**4. Conclusions**

The enhancement of the oxidative stress resistance of *C. elegans* induced by Q could be explained, at least in part, by the modulation of the insulin signaling pathway, namely the genes *age-1, akt-1, akt-2,* *daf-18, sgk-1, daf-2, skn-1*, and *hsp-16.2*. However, this effect would be independent of *daf-16* and *hsf-1*. The implication of *daf-2* and not of *daf-16* in the observed effects seems to reinforce the idea that there are signals regulated by *daf-2* independent of *daf-16* that are also essential for the extension of longevity and resistance to stress.

The expression of *skn-1* was not changed by the treatment with Q when worms were submitted to stress, but the results obtained in the *skn-1* mutants pointed out that it was necessary to mediate the resistance to thermal stress. It has been reported that SKN-1 promotes longevity by a different mechanism to the protection against oxidative damage [29,30], and also that the increase of lifespan by Q was maintained in *skn-1* mutants [11]. This could explain why quercetin does not alter *skn-1* expression under normal conditions, while this gene seems involved in the increased resistance to stress induced by Q.

The studies with transgenic strains showed that Q did not produce an increase in the expression of the antioxidant enzymes GST-4 and SOD-3, nor of the heat shock proteins HSP-16.2 and HSP-70 in young worms, whereas an increase was produced in the expression of HSP-16.2 in aged worms. This observation could be important to explain, at least in part, the effects of enhanced lifespan and greater resistance to stress produced by Q in *C. elegans*, since the expression of many heat shock proteins diminishes throughout worm life, a process that might be counteracted by this flavonol.

In summary, the network of signaling pathways that could be modulated by quercetin and other flavonoids is diverse and complex, and the molecular mechanisms of action can vary depending on the compound. For instance, while DAF-16 and HSF-1 transcription factors were previously demonstrated to be involved in the effects of epicatechin [31], they do not seem to be required in the stress resistance effects of Q. In this work, just the insulin signaling pathway has been explored, but other routes are surely involved, which should be considered in future studies.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/8/12/585/s1, Table S1: Oligonucleotide sequence of primers used to determine the expression levels of *C. elegans* genes by RT-QPCR.

**Author Contributions:** Conceptualization, C.S.-B. and A.M.G.-P.; Methodology B.A.-D., S.G.-M. and M.R.R.; Formal Analysis, B.A.-D., E.S.-H., M.D. and S.G.-M.; Resources, C.S.-B., A.M.-V, M.R.R. and A.M.G.-P.; Writing—Original Draft Preparation, B.A.-D. and C.S.-B.; Writing—Review and Editing, B.A.-D., C.S.-B., A.M.G.-P. and A.M.-V.; Supervision, C.S.-B., A.M.G.-P. and A.M.-V.; Project Administration, C.S.-B., A.M.G.-P. and A.M.-V.; Funding Acquisition, C.S.-B., A.M.G.-P. and A.M.-V.

**Funding:** This research was funded by the Spanish Ministerio de Economía y Competitividad (MINECO Projects AGL2015-64522-C2 and BFU2015-64408-P) and Fondo Europeo de Desarrollo Regional (FEDER)-Interreg España-Portugal Programme (Project ref. 0377\_IBERPHENOL\_6\_E).

**Acknowledgments:** The authors are thankful to Francisco J. Martín-Vallejo for statistical advice.

**Conflicts of Interest:** The authors declare no conflict of interest.
