Fig 21 : Functional correlation analysis to integrate dilp 2-3,5 and mNSC-ablation models.
Heatmap representing correlations between log-fold-changes of proteins annotated with GO terms in fat, gut, and brain tissue. Log-fold-changes were derived from the following comparisons: dilp 2-3,5 /Wol- vs. w Dah /Wol- (control),
dilp 2-3,5 /Wol+ vs. w Dah /Wol+ (long-lived), w Dah /Abl vs. w Dah (long-lived), dfoxo Δ/Δ /Abl vs. dfoxo Δ/Δ (control). Correlations
were z-transformed using empirical distributions obtained by permutation. Up to seven representative, high scoring GO terms (z-value ≥ 2 in central column) were selected in each tissue and sorted in descending order of their maximal difference to controls.
To discriminate between parts of the insulin response that are conserved in long-lived dilp 2-3,5
differences between long-lived flies and normal lived controls in each of the two models ( w Dah /Abl vs. w Dah , and dilp 2-3,5 vs. w Dah ). We selected the log-fold-changes of proteins annotated
within a size-filtered list of GO terms (between 10 and 400 annotated genes). A coordinated, conserved insulin response of a func onal term will manifest in a high posi ve correla on coefficient between the long-lived proteome responses.
To reduce redundancy of GO terms and increase interpretability, we employed an itera ve filtering scheme: descending the list of GO terms from most highly correlated between
long-lived responses to most nega vely correlated, we added all annotated proteins from each term to an exclusion list. As correla ons show higher variances at lower numbers of
observa ons, this procedure will select smaller, more specific GO terms first. We then required each subsequent term to contain at least 10 proteins that were not observed in any previous term (i.e. on the exclusion list), or be discarded. To make terms of different sizes directly comparable and account for prior posi ve correla on of log-fold-changes, empirical
distribu ons of correla ons for terms of any observed size (number of proteins in the term) were calculated by repeated shuffling of all observed log-fold-changes in each comparison. The actually observed correla ons were then z-transformed using the parameters of the empirical distribu on. We further selected terms that showed a high correla on, i.e. z-scores in excess of 2 standard devia ons above the mean ( File 2 ). In addi on to the correla ons between
long-lived comparisons, comparisons performed in two control backgrounds were then included in the analysis: dilp 2-3,5 vs. w Dah in absence of Wolbachia , as well as mNSC abla on vs. w Dah in a
dfoxo -null background. Both the absence of Wolbachia and dfoxo-knockout lead to an
abroga on of the lifespan-extension phenotype in their respec ve model system, but may play limited roles in pleiotropic phenotypes (Slack et al. 2011) . A high correla on to these controls does not exclude an involvement in lifespan extension en rely, but a low correla on lends addi onal confidence that a func onal term is involved in the extension of lifespan observed in the respec ve model.
In the fat body, we observed several func onal terms that were correlated between the long-lived insulin responses, but not with regard to either of the controls. These included regula on of the cytosolic ribosome ( Fig 21 ), suppor ng our observa on that fat body transla on is modulated specifically in long-lived flies ( Fig 8 ). The concordant regula on of ncRNA processing suggests that non-coding RNA may play a role in the longevity-specific insulin response ( Fig 8 ). Interes ngly, while carbohydrate metabolism was longevity-specifically
regulated, proteins annotated with lipid homeostasis and starva on response were induced in a correlated manner independently of Wolbachia ( Fig 21 ).
In the gut , several func onal terms were closely associated between long-lived comparisons, but dysregulated in normal lived controls. These included protein polyubiqui na on, a process that is integral to protein turnover and recycling by the proteasome. We have previously shown
gut of mNSC-ablated flies (Tain et al. 2017) . We could confirm that this was also the case in
dilp 2-3,5 mutants (Tain et al. 2017) . In addi on to that, septate junc on proteins were regulated
in a consistent manner between long-lived fly models.
In the brain , proteins belonging to the insulin receptor signalling pathway showed a
coordinated response in long-lived phenotypes, reflec ng the similarity of the interven ons that result in the loss of dilp expression in both model systems ( Fig 21 ). This pa ern was independent of Wolbachia and dfoxo status, as was regula on of cell growth ( Fig 21 ). Regula on of the tricarboxylic acid cycle was consistent between long-lived contrasts
independently of Wolbachia , but altered in the dfoxo -knockout ( Fig 21 ). The same was true for transla onal elonga on. We failed to detect a change in transla on rate in heads of long-lived mNSC-ablated flies ( Fig 8 ); however, a more precise essay using only brain ssue might yield different results. Addi onally, ac n filament polymeriza on was highly correlated between long-lived responses and with regard to dfoxo- null controls, but an -correlated in
Wolbachia -nega ve controls ( Fig 21 ). Wolbachia relies on the host ac n cytoskeleton for
effec ve transmission and can directly manipulate it (Newton et al. 2015; Sheehan et al. 2016) . In par cular, we observed Wolbachia- dependent up-regula on of the ac n interac ng protein Flr and WASp, as well as down-regula on of the highly conserved Arp2/Arp3 complex (Kang et al. 2010) ( File 2 ). Arp2/3 induc on facilitates Akt membrane recruitment upstream of dFOXO (Zhao et al. 2015) , linking ac n cytoskeleton organiza on to insulin signalling. This suggests that
Wolbachia may modulate insulin signalling through manipula on of cytoskeleton components
in the brain.
In the thorax, the longevity-specific concordant regula on of proteins involved in cell adhesion, myosin binding, and structural organiza on point towards an effect on muscle func on. We observed a beneficial effect of reduced insulin on climbing ability of ageing dilp 2-3,5 mutants, as
shown earlier in this thesis ( Fig 17 ). While our essay did not encompass dfoxo- null flies, our correla on analysis supports dfoxo- dependence of improved muscle func on upon reduced IIS, corrobora ng previous reports (Demon s & Perrimon 2010) .