**1. Introduction**

Quercetin (Q) is the most abundant flavonol in the human diet, being present in a wide variety of plant-derived foods, such as nuts, grapes, onions, broccoli, apples, or black tea. Dietary quercetin consumption has been associated to different health benefits, including antioxidant and anti-inflammatory effects and protection against aging-related diseases, such as cardiovascular pathologies, cancer, and neurodegenerative disorders [1–3]. Classically, these actions have been explained, at least in part, by its antioxidant and free-radical scavenging properties, as demonstrated in in vitro studies [4,5], although the actual mechanisms through which quercetin and other flavonoids

exert their in vivo effects remain unresolved. Understanding the molecular mechanisms by which Q can exert its biological activity is important in order to develop strategies to modulate the physiological changes associated with aging that lead to chronic diseases. Although studies with quercetin and other flavonoids using in vivo models have increased in recent years, most of the knowledge on their biological activity still derives from in vitro or ex vivo findings, while assays that consider complex interactions of various processes, such as absorption, metabolism, and interaction with organs and tissues are more limited [6].

Some important molecular pathways in complex organisms can be explored using the model organism *Caenorhabditis elegans*. There is a high degree of homology between *C. elegans* and human genes involved in different processes, such as aging, apoptosis, cell signaling, cell polarity, metabolism, or cell cycle, which are conserved between mammals and the nematode [7]. In fact, *C. elegans* offers promising possibilities to study mechanisms of action and effects of secondary compounds of foods and plants, due to its simplicity of handling, conservation of metabolic pathways, and the possibility of manipulating cell signaling routes by biotechnological methods. In addition, it is not pathogenic, and no ethical boundaries exist to its experimental usage.

Different studies have evaluated the biological effects of quercetin and related compounds in *C. elegans*. In a previous work, we have shown that growing *C. elegans* in the presence of 200 μM of Q or its 3- - and 4- -*O*-methylated metabolites significantly prolonged the lifespan and increased the resistance against thermal- and juglone-induced oxidative stress [8]. Further support was obtained by checking the oxidation status of proteins in the nematode, which was greatly reduced [8]. Similar observations were also made by other authors [9–13]. Treatment with Q was also seen to lead to a reduction in the intracellular levels of reactive oxygen species (ROS), either in nematodes subjected to or not subjected to stress [11,13–15]. In general, these studies show that Q possesses a relevant biological activity in an in vivo system, which results in greater protection against oxidative processes. Regarding worm lifespan, an extension has been found in worms grown in the presence of concentrations of Q between 70 and 200 μM, while no lifespan extension was observed for Q concentrations outside that range. On the contrary, a hormetic response of the worm to this phytochemical has been demonstrated, so that the increase in the concentration of quercetin above certain levels results in decreased survival [16]. Nevertheless, the molecular mechanisms through which these effects are produced are still unclear, and different explanations, sometimes apparently contradictory, have been offered by distinct authors [9–14].

The response to oxidative stress in *C. elegans*is regulated through several pathways, including those of insulin/IGF-1 (IIS), c-Jun N-terminal kinases (JNK), and the signaling p38 MAPK (mitogen-activated protein kinases) pathway [17–19]. The IIS pathway (Figure 1) controls many important biological processes, including development, reproduction, metabolism, somatic maintenance, and stress resistance [20]. Insulin-like peptides binding to DAF-2, the orthologue of the insulin/IGF-1 receptor in *C. elegans* [21], activate its tyrosine kinase activity. This activation triggers a cascade of phosphorylation events through different kinases (AGE-1/PI3k, PDK-1, AKT-1/2, and SGK-1) that promote the phosphorylation-dependent cytoplasmic sequestration of the factors DAF-16/FoxO, HSF-1, and SKN-1/Nrf, preventing their transcriptional activity [22]. On the other hand, loss of insulin signaling in *C. elegans* results in several cytoprotective phenotypes resistant to both thermal and oxidative stress and also increases pathogen resistance and lifespan [23]. Flavonoids could influence cellular systems changing the expression of different genes through the modulation of distinct transcription factors, acting simultaneously on various signaling pathways, including the IIS pathway [24].

**Figure 1.** Scheme of the insulin/IGF-1 signaling pathway (IIS) in *C. elegans.* The components of the pathway that promote IIS are colored red and those that either antagonize IIS or are antagonized by IIS are colored green.

In order to contribute to elucidate the mechanisms involved in the effects of flavonoids, in this work, the ability of quercetin to modulate the insulin/IGF-1 signaling pathway (IIS) has been explored. Specifically, the influence on the resistance to thermally-induced oxidative stress has been assessed using *C. elegans* strains with loss-of-function mutations in genes of the IIS pathway (i.e., *daf-2, age-1, daf-16, akt-1, akt-2; sgk-1, hsf-1, skn-1*, and *daf-18*). Additionally, the ability of Q to modify the expression of some genes related to stress, namely *daf-16, hsf-1, skn-1, hsp-16.2, hsp-70, sod-3*, and *gst-4*, has also been determined by RT-qPCR or using the GFP fluorescent reporter in *C. elegans* transgenic strains.
