3.2.2. Gryllus Bimaculatus Cricket and Silkworms

The two-spotted cricket *Gryllus (G.) bimaculatus*, a member of primitive group *Polyneoptera*, is another kind of species that were used to examine the longevity effect of RJ. RJ dietary supplementation to *G. bimaculatus* during early nymph stage significantly decreased developmental time, extended lifespan, and increased the body size of both males and females in a dose-dependent manner (8−15% *w*/*w*) compared with the control high protein, sugar, and lipid diet. The prolonged lifespan of RJ was not due to an extended nymph stage since the adult stage emerged earlier in crickets fed RJ than those fed the control diet. Similarly, RJ administration increased body size and egg size in female silkworms. The authors concluded that the effects of RJ were not attributed to the nutritional supplement itself; however, they did not examine the molecular mechanism behind them [65].

## 3.2.3. Caenorhabditis (C.) Elegans Nematodes

*Caenorhabditis (C.) elegans* nematodes have been used in several instances to test the longevity-promoting activity of RJ. Japanese researchers found that RJ, protease-treated RJ (pRJ), pRJ-Fraction 5 (pRJ-Fr.5), and a derivative of pRJ-Fr.5—10-HDA (the main lipid of RJ)—extended the lifespan of *C. elegans.* RJ 10 μg/mL was an optimal dose for enhancing longevity by 7−9%, while RJ 1 or 100 μg/mL had no effect. Meanwhile, all pRJ concentrations significantly prolonged the mean lifespan—though the greatest effect was noticed at 10 μg/mL, which increased mean lifespan by 7–18%. The longevity effect of combined pRJ-Fr.5 and 10-HDA was greater than that induced by each treatment on its own. DNA microarray and RT-PCR showed that the longevity-promoting effect was attributed to reduction of the *insulin*/*IGF-1* signaling—pRJ-Fr.5 upregulated the expression of dod-3 gene and downregulated the expression of *ins-9*, an insulin-like peptide gene, along with *dod-19*, *dao-4*, and *fkb-4* genes (further details are shown below in the mechanism section) [52]. In two subsequent studies RJ, pRJ, and 10-HDA enhanced longevity and increased stress resistance of *C. elegans* against thermal, irradiation, and oxidative stress [66,67]. Intact, deglycosylated, and mildly heat-treated royalactin extended mean lifespan of *C. elegans* by 18–34%—higher concentrations produced the vastest lifespan-extending effect. Royalactin also enhanced locomotion, which indicates promotion of healthy aging [68].

#### 3.2.4. Mice

Few studies investigated the lifespan extending activity of RJ in mice. In an early study, intermediate and high doses (50 and 500 ppm) of RJ significantly prolonged the mean lifespan of C3H/HeJ mice by 25%, whereas RJ at a low dose (5 ppm) yielded no significant effect. Meanwhile, all doses of powdered RJ, contrary to bee and *Drosophila* studies, had no effect on mice growth, food intake, or appearance compared with control mice. RJ treatment significantly lowered kidney DNA and serum levels of 8-hydroxy-2-deoxyguanosine, a marker of oxidative stress that increases with

aging [2]. Similarly, long-term intragastric administration of RJ and pRJ to a d-galactose-induced aging mice model resulted in numerous anti-aging and healthspan effects: preventing aging-related weight loss, improving memory and motor performance, and delaying aging-related atrophy of thymus, thus preventing diminution of the immune function compared with control animals. The effects were attributed to inhibition of lipid peroxidation and improvement of levels of antioxidant enzymes [69]. Likewise, dietary supplementation of RJ and pRJ (0.05% or 0.5%, *v*/*v*) to genetically heterogeneous head tilt mice—which exhibit vestibular dysfunction, imbalanced position, and inability to swim—could not prolong lifespan but significantly delayed age-related impairment of motor functions, positively improved physical performance of treated mice on four tests (grip strength, wire hang, horizontal bar, and rotarod), lowered age-related muscular atrophy, increased markers of satellite cells (muscle stem cells), and suppressed catabolic genes [70]. Another study examined the survival-expanding time (not lifespan extension) of oral RJ treatment (75, 150, and 300 mg/kg body wt/day for 13 consecutive days) following NaNO2 intraperitoneal injection or decapitation as models of brain hypoxia and complete brain ischemia [71]. Findings indicated that the intermediate dose of RJ (150 mg) significantly expanded survival time, whereas RJ 75 mg had no significant effect, meanwhile RJ 300 mg significantly decreased survival time. The author suggested that low pH of RJ (3.6 to 4.2) in mice treated with high doses of RJ induced activation of acid-sensing channels, increased acidosis of extracellular fluid, and aggravated brain ischemia [71].
