Phenotypes associated with lifespan extension and stress resistance are observed in <i>hyl-1;lagr-1</i>.

<p>(A) Pumping rates of N2, <i>hyl-1;lagr-1,</i> and an <i>eat-2</i> mutant. The latter displays a pronounced reduction in pumping rate and is commonly used as a genetic model for dietary restricted animals. Bars represent the mean number of pumps per minute. Compared to N2 displaying a mean pumping rate of 201±2 pumps/min, <i>hyl-1;lagr-1</i> shows a 17.4% decrease with a mean pumping rate of 166±2, P<0.0001, while <i>eat-2</i> shows a 67.2% decrease with a mean pumping rate of 66±3, P<0.0001. The data represents an mean ± SEM of 15 measurements in 5 worms of each genotype. (B) Quantification of fluorescent beads in the pharynx and the anterior part of the intestine following a feeding period of 30 minutes. Compared to N2, <i>hyl-1;lagr-1</i> displays 59% less fluorescence, P = 0.0026. Mean ± SEM is shown, n indicates the number of worms. (C) Mean total brood size of N2 and <i>hyl-1;lagr-1</i>. Compared to N2 which displays a mean brood size of 304±9, <i>hyl-1;lagr-1</i> shows a 33% decrease with a mean brood size of 204±6, P<0.0001. Mean ± SEM is shown, n = number of worms examined. (D) Survival curves of N2 and <i>hyl-1;lagr-1</i> subjected to heat-shock at 37°C. Compared to N2, <i>hyl-1;lagr-1</i> shows increased resistance, P = 0.0016. A total of 60 worms of each strain were assayed. Mean ± SD of 3 experiments is shown. N2-worms (6) and <i>hyl-1;lagr-1</i> worms (22) were censored but are incorporated in the analysis until the time they were censored. (E) Bars represent mean median heat shock survival from the 3 experiments shown in D. Compared to N2 which has a median survival of 7 hours, <i>hyl-1;lagr-1</i> displays a 29% increase in heat shock resistance with a median survival of 9 hours. Error bars represent ± SD.</p

Autophagy is increased in <i>hyl-1;lagr-1</i> and the response mechanism differs from that of wild type.

<p>LGG-1 is part of autophagosomal membranes and widely used as an indicator of autophagy in <i>C. elegans</i>. Bars represent the mean number of LGG-1::GFP-containing puncta per seem cell in non-starved wild type and <i>hyl-1;lagr-1</i> worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi. The number in each bar indicates the total number of seam cells observed. (A) Knock-down of <i>atg-12</i> lowers the level of autophagy in both wild type and <i>hyl-1;lagr-1</i>. (B) Knock-down of <i>pha-4</i> does not change the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (C) Knock-down of <i>daf-16</i> lowers the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (D) Knock-down of <i>skn-1</i> lowers the increased level of autophagy in <i>hyl-1;lagr-1</i> but increases autophagy in wild type. (E) Knock-down of <i>daf-2</i> increases autophagy to the same extent in wild type and <i>hyl-1;lagr-1</i>. (F) Knock-down of <i>sphk-1</i> increases the level of autophagy in <i>hyl-1;lagr-1</i> beyond wild type level. Statistical analyses were performed by unpaired two-tailed t-test (with Welch’s correction if variances were significantly different) using GraphPad Prism version 6.0 (GraphPad Software). The Bonferroni method was used to correct for multiple comparisons and P values below 0.0125 were considered statistically significant equivalent to a significance level of 0.05. (*) P≤0.0125, (**) P≤0.001, and (***) P≤0.0001. N used for analysis is the total number of worms observed for each treatment (23–45 worms, two trials). Mean ± SEM is shown.</p

Knock-down of PHA-4, DAF-16, SKN-1, ATG-12, or SPHK-1 affect the extended longevity of <i>hyl-1;lagr-1.</i>

<p>Cumulative survival curves of N2 and <i>hyl-1;lagr-1</i> worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi from the early adult stage. (A) When subjected to <i>atg-12</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>atg-12(RNAi)</i> control animals, P = 0.3053. (B) When subjected to <i>pha-</i>4 RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>pha-4(RNAi)</i> control animals, P = 0.2369. (C) When subjected to <i>daf-16</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is decreased beyond the extent of <i>daf-16(RNAi)</i> control animals, P = 0.0002. (D) When subjected to <i>skn-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>skn-1(RNAi)</i> control animals, P = 0.5476. (E) When subjected to <i>daf-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is further extended compared to both <i>hyl-1;lagr-1</i> control animals, P<0.0001, and <i>daf-2(RNAi)</i> control animals, P<0.0001. (F) When subjected to <i>eat-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> control animals, P = 0.0002, while the lifespan of <i>eat-2(RNAi)</i> animals is extended compared to wild-type control animals, P<0.0001. (G) When subjected to <i>aak-2</i> RNAi, <i>hyl-1;lagr-1</i> lifespan is decreased compared to <i>hyl-1;lagr-1</i> animals, P = 0.0009, while no lifespan effect is seen when comparing <i>aak-2(RNAi)</i> animals to wild-type control animals, P = 0.0975. (H) When subjected to <i>sphk-1</i> RNAi, the extended lifespan of <i>hyl-1;lagr-1</i> is normalized to the extent of <i>sphk-1(RNAi)</i> control animals, P = 0.8002. For additional details about these experiments, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone-0070087-t001" target="_blank">Table 1</a>.</p

Adult lifespan of <i>hyl-1;lagr-1</i> and N2 control worms subjected to empty vector control or the indicated RNAi at 20°C.

a<p>Median/mean RNAi lifespan of N2 and <i>hyl-1;lagr-1</i> fed the specified RNAi-bacteria.</p>b<p>Some animals were censored as they crawled of the plate, ruptured, or died as a “bag of worms”, however they are incorporated in the data set up until the day they were censored. The number of individual trials is in parentheses.</p>c<p>Median/mean control lifespan fed vector-only control bacteria.</p>d<p>P-values were determined using the Gehan-Breslow-Wilcoxon test using GraphPad Prism version 6.0 (GraphPad Software). The Bonferroni method was used to correct for multiple comparisons and P- values below 0.0125 are considered statistically significant equivalent to a significance level of 0.05 with four pair-wise comparisons. Cumulative statistics is shown in this table as experimental animals subjected to the same treatment behaved similarly between trials. Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone-0070087-g001" target="_blank">Figure 1</a>.</p

Lipidomic analysis reveals a modified sphingolipid composition in <i>hyl-1;lagr-1</i>.

<p>Relative abundance of detected sphingolipid species showing significant changes in <i>hyl-1;lagr-1</i>. Sphingolipids containing C24-26 fatty acids and sphingomyelin species containing C16-18 fatty acids are lowered in <i>hyl-1;lagr-1</i>, while sphingolipids containing C21-22 fatty acids acids are more abundant. <i>C. elegans</i> sphingolipids predominately contain C17 long-chain bases, thus making the fatty acid chain length readily deducible. The number of carbon atoms indicated is without head groups. (A) Sphingomyelins containing C16-18 fatty acids are significantly reduced in <i>hyl-1;lagr-1</i>. (B) All significantly changed sphingolipid species containing C21 fatty acids are more abundant in <i>hyl-1;lagr-1</i>. (C) All significantly changed sphingolipid species containing C22 fatty acids are more abundant in <i>hyl-1;lagr-1</i>. (D) The only significantly changed sphingolipid species containing C23 fatty acids is more abundant in <i>hyl-1;lagr-1</i>. (E) All significantly changed sphingolipid species containing C24 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. (F) All significantly changed sphingolipid species containing C25 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. (G) All significantly changed sphingolipid species containing C26 fatty acids are less abundant in <i>hyl-1;lagr-1</i>. Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism version 6.0 (GraphPad Software). Results from two biological experiments are shown for each strain. Mean ± SD is shown. All species shown are at least significant different at the P<0.05 level. The entirety of detected sphingolipid species can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone.0070087.s007" target="_blank">Figure S7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070087#pone.0070087.s008" target="_blank">Figure S8</a>.</p