Perceptive costs of reproduction drive ageing and physiology in male Drosophila

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1 In the format provided by the authors and unedited. Perceptive costs of reproduction drive ageing and physiology in male Drosophila Zachary M. Harvanek 1,2, Yang Lyu 1, Christi M. Gendron 1, Jacob C. Johnson 1, Shu Kondo 3, Daniel E. L. Promislow 4,5 and Scott D. Pletcher 1,6 * VOLUME: 1 ARTICLE NUMBER: 0152 NATURE ECOLOGY & EVOLUTION DOI: /s

2 Figure S1: The physiological effects of pheromone perception are robust across laboratory strains. A second genotype (w 1118 ) responded similarly in terms of triacylglyceride (TAG) stores to mating in the presence and absence of pheromones. n = 10 biological replicates of 5 flies each. The box represents SEM (Standard Error of the Mean, centered on the mean), whiskers represent 10%/90%, and the horizontal line represents the median. Figure S2: Without competition, males mate with females at a similar rate regardless of pheromone profile. (A) For those males that mated with both females, males that first mated with masculinized females were significantly faster to remate with wild type females (P = 0.002, two sided t test). n = 41 males. (B) In non competitive environments, time to mating was similar for males housed with females expressing either male or female pheromone profiles (maximum likelihood test for identical rates, P = 0.56). In both sets of flies, over 70% mated during the course of the 6 hour experiment. n = 50 males per group. (C) When presented with a single female, males had similar success at fertilizing that female regardless of the pheromone profile of the female. Bars represent percent fertilized in a single experiment. Error bars represent 95% confidence intervals. N = 46 for wild type female group, N = 49 for masculinized female group, Fisher s Exact test, P = (D) Total time (in minutes) experimental males spent interacting with donor animals of the respective exposure treatment. Samples were taken from random 10min intervals within an 8 hour observation period. Each point represents a single male fly. Non parametric ANOVA among all treatments P < Relevant pairwise P values are shown. The top bar represents the observation that all comparisons among female vs. male pheromone treatments yielded P < ANOVA P values were calculated using nonparametric Kruskal Wallis rank sum test. Pairwise P values were calculated by non parametric permutation t test. (E) The number of interactions for those flies presented in panel D. ANOVA among all treatments P = P values calculated as in panel D. (F) Assessment of wing damage for individual experimental male flies following 14 days of exposure to treatment animals. 0 = no damage, 1 = mild damage (estimated < 10%), 2 = moderate damage (10 50%), and 3 = excessive damage (> 50%). ANOVA among all treatments P = P values calculated as in panel D. For all box plots, box represents SEM (Standard Error of the Mean, centered on the mean), whiskers represent 10%/90%, and the horizontal line represents the median. Figure S3: Costs of reproduction require the pheromone receptor ppk23. (A) w 1118 control males exposed to females expressing female pheromones lived significantly shorter than males exposed to either males (P < ) or females expressing male pheromones (P < ). n = 100 flies per group. (B) In the presence of females expressing female pheromones, the ppk23 mutation extended the lifespan of males because it eliminates self imposed costs of reproduction. n = 100 flies per group. (C) The ppk23 mutation did not influence lifespan independent of pheromone perception because ppk23 and w 1118 males exhibited statistically NATURE ECOLOGY & EVOLUTION DOI: /s

3 indistinguishable survival patterns when housed with wild type males. n = 100 flies per group. (D) ppk23 mutants were resistant to the negative effects of pheromone exposure on starvation resistance (P = 0.91 comparing ( ) and ( ) exposures, P = 0.15 comparing ( ) and ( ) exposures, P = 0.35 comparing ( ) and ( ) exposures, n = 50 flies per group). (E) w 1118 control male flies exposed to females expressing female pheromones lived significantly shorter under starvation than those exposed to masculinized females (P < ) or to males (P < ). There was no difference between males exposed to males or masculinized females (P =.61). n = 50 flies per group. P values were obtained by Cox regression. Figure S4: Reproductive output is not decreased in ppk23 mutants. Ppk23 mutants produced more offspring than w 1118 controls on day 7, 21, and 35 ( P < , P < , and P = 0.013, respectively, by Tukey test), and on day 35 this difference was due largely to an interaction between genotype and exposure (P = by 2 way ANOVA interaction between genotype and exposure). This interaction suggests that later in life, pheromone perception may result in decreased reproductive output. Females with female pheromone profiles produced more offspring than masculinized females during the first week (P = by Tukey test) but not during subsequent weeks (P = 0.11 and P = 0.19 for days 21 and 35, respectively). This was true when housed with both types of males except for the interaction on day 35 when the presence of and ability to perceive female pheromones combined to decrease offspring production. This suggests the differences are either due to the females themselves or other pheromone signals not transmitted by ppk23. n = 20 vials per group per time point. Error bars represent SEM. Figure S5: Pheromone perception and mating alter the neurometabolome. (A) Distribution of Z scores (white bars) for randomized data when separating pheromone exposure (left) and mating status (right) and the Z scores of principal components that best account for those effects (red lines). N = 10,000 permutations, more details can be found in the methods section Data input and PCA analysis. (B) Negative mode data demonstrating separation of mating status (PC2) and pheromone exposure (PC3). (C) Distribution of ANOVA P values of candidate metabolites that are different between the pheromone exposure groups (top, red line) or mating status group (bottom, blue line). We selected top 20 metabolites that best explain the pheromone exposure groups (top), or mating status group (bottom), based on their coefficient from PC3 or PC2, respectively. P values from all the metabolites are plotted in grey shadow, as the background. A significant difference between candidate metabolites and background is observed (P < 0.05) Kolmogorov Smirnov Test). Figure S6: data from crz + neuronal inhibition and activation studies. animals for this experiment (w 1118 x crz Gal4) exhibited lifespans characteristic of those reported for other laboratory strains (See Fig. 1B). Male flies exposed to feminized males lived shortest (P < NATURE ECOLOGY & EVOLUTION DOI: /s

4 compared to animals exposed to each of the other exposures); males exposed to females with female pheromones lived marginally shorter than those exposed to males with male pheromones (P = 0.064); and males exposed to masculinized females lived similar lifespans to those exposed to control males (P = 0.34). Lifespans are shorter, and exposure effects are compressed, in these experiments, which were conducted at a high temperature for neuronal manipulations (29 C). n = 100 flies per group. Figure S7: The costs of reproduction are mediated through dfoxo signaling. dfoxo w24 mutants were resistant to the effects of pheromones on all measured phenotypes, including (A) starvation (P = 0.12 for dfoxo mutants, P = for controls, n = 50 flies per group), (B) TAG storage ( n = 10 biological replicates for control genotype groups, 6 biological replicates for foxo genotype groups, each composed of 5 flies), and (C) lifespan ( n = 100 flies per group), where the effects of pheromones are significantly reduced (P < for interaction term by Cox regression), though not completely eliminated (P < for pheromone effects on both dfoxo w24 and control animals). (D) Male flies trans heterozygous for the two dfoxo alleles (w ; +;dfoxo w24 /dfoxo Δ94 ) were immune to the effects of pheromones on starvation resistance (P = 0.82), while controls (w ;+;+/dfoxo Δ94 ) responded as expected (P < ). n = 50 flies per group. (E) yw males exposed to feminized males have significantly lower Thor (d4ebp) expression than animals exposed to control males (two experiments, each significant independently by twotailed t test: P = and P = 0.049, and combined by Tukey: P = 0.023; no interaction between experiment/exposure by ANOVA: P = 0.20). Data represent one of the experiments. n = 4 biological replicates (one lost due to failure to amplify) for feminized male exposure, and n = 5 biological replicates for wild type male exposure, with 25 flies per replicate. For the box plot in panel B, the box represents SEM, whiskers represent 10%/90%, and the horizontal line represents the median. Figure S8: The costs of reproduction do not require TOR or Sir2. (A) Rapamycin treatment did not prevent the effects of pheromone exposure on lifespan (P < for both drug and vehicle control). (B) Sir2 / mutant males were affected similarly by pheromone exposure as were wild type males (P = 0.73 for interaction between genotype and exposure, P < comparing exposures for both genotypes). n = 100 for each group. NATURE ECOLOGY & EVOLUTION DOI: /s

5 Table S1. Pheromone exposure and mating elicit specific, distinct changes in the neurometabolome. The top 20 metabolites, based on their loadings on PC2 (mating) or PC3 (pheromone exposure), were identified whose abundances were associated with the effects of mating or pheromone exposure. Mating Pheromone Exposure Positive mode Negative mode Positive mode Negative mode Nupharamine C22H26NO6S C7H3S 2,4- Dinitrophenylhydrazine Glucoarabin Hordatine A C6H7Cl O S Haloxyfop methyl Streptozocin Metaflumizone C17H40Cl N5 O S2 C6H6N6O6 DG(20:0/20:4(8Z,11Z,14Z,17Z)/0 C21H41N9O3 Granisetron Threonate :0) @ Palatinose C5H3Cl3N O6S2 MG(18:0/0:0/0:0) D-Fructosyl-D-fructofuranose Lappaol B C6H14O4 Neomycin B C19H39N4O3 L-beta-aspartyl-Lglutamic Chlorphenesin Nonate acid L-Glutamate 4-Hydroxy-L-threonine C20H41N4 Cellopentaose C21H43N4O3 Phosphoric acid Tiocarbazil Fluconazole C21H41N4O3 Apholate Varanic acid @ PE(20:3(8Z,11Z,14Z)/20:1(11Z)) L-beta-aspartyl-Lglutamic phorbol 13-acetate 12-myristate C36H26N15O5 acid @ LysoPE(0:0/18:3(9Z,12Z Dichloroarcyriaflavin A C8H16O S3,15Z)) Levofuraltadone Prenyl caffeate Falcarindiol C22H26N O6S C5H6N5O2 Hygromycin A C42H56N4O6 Beta-d-lactose C23H18N5O22 Diglykokoll PE(15:0/14:0) alpha-l- Rhamnopyranosyl-(1-2) @ Phospho-L-serine 3-Octaprenyl-4-hydroxybenzoate C13H16Cl2N6O7S5 C34H61N13 N-Formyl-L-glutamate Ampalex 3,5-Di-O-galloyl-4-Odigalloylquinic acid Dimethirimol PPPi C7H12N7 Diethanolamine Inosine Terpenoid EA-I Nupharamine Chlorobenzilate C23H19N2O24 N-Carbamoylsarcosine O-Phosphorylethanolamine C7H2O11S NATURE ECOLOGY & EVOLUTION DOI: /s

6 Table S2: Targeted survey of potential downstream effectors for the costs of reproduction. We surveyed the effect of several candidate genes or interventions on the pheromone response to either TAG or starvation resistance. In this analysis we identified dfoxo as being required for a significant effect of pheromones on the relevant phenotype (at P < 0.10). Manipulation or Gene P AkhR Apoltp 7.E 13 CG E 09 CG E 14 CG E 09 Diet, high (15%S/Y) 1.E 08 Diet, low (5%S/Y) 5.E 10 Diff <1E 16 Foxo GstD2 2.E 04 ImpL2 6.E 07 Retinoic Acid <1E 16 Pepck 2.E 11 Relish Tudor (no germline) 4.E 12 Sex Peptide Sodh2 1.E 06 sug 4.E 06 Rapamycin 2.E 05 UPD3 4.E 07 NATURE ECOLOGY & EVOLUTION DOI: /s

7 Supplementary File 1: Neurometabolomic changes upon pheromone exposure and mating (excel file, 1209 kb). This contains data for both independent neurometabolomic experiments ( exp1 and exp2 ). In the data, wm refers to wild type male exposure, wf refers to wild type female exposure, fm refers to feminized male exposure, and mf refers to masculinized female exposure. Other lanes are for QC. NATURE ECOLOGY & EVOLUTION DOI: /s

8 P = 2E-5 TAG (ug/fly) P = P = 0.37 Figure S1 NATURE ECOLOGY & EVOLUTION DOI: /s

9 A Never Remated 250 Remating time (min) P = First Female Time to Mate (min) B P = 0.56 Female C Percent fertilized100 P = 0.10 Female Interaction Time (min) D P = 0.48 P < P = 0.25 Interactions E P = P = 0.12 P = Wing Damage F P = P = P < P = Figure S2 NATURE ECOLOGY & EVOLUTION DOI: /s

10 A. B. P < P < Experimental Flies ppk23 w 1118 : C. D. P = 0.81 Experimental Flies ppk23 w 1118 : ppk23 P = 0.40 E. Starvation Time (hrs) P < Figure S3 Starvation Time (hrs) NATURE ECOLOGY & EVOLUTION DOI: /s

11 Offspring/vial ppk23 Donor Figure S4 NATURE ECOLOGY & EVOLUTION DOI: /s

12 A. Count B. PC2 (10.8%) max(z-score pheromones) 2000 P = P = PC3 PC PC3 (9.4%) C. Density Density max(z-score mating) Pheromones P < Log10 (ANOVA p-value) Mating P < Log10 (ANOVA p-value) Figure S5 NATURE ECOLOGY & EVOLUTION DOI: /s

13 Crz Neurons Figure S6 NATURE ECOLOGY & EVOLUTION DOI: /s

14 A. dfoxo w24 B. TAG (ug/fly) P < P = 0.15 C. Starvation Time (hrs) D. dfoxo w24 dfoxo trans dfoxo w24 E. Starvation Time (hrs) Expression (A.U.) P=0.02 Figure S7 NATURE ECOLOGY & EVOLUTION DOI: /s

15 A. Vehicle Rapmycin B. Sir2 -/- Figure S8 NATURE ECOLOGY & EVOLUTION DOI: /s

Analysis of variance (ANOVA) Comparing the means of more than two groups

Analysis of variance (ANOVA) Comparing the means of more than two groups Example: Cost of mating in male fruit flies Drosophila Treatments: place males with and without unmated (virgin) females Five treatments