Fatty acid beta-oxidation, but not desaturation, plays a role in ARD entry.

(A) NHR-49 regulates both fatty acid beta-oxidation (ACS-2) and desaturation (FAT-7) via genetically separable pathways that are known to generate distinct phenotypic outputs. (B) ARD entry was significantly impaired with acs-2(ok2457) but not with the fat-7(wa86) mutant. The double mutant acs-1(ok2457); ogt-1(ok1474) was also significantly impaired for ARD entry. P-value **** = <0.0001 in comparison with N2 as determined using one-way ANOVA, NS = not significant. (C) Lifespan under standard conditions and with refeeding at day 30 of ARD was not significantly altered in these mutants as determined by one-way ANOVA. Error bars represent standard deviation.</p

Lifespan of mutant strains in recovery after 30 days of ARD and summary model.

Lifespan of wild-type and mutant strains in control husbandry conditions (blue bars) compared to those recovered after 30 days of ARD (gray bars). Lifespan after recovery from ARD was significantly defective in aak-2(ok524), daf-16(mu86), and rsks-1(ok1255). The mutant skn-1(zj15) also had a significantly reduced lifespan post-ARD but not as strongly as the others. A minimum of 8 worms from independent experiments were analyzed for lifespan and statistics were performed in Graph Pad Prism using one-way ANOVA. P-value **** = nhr-49, oga-1, ogt-1, and sir-2.1 play a role in ARD initiation, daf-16, rsks-1, and skn-1 function primarily in recovery from ARD, with aak-2 playing a role in the regulation of both states. Conversely, none of the alleles that we explored significantly impacted maintenance of the ARD.</p

Germline shrinkage during ARD maintenance and regrowth during recovery.

Representative images (with magnified panels for detail] showing the germline (outlined in yellow] of worms starved (left panel] at day 30 of ARD, with uterine embryos noted with white arrows. The right panel shows worms that have been re-feed for 48 hours after 30 days of ARD, with white dashed lines to indicate the germline, where healthy embryos and/or oocytes can be seen. All strains showed robust shrinkage of the germline during starvation and regrowth after feeding. The 50 μm scale bar shown in the top row applies to all images in each column.</p

Additional strains stained for ORO after 30 days of ARD.

In addition to the ogt-1-dependent pathway, we also looked at strains with diverse phenotypes to see if changes were unique to the ogt-1 pathway. We observed that outside of the ogt-1 pathway, strains that did not influence either entry or exit (age-1(hx546)), strains that also only influenced entry (sir-2.1(ok424)), and strains that influenced only recovery (skn-1(zj15)) had a marked increase in ORO staining/TAG stores compared to both wildtype and the ogt-1 pathway. We also observed this same pattern with genes downstream of nhr-49, such that acs-2 (with a defective ARD entry) had reduced ORO staining but fat-7 (no defect in ARD entry) had a stronger ORO signal. Arrows indicate retained embryos, which show high ORO staining. For easier comparison to control, the image of N2 worms in ARD from Fig 5 is included here. (TIF)</p

Changes in glycogen and trehalose levels before and after ARD.

(A) Carminic acid staining (indicative of glycogen and trehalose levels) varied greatly between strains. As we have previously reported [22], ogt-1(1474) showed slightly higher levels of carminic acid staining than other strains in standard husbandry conditions, though in this study this change did not reach significance. After 30 days of ARD (lower panels) we noted that nhr-49(nr2041) had a dramatic increase in staining, while wild type and ogt-1(ok1474) did not. These results did not correlate with the observed defect in ARD entry for these strains. (B) ImageJ based quantification of carminic acid fluorescence by pixel intensity. P-value **** = (TIF)</p

24 hours of ARD results in noticeable decrease in ORO staining.

(A) Decrease in ORO signal were observable at 24 hr after worms were placed on ARD plates (as compared to control fed worms in Fig 5), as shown with representative strains. (B) Strains were stained with DAPI to demonstrate efficient small-molecule penetrance of cuticles across strains, indicating that changes in ORO staining are not related to differences in cuticle penetrance between strains. (TIF)</p

Model of genetic modifiers of ARD phases.

Specific nutrient-sensing pathways contribute to ARD entry and exit. The initial sensing and dissemination of the signal to enter ARD (green pathways) includes genes encoding proteins involved in O-GlcNAc cycling (ogt-1, oga-1, with dotted arrows showing their biochemical interaction), genes that regulate fatty acid metabolism (nhr-49, acs-2) and sirtuins (sir-2.1). The dashed green line indicates an allele-specific effect on ARD entry. Energy sensing, mediated by the product of aak-2, uniquely acts in both ARD initiation and recovery (yellow pathway). The two members of the insulin/IGF signaling pathway tested in this study showed differing results with age-1 not affecting ARD (grey pathway), while the downstream daf-16 affected recovery (red pathway). ARD recovery additionally relies on the TOR pathway (rsks-1), which may also interact with daf-16. Stress signaling influences ARD recovery as well, with skn-1 playing a minor role, and another possible pathway interacting with daf-16.</p

Brood size variability.

The table shows the average brood size under normal husbandry (control) or re-fed animals following 5 or 10 days in ARD. A high degree of variability was observed between and within experimental replicates for the average brood size (SEM). These brood sizes did not correlate to fate adoption of each strain nor with influence on any of stages of ARD. Red indicates a decrease compared to wildtype whereas green indicates an increase vs wildtype. (PDF)</p

DAPI staining of spermatheca during ARD reveal changes in number of sperm nuclei during ARD.

(A) DAPI staining of worms after 15 days in ARD reveals fewer sperm in the spermatheca of ogt-1(1474), and nhr-49(nr2041) compared to wild-type N2 worms. In the oga-1(ok1207) worms, more sperm were observed. White arrows indicate examples of sperm nuclei. (B) Whereas most of the strains analyzed saw a decrease in the number of sperm nuclei present at day 30 of ARD, oga-1(ok1207) had an overall increase compared to wildtype. However, these dynamics did not correlate with changes in brood size among selfed individuals. Green arrows indicate increased brood numbers vs wildtype and red arrows indicate decreased brood vs wildtype. (TIF)</p

Oil Red O staining reveals redistribution of lipid stores in ARD.

(A) Representative images showing the distribution of Oil Red O in control (top) and starved animals after day 30 of ARD (bottom) in the ogt-1-dependent pathway. Retention of TAGs appeared to be strongest in the retained embryos in all mutant strains (white arrows). Scale bar indicates 50 μm and is consistent across each row. (B) Fiji Image analysis-based quantification of ORO staining (see S1 Methods) of wildtype and the mutant strains of the ogt-1-dependent pathway in normal husbandry (control) and in day 30 of ARD. Using two-way ANOVA analysis, both variables (genotype and feeding condition) were found to have significant effects, and these variables interact significantly. For each strain, mean ORO density decreased after 30 days of ARD: most dramatically for nhr-49(nr2041) and not significantly for ogt-1(ok1474). In normal husbandry conditions, ogt-1(1474) showed significantly less ORO signal than did N2 worms. P-value **** = <0.0001, *** = <0.001, ** = <0.01. Error bars represent standard deviation.</p