*3.1. Assays in Wild Type and Mutant Worms*

An enhancement in the survival was observed in N2 wild-type worms treated with Q 200 μM after being subjected to thermal stress compared with non-treated controls. Specifically, in the assays carried out on the second day of adulthood, the average proportion of living worms after stress was 64.78% in the control group and 81.11% in Q-treated worms (*p* = 0.000), while on the ninth day of adulthood, the survival rate was 35.1% in untreated worms and 47.2% in treated animals (*p* = 0.001) (Figure 2).

The molecular mechanisms involved in this enhancement of thermotolerance were explored, checking the ability of Q to modulate the stress resistance in *C. elegans* mutant strains. Initially, the effect of Q on thermal stress resistance was evaluated in worms carrying loss of function mutations in genes of the IIS pathway, namely *age-1(hx546)*, *akt-1(mg306)*, *daf-2(e1370),* and the double mutant *akt-2(tm812); sgk-1(ft15)*, all of them long-lived and with greater resistance to stress than the wild-type strain. Similar to the wild-type, mutant worms were subjected to a thermal shock (35 ◦C, 6–8 h) on days 2 and 9 of adulthood. The obtained results showed that the stress resistance was not increased in any of the mutant worms treated with quercetin at either day 2 or 9 (Figure 3). These results suggested that *age-1, akt-1, akt-2, sgk-1*, and *daf-2* were necessary to mediate stress resistance induced by Q in *C. elegans*.

**Figure 2.** Percentages of survival following thermal stress (35 ◦C, 8 h) applied at days (**A**) 2 and (**B**) 9 of adulthood in N2 wild-type *C. elegans* strain not treated (controls) and treated with Q (200 μM). Statistical significance was calculated using the Chi Square Test (200–300 individuals per assay in both controls and treated worms). The asterisks (\*\*\*) indicate significant differences at *p* < 0.001.

Pietsch et al. [11] also studied the effect of Q in some of these mutants. As in our case, they did not find an improvement in the survival after thermal stress in *daf-2* and *age-1* mutants treated with 200 μM quercetin, concluding that that those genes were required to mediate the stress-protective effects of the flavonol. However, they found that the treatment with Q unexpectedly produced an improvement in the resistance to stress in the *akt-2* mutant, a gene located downstream of DAF-2 and AGE-1 in the IIS pathway. They proposed that this could be explained because SGK-1 was more important for resistance to stress than the AKT kinases [11]. In the present study, no increase in the survival after thermal stress was observed in the double-mutant *akt-2;sgk-1* treated with Q (Figure 3), suggesting that *sgk-1* could actually be necessary to improve the resistance to stress by quercetin. Similarly, no changes in the survival after stress were produced in Q-treated *akt-1* mutants, a gene that encodes an ortholog of serine/threonine kinase AKT/PKB and interacts with the IIS pathway. This indicates that the kinase *akt-1* was also necessary for the improvement in the resistance to stress in *C. elegans.*

The resistance to thermal stress in response to Q was also studied in *daf-16, hsf-1, skn-1, daf-18*, and *hsp-16.2* mutant worms. The transcription factors DAF-16, HSF-1, and SKN-1 produce changes in the expression of several genes in response to a reduced IIS pathway. The genes regulated by these transcription factors are functionally relevant, including stress response genes, such as catalases, glutathione-*S*-transferases, metallothioneins, and genes that encode antimicrobial peptides, chaperones like *hsp-16.2*, apolipoproteins, and lipases [27]. As it can observed in Figure 4, the treatment with Q did not improve the survival in *daf-18, skn-1*, and *hsp-16.2* mutants exposed to thermal stress, suggesting that these genes would be necessary to explain the effect of Q in worm resistance against stress. However, the treatment with Q continued to produce an improvement in the resistance to thermal stress in *daf-16* and *hsf-1* mutants, indicating that the effect of Q in *C. elegans* was independent of these genes. Similar results on the influence of Q were obtained in young and older adults in the studied mutants (Figure 4).

**Figure 3.** Percentages of survival following thermal stress (35 ◦C, 8 h) applied at days 2 (**A–D.1**) and 9 (**A–D.2**) of adulthood in different long-lived *C. elegans* mutants from the IIS pathway: *daf-2(e1370)* (**A**), *age-1(hx546)* (**B**), *akt-1(mg306)* and *akt-2(tm812)* (**C**); *sgk-1(ft15)* (**D**) cultivated in the absence (controls) and presence of Q (200 μM). Statistical significance was calculated using the Chi Square Test. In both controls and treated worms, ten plates were used per assay containing 20 worms per plate (i.e., 200 worms in total). The asterisk (\*) indicates significant differences at *p* < 0.05.

**Figure 4.** Percentages of survival following thermal stress (35 ◦C, 6 h; except for *hsp-16.2* (35 ◦C, 4 h)) applied at days 2 (**A–E.1**) and 9 (**A–E.2**) of adulthood in *daf-16 (mu86)* (**A**), *hsf-1(sy441)* (**B**), *skn-1(zu67)* (**C**), *daf-18(e1375)* (**D**) *and hsp-16.2(gk249)* (**E**) mutants cultivated in the absence (controls) and presence of Q (200 μM). Statistical significance was calculated using the Chi Square Test. In both controls and treated worms, ten plates were used per assay containing 20 worms per plate (i.e., 200 worms in total), but in the case of the *skn-1* mutant, 300 worms (20 worms per plate/15 plates) were used. The asterisk (\*) indicates significant differences at *p* < 0.05 on comparing with control.
