Paramylon from Euglena gracilis EOD-1 may extend lifespan by activating antioxidant defenses through clec-196 in Caenorhabditis elegans

For initial confirmation of whether C. elegans ingests particulate PM, worms were exposed to PM suspended in S basal buffer [100 mM NaCl, 50 mM potassium phosphate (pH 6.0)]. During feeding, C. elegans pumps liquid into the intestinal lumen through rhythmic contractions of the pharynx. Observations indicated that PM, in its particulate form, was taken up gradually into the digestive tract via the pharynx (Fig. 1a). Next, to investigate PM effects on the C. elegans lifespan, 5-day-old adult worms were transferred to NGM plates supplemented with PM at concentrations of 10 mg/mL and 30 mg/mL, prepared as described in Materials and Methods reported herein. As shown in Fig. 1b and Supplementary Table 1, PM at concentrations of 10 mg/mL and 30 mg/mL respectively increased the mean lifespan of C. elegans by 14% and 16%. Until day 10, no notable difference was found among the survival rates of the three groups. However, from day 12 onward, the survival curves of the two PM-treated groups began to diverge from those of the control group fed only OP50.

Caloric restriction is known to extend the lifespan of various organisms, and of C. elegans^28,29. Worms subjected to caloric restriction typically exhibit reduced body size and brood size³⁰. To ascertain whether the lifespan extension induced by PM was attributable to caloric restriction, the body sizes and brood sizes of PM-fed worms were compared with those of control worms fed OP50 alone. PM supplementation did not affect the worm growth curve (Fig. 2a and Supplementary Table 2). Similarly, no significant differences were found in brood size (Fig. 2b and Supplementary Table 2). Furthermore, measurement of the developmental time until the onset of egg laying showed that PM supplementation had no observable effect (Supplementary Fig. 1 and Supplementary Table 7). These findings suggest that the lifespan extension conferred by PM occurs independently of caloric restriction.

Lipofuscin accumulation and muscle function are known to correlate with the C. elegans aging process³¹. Lipofuscin, a byproduct of lipid peroxidation, is commonly assessed via autofluorescence intensity. The autofluorescence intensity of worms fed with PM was significantly lower than that observed in worms fed OP50 alone (Fig. 3a and b and Supplementary Table 3). To evaluate muscle function further, locomotory scoring was performed. Locomotory scores were assessed every 48 h at the time points presented in Fig. 3c. Throughout the experiment period, the proportions of worms exhibiting vigorous movement (class A) were consistently higher in the PM-treated group than in the control group, indicating improved locomotor ability.

When ingested by mice, PM granules, which are resistant to various digestive enzymes, are known to be excreted in their intact particulate form without being digested¹⁷. As presented in Fig. 1a, PM was also observed as particulate matter within the intestinal lumen of C. elegans, suggesting that it might exert its biological functions without undergoing either digestion or absorption. To elucidate the molecular mechanisms underlying PM-mediated lifespan extension, we examined the expression of clec-196, an ortholog of β-glucan receptor DECTIN-1, f52e12, an ortholog of DECTIN-2, and pezo-1, an ortholog of mechanosensitive ion channel PIEZO-1. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of clec-196 was upregulated upon PM administration (Fig. 4a and Supplementary Table 4). In contrast, no marked change was observed in the expression level of f52e12 or of pezo-1 following PM treatment. To investigate further whether clec-196 is involved in PM-mediated lifespan extension, RNAi via the feeding method was conducted. As shown in Fig. 4b and Supplementary Table 4, worms fed with Escherichia coli HT115 (HT115) transformed with a vector expressing clec-196 dsRNA exhibited approximately 90% suppression of clec-196 expression compared to the control worms. Subsequently, we assessed the PM effects on the lifespans of clec-196 knockdown worms and control worms. In control worms, similarly to wild-type animals, PM supplementation extended the lifespan considerably: a 10% increase in mean lifespan (Fig. 4c and Supplementary Table 1). In contrast, this lifespan-extending effect of PM was not found in clec-196 knockdown worms (Fig. 4d and Supplementary Table 1). These results indicate that clec-196 is necessary for pro-longevity effects of PM in C. elegans.

The DAF-2/DAF-16, JNK-1/DAF-16, and p38 MAPK/SKN-1 signaling pathways are known to contribute to stress resistance, which is associated in turn with lifespan extension^32–34. To ascertain whether these pathways are involved in the lifespan-extending effects of PM, or not, we used qRT-PCR to examine the expression of genes associated with these three pathways. As shown in Fig. 5a and Supplementary Table 5, the expression levels of jnk-1 and daf-16 were upregulated in response to PM treatment. It is noteworthy that these upregulations were suppressed by knockdown of clec-196 (Fig. 5b and Supplementary Table 5). In contrast, PM supplementation had no apparent effect on the expression of daf-2 or age-1, which are involved in the DAF-2/DAF-16 pathway, or on expression of sek-1, pmk-1, or skn-1, which are components of the p38 MAPK/SKN-1 pathway. Taken together, these results suggest that the pro-longevity effect of PM is mediated through activation of the JNK-1/DAF-16 signaling pathway via clec-196.

As a biological validation of the gene expression analysis results and for further evaluation of whether PM-mediated lifespan extension is associated with increased daf-16 expression, we examined C. elegans strains TJ356 and CF1038. The lower panel of Fig. 5c portrays a representative image of a TJ356 worm categorized as "nuclear," in which strong fluorescence is observed within the cell nuclei, whereas no fluorescence is detected in the soma, which indicates that the transcription factor DAF-16::GFP has translocated into the nuclei. By contrast, worms classified as "cytosolic" display weak, diffusely distributed fluorescence throughout the body (Fig. 5c, upper panel), suggesting that DAF-16::GFP remains in the cytoplasm. In the "intermediate" type, bright nuclear fluorescence is observed along with faint background fluorescence (Fig. 5c, middle panel), indicating that the transcription factor has partially translocated into the nuclei, whereas a portion remains in the cytoplasm. Multiple fluorescence images were acquired from TJ356 worms treated with PM. Each worm was categorized according to the classification criteria presented above. Based on these observations, the nuclear translocation rate of DAF-16 was calculated. Although no worm in either the control or PM-treated group was classified as "nuclear," the proportions of worms categorized as "intermediate" were markedly different between the two groups: 6.5% in control worms and 39.1% in PM-treated worms (Fig. 5d). For further confirmation of the involvement of DAF-16 in PM-mediated lifespan extension, we assessed the effect of PM treatment on the lifespan of the DAF-16 mutant strain CF1038. The results demonstrated that the lifespan of CF1038 worms fed PM was comparable to that of control CF1038 worms fed OP50 alone (Fig. 5e and Supplementary Table 1). These findings suggest that the lifespan-extending effect of PM is dependent on the DAF-16 signaling pathway.

To investigate further whether the DAF-16 signaling pathway is involved in PM-mediated lifespan extension, or not, we measured the expression levels of antioxidant-related genes downstream of DAF-16. As Fig. 6a and Supplementary Table 6 show, the expression levels of sod-3, sod-4, sod-5, ctl-1, and ctl-2 were elevated in worms fed PM compared to those fed OP50 alone. In contrast, the expression of sod-1 and sod-2 was unaffected by PM treatment. Subsequently, worms fed PM were subjected to staining with BES-H₂O₂-Ac, a fluorescent probe for hydrogen peroxide (H₂O₂) detection. The intestinal tract and surrounding tissues exhibited a marked degree of fluorescence in control worms fed OP50 alone, whereas this fluorescence was much lower in worms fed PM (Fig. 6b and c and Supplementary Table 6). These findings suggest that the lifespan-extending effect of PM is associated with activation of an antioxidant pathway mediated by DAF-16.

The wild-type C. elegans strain Bristol N2 and its derivative mutant strains were obtained from the Caenorhabditis Genetics Center (CGC; MN, USA). The mutants used for this study were NL2099 rrf-3 (pk1426) II, CF1038 daf-16 (mu86) I, and TJ356 zls356(DAF-16::GFP + rol-6). Worms were maintained using standard techniques on 9 cm nematode growth medium (NGM) plates seeded with OP50 an incubator at 20 °C⁴⁶. NGM plates contained 2.0% (w/v) peptone, 1.8% (w/v) agar, 25 mM potassium phosphate, 1.0 mM MgSO₄, 1.0 mM CaCl₂, and 5.0 µg/mL cholesterol. OP50 was grown in Luria–Bertani (L.B.) broth overnight at 37 °C. Eggs were recovered from young adult worms by exposure to a lysis solution (0.6% sodium hypochlorite, 200 mM sodium hydroxide). The egg suspension was incubated 12–14 h at 20 °C to facilitate hatching and growth on NGM plates seeded with OP50 for 3 days. Some of the experiments were conducted with the approval of the Institutional Committee for Biosafety of Recombinant DNA Experiments at Ishikawa Prefectural University (Approval No. 24 − 13).

For this study, PM derived from the E. gracilis EOD-1 (Kobelco Eco-Solutions Co., Ltd., Kobe, Japan) was used. E. gracilis EOD-1, a high-PM-accumulating strain, stores 70–80% PM within its biomass. Given that PM from the E. gracilis EOD-1 exists as granules of approximately 3 μm diameter⁴⁷, its administration to C. elegans was performed in accordance with protocols established in earlier studies involving microplastic particle exposure³⁵. Briefly, 4-day-old or 5-day-old young adult worms were transferred onto NGM plates coated with a PM suspension that had been prepared by dispersing PM in an OP50 culture to achieve final concentration of 10 or 30 mg/mL. These concentrations were determined based on our earlier studies in which indigestible oligosaccharides were administered to C. elegans⁴⁸. In addition, regarding the upper concentration limit, we found that observation with a stereomicroscope was technically difficult at concentrations above 50 mg/mL; therefore, we referred to the study by Gao et al. using oat powder containing b-glucan and set the upper concentration at 30 mg/mL⁴⁹.

Four-day-old young adult worms were treated using 5-fluorodeoxyuridine (0.5 mg/mL) to prevent progeny production⁵⁰. The resultant synchronized hermaphrodites were transferred to NGM plates (10 worms per 35 mm plate) covered with OP50 alone or OP50 plus PM (10 or 30 mg/mL). The plates were incubated at 20 °C. Then live and dead worms were counted three times a week. Worms were considered "dead" when they failed to respond to a gentle touch with a worm picker. Worms that crawled off the plate or showed non-natural death, such as internal hatching or adhering to the wall of the plate, were considered "lost." Experiments were performed at least in triplicate, and the "lost" individuals were excluded from the analysis of lifespan.

Four-day-old young adult worms were placed on NGM plates (one worm per 35 mm plate) covered with OP50 alone or OP50 plus 30 mg/mL PM. The live worm body sizes were measured every 24 h until 7 days of age. Then they were analyzed as reported earlier⁴⁴. For brood size measurement, the parental worms were transferred every 24 h to fresh NGM plates until the end of the reproductive period. After the resulting progenies were left to develop for 2 days, they were counted.

The time from egg to the first egg-lay was defined as the developmental time of the worm. Developmental time was measured using the previously described method with minor modifications⁵¹. Four-day-old young adult worms were placed on NGM plates seeded with OP50 and allowed to lay eggs for 30 min. Individual eggs were then transferred to separate NGM plates seeded with OP50 alone or OP50 plus 30 mg/mL PM. Beginning at 67 h after egg laying, worms were examined every hour to determine the onset of egg laying, which was recorded as the developmental time.

The autofluorescence of intestinal lipofuscin was measured as an index of senescence. Four-day-old young adult worms were placed on NGM plates covered with OP50 alone or OP50 plus 30 mg/mL PM until they became 14-day-old adult worms. After randomly selected worms were washed three times with S basal buffer, they were anesthetized with 100 mM sodium azide. Lipofuscin autofluorescence images were detected with excitation at 542 nm and emission at 593 nm using a microscope (EVOS M7000; Thermo Fisher Scientific Inc., MA, USA). To calculate the lipofuscin-positive area, densitometry measurements were taken using ImageJ software.

Four-day-old young adult worms were placed on NGM plates (one worm per 35 mm plate) covered with OP50 alone or OP50 plus 30 mg/mL PM. Locomotory assay of worms was performed every 48 h until 18 days of age using a scoring method described in an earlier report⁵². Worms were classified according to a four-point scale: class "A" worms showed spontaneous movement or vigorous locomotion in response to prodding; class "B" worms did not move unless prodded or appeared to have uncoordinated movement; class "C" worms moved only their head or tail in response to prodding; and class "D" worms were dead.

Four-day-old young adult worms were placed on NGM plates covered with OP50 alone or OP50 plus 30 mg/mL PM for 24 h. Worms were collected and washed three times with sterilized S basal buffer. Then, total RNA was extracted and reverse-transcribed. The resultant cDNA was subjected to qRT-PCR using each specific primer described in Table 1. PCR was performed using a PowerUP SYBR Green PCR Master Mix and a real-time PCR system (StepOnePlus; Applied Biosystems Inc., CA, USA). PCR conditions were denatured as 95 °C for 15 s, with primer-annealing and elongation at 60 °C for 1 min, with subsequent melting curve analysis for which the temperature was increased from 60 °C to 95 °C. The Ct values were transformed into relative quantification data using the 2^{− ΔΔCt} method. Data were normalized to the act-1 endogenous control.

RNA interference was conducted based on a method reported earlier, with minor modifications⁵³. Briefly, HT115 harboring either the L4440 vector expressing clec-196 dsRNA (Clone: sjj_F26D10.12; DNAFORM Inc., Yokohama, Japan) or the empty L4440 vector (#1654; Addgene Inc., MA, USA) as a negative control was cultured overnight in L.B. broth. Subsequently, 1.0 mL of the bacterial suspension was spread onto NGM plates supplemented with 100 µg/mL ampicillin and 1.0 mM isopropyl-β-D-thiogalactopyranoside. The plates were left at room temperature overnight. Synchronized L1 larvae of the RNAi-sensitive C. elegans strain NL2099 were then transferred to the prepared NGM plates and were cultivated until 4-day-old young adult worms. The worms were subsequently used for the respective assays. To assess the knockdown efficiency, total RNA was extracted from 4-day-old young adult worms and was analyzed using qRT-PCR.

Four-day-old young adult worms of TJ356 strain were placed on NGM plates covered with OP50 alone or OP50 plus 30 mg/mL PM for 24 h. Randomly selected worms were washed three times with S basal buffer and were anesthetized with 100 mM sodium azide. Changes in GFP fluorescence in DAF-16::GFP worms were observed with excitation at 470 nm and emission at 510 nm using the imaging system (EVOS M7000; Thermo Fisher Scientific K.K.). GFP expression patterns were classified into three categories: cytosolic, intermediate, and nuclear, as described in an earlier report⁵⁴. The distribution ratio of each GFP expression pattern was calculated for each treatment group. As a positive control for the nuclear pattern, 5-day-old worms subjected to heat stress at 35 °C for 2 h were used.

To detect cellular H₂O₂ levels in worms, fluorescent probe BES-H₂O₂-Ac (Fujifilm Wako Pure Chemical Corp., Tokyo, Japan) was used. For use as a staining solution, the BES-H₂O₂-Ac was diluted to a final concentration of 200 µM using S basal buffer. Four-day-old young adult worms were placed on NGM plates covered with OP50 alone or OP50 plus 30 mg/mL PM until they developed to 14-day-old adult worms. After randomly selected worms were washed three times with S basal buffer, they were treated with 450 µL of staining solution for 1 h. After washing with S basal buffer, the worms were anesthetized using 100 mM sodium azide. The H₂O₂ levels were observed with excitation at 470 nm and emission at 510 nm using an imaging system (EVOS M7000; Thermo Fisher Scientific K.K.).

After C. elegans survival was calculated using the Kaplan–Meier method, survival differences were tested for significance using the log-rank test. For comparisons between the Control and PM groups, normality was first assessed using the Shapiro–Wilk test. When the data did not follow a normal distribution, significance was determined using the Mann–Whitney U test; otherwise, Student's t-test was applied. Comparisons among four groups were performed using one-way analysis of variance (ANOVA) with Tukey's post hoc testing. Statistical analyses were performed using software (GraphPad Prism ver. 8.0.1; GraphPad Software Inc., CA, USA). All results are expressed as mean ± SE. Differences for which p < 0.05 were inferred as significant.