski et al. (2012). Adult flies separated by age, genotype, and or treatment were dissected, ventral side up, in room temperature PBS. Having exposed fat bodies, partially dissected flies were rinsed 1X in fresh PBS. Lysotracker green (Molecular Probes, Eugene, OR) was diluted to 0.01µM in PBS and applied to dissected preps for 30 seconds. Samples were washed 3 times in fresh PBS. Stained fat bodies were subsequently removed and mounted in Vectashield reagent (Vector Laboratories, Burlingame, CA, USA). Confocal work was done at the Microscopy, Imaging and Cytometry Resources Core at Wayne State University, School of Medicine on a Zeiss Laser Scanning LSM 780 (Jena, Germany) using a 100X oil immersion objective. Images were analyzed using ImageJ. A minimum of 10 samples were analyzed for each sample and duplicate or triplicate biological cohorts were assessed for each group. Data were subjected to student t- test following quantification. Spontaneous Activity The DAM5 activity monitor (Trikinetics) was used to record spontaneous activity in individual flies as in (Chiu et al., 2010) with the following modifications: Flies were assessed at 5 days of age or 25 days of age and monitored on 10% sucrose/10% yeast food. At least 16 flies were used per group, and triplicate biological samples were assessed. Data were collected in 2 second bins for 3 hours on 3 consecutive days (day 4-6 or day 24-26). Experiments were performed at 25ºC, 20% humidity and carried out at the same time of day to control for variations in environment and/or circadian rhythms. Data are presented as number of beam crosses per hour. Graphing and statistical analysis (single-anova with Bonferroni post-hoc comparison) were performed in GraphPad Prism.

19 Supplemental References Chiu, J.C., Low, K.H., Pike, D.H., Yildirim, E., and Edery, I. (2010). Assaying locomotor activity to study circadian rhythms and sleep parameters in Drosophila. J Vis Exp. Jenett, A., Rubin, G.M., Ngo, T.T., Shepherd, D., Murphy, C., Dionne, H., Pfeiffer, B.D., Cavallaro, A., Hall, D., Jeter, J., et al. (2012). A GAL4-driver line resource for Drosophila neurobiology. Cell reports 2, Natarajan, N., Ramakrishnan, P., Lakshmanan, V., Palakodeti, D., and Rangiah, K. (2015). A quantitative metabolomics peek into planarian regeneration. Analyst 140, Pfeiffer, B.D., Jenett, A., Hammonds, A.S., Ngo, T.T., Misra, S., Murphy, C., Scully, A., Carlson, J.W., Wan, K.H., Laverty, T.R., et al. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 105, Piazza, N., Hayes, M., Martin, I., Duttaroy, A., Grotewiel, M., and Wessells, R. (2009). Multiple measures of functionality exhibit progressive decline in a parallel, stochastic fashion in Drosophila Sod2 null mutants. Biogerontology 10, Wessells, R.J., Fitzgerald, E., Cypser, J.R., Tatar, M., and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nat Genet 36,

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