To investigate the protective effect of TWE against heat stress in C. elegans wild type N2, the treated nematodes were exposed to higher temperature (30 or 35 °C) and survival rate was observed. TWE at a concentration of 300 μg/mL showed the largest positive effect and enhanced thermo tolerance in the worm at both temperatures (data not shown). When the adult worms were exposed to mild heat stress (30 °C), about 60% enhancement in mean life span was observed in TWE-treatment. Similarly, TWE treatment also had a positive effect (P < 0.05) on survival rates of worms at higher temperature (35 °C). At the time when all of the control worms died, 48% of the nematodes treated with TWE (300 μg/mL) were alive. The mean survival rate was significantly increased (68%) by 300 μg/mL TWE treatment compared to the control.

In C. elegans the transcription factor daf-16 mediates the expression of a number of genes that leads to stress tolerance and improved longevity. To study whether daf-16 was involved in TWE-elicited thermal tolerance, the effect of TWE on sub-cellular localization of daf-16 was examined in a transgenic nematode strain TJ356. In this strain, daf-16 is fused to a green fluorescent protein (GFP) making it possible to observe sub-cellular localization of daf-16 in the living worm. Under normal growth conditions, daf-16 is localized primarily in the cytosol, but under heat stress it is rapidly trans located into the nucleus [12]. Our results clearly showed that the daf-16::gfp protein was trans-located into the nucleus of the intestinal cells under heat stress (Figure 1). TWE treatment did not affect nuclear translocation of daf-16::gfp. The translocation of the fusion protein was similar in both the control and TWE treated worm (Figure 1).

To further rule out the possible involvement of daf-16 pathway in the TWE-induced thermal stress tolerance, the response of the wild type strain (Bristol N2) was compared to a daf-16 mutant (mgDf50). The wild type and the daf-16 mutant (mgDf50) nematode were exposed to heat stress (35 °C). The result showed that TWE treatment enhanced the thermal tolerance of both the wild type and the daf-16 mutant with a mean increase of 1.3 and 2.2-fold, respectively (Figure 2). This indicates that the effect of TWE on heat tolerance in C. elegans is independent of daf-16.

To determine the effect of TWE on lifespan of C. elegans N2 strain, the worms were treated with different concentrations of TWE. Low concentration (20 μg/mL) of TWE had no effect on longevity (Table 1; Figure 3). However, the higher concentrations (100 and 300 μg/mL) significantly extended the mean life span by 2 days or more. On the other hand, higher concentrations (e.g., 600 and 1000 μg/mL) had no significant effect on the life span of C. elegans as compared to the 300 μg/mL (Figure 3; Table 1).

The most effective concentration TWE 300 μg/mL not only extended the mean life span of C. elegans by 3 days (indicative of an extension of mean life span by 17%), but also increased the maximum life span by approximately 17% (5 days) (data not shown). Because TWE treatments were present throughout the entire life stages of C. elegans (starting from the egg stage and onward), the question arose whether the TWE extended life span when the nematode was exposed at a later life stage. Interestingly, the median concentration TWE (300 μg/mL) extended the mean life span (17.76 days) (P < 0.05) compared to the control (16.13 days) (Figure 4).

TWE treatment did not affect body length of nematodes. Average body length was 1.23 mm in TWE-treated worms compared to 1.30 mm in the control (data not shown). Similarly, the TWE treatment had no effect on body width. The average width was 66.0 μm in controls and 65.6 μm in the TWE-treated worms (data not shown).

The pharyngeal pumping rate in TWE-treated worms and controls were measured to examine the possibility that TWE treatment may have perhaps reduced the food intake of C. elegans, which in turn may lead to caloric restriction and as a result increased life span. C. elegans uses the pharynx to pump in the bacteria upon which it feeds and transports them to the intestine. The speed of pharyngeal contraction declines with age and the reduction in pumping rate has been applied as one of the indices of the decline of overall physiological functions. The age-dependent decline in pumping rate from day 3–9 of adulthood was apparent in the control. However, the control nematodes exhibited a significant decline in pharyngeal pumping rate as compared to TWE-treated on day 6 and 9 of adulthood (Figure 5).

To test the effect of TWE treatment on C. elegans reproduction, the distribution of daily reproductive output and overall brood size were recorded. Reproduction was observed on day 1 and lasted for 4 days of adulthood. No significant differences was found in the brood size, averaging 230 (control) and 215 (TWE-treated) per worm. However, it was interesting to note that TWE-treated nematodes showed a significant delay in fecundity with a >2 fold decrease on day 2, but showed a surprising >4 fold increase in fecundity on day 4 (Figure 6).

To elucidate whether TWE treatment elicits stress related molecular responses in C. elegans, we studied transgenic lines of superoxide dismutases (sod-3), antioxidant enzymes also important regulators of life span and stress resistance, stress-responsive glutathione S-transferase (gst-4) that confers resistance to oxidative stress and hsp-16.2. Transgenic strains of nematode sod-3::gfp (cf1553), gst-4::gfp (cl2166) and hsp-16.2::gfp (cl2070) were treated with TWE and exposed to thermal stress. The expression of sod3, gst4 and hsp16.2 was examined by observing the intensity of green fluorescent protein. TWE treatment significantly reduced the fluorescence in nematode carrying the sod-3::gfp (60% less compared to the control, Figure 7), indicating that TWE-treatment did not increase the expression of the sod-3 gene. Similarly, TWE treatment significantly reduced the fluorescence in the hsp-16.2::gfp strain after a 2 h heat-shock (at 35 °C), followed by 4 h recovery at 20 °C. TWE (300 μg/mL) concentration reduced fluorescence in C. elegans strain gst-4::gfp as compared to untreated control (Figure 7).

Because the heat stress effect and aging is related to oxidative stress, we measured the concentration of intracellular ROS in C. elegans. In the TWE-treated wild type N2 worms, TWE treatment significantly reduced the concentration of ROS under oxidative stress (300 μM juglone) compared to worms without TWE at 2 h, 4 h and 6 h of treatment (Figure 8).

TWE treatment altered the expression of stress-related genes (hsp-16.2, and skn-1) (Figure 9). The expression of hsp-16.2, increased considerably with heat stress and was higher in TWE-treated worms compared to the control. Similarly, skn-1 was up- regulated in TWE treatment. In contrast, the expression of daf-2, sod-3, and daf-16 genes remained unchanged under heat stress in both the TWE treatment and the control.

Proteomic changes induced by TWE treatment under thermal stress was studied by 2D gel electrophoresis analysis of the total protein (Figure 10). Most C. elegans proteins resolved at pH 4–7 and about 1000 spots were detected on a SDS-PAGE. Protein spots of TWE-treated and control worms were compared under thermal stress and non-stressed conditions. Differentially expressed protein spots were then isolated, digested with trypsin, treated with POROS R2 and R3 resins, and analyzed by MALDI-TOF-MS or MALDI-TOF-MS/MS. The location of the corresponding peak for each protein and its mass and pI were confirmed (Table 2). Analysis of C. elegans total proteome under heat stress and under non-stressed conditions revealed 40 differentially expressed proteins. Among the 40 proteins, 34 proteins were up-regulated and 6 were down-regulated (Table 2). The TWE specific differentially expressed proteins of C. elegans were functionally characterized and listed in Table 2.

To study the probable function of the identified proteins, a standard protein BLAST search was performed using NCBI protein database [13] (Table 2) according to the Worm pep database. Interestingly, of the 40 proteins identified in this study, more than 80% of the proteins were “hypothetical proteins”. Analysis of PMF data of 40 proteins derived by MS analysis using the MASCOT search algorithm showed functional homology to the following proteins: CAEEL Putative uncharacterized protein; putative aldehyde dehydrogenase family 7 member A1 homolog; heat shock protein Hsp-16.11/16.48/16.49/25; 40S ribosomal protein SA OS; saposin-like protein family protein 20; carbamoyl-phosphate synthase L chain ATP-binding protein OS; superoxide dismutase OS; sensory axon guidance protein 7; myosin regulatory light chain 1 OS; cytochrome c oxidase subunit 5A; ATP synthase subunit beta, mitochondrial OS; glutathione peroxidase R05H10.5 OS; galectin OS; 60S ribosomal protein L18 OS; and peroxiredoxin protein 2. Based on descriptions derived from three separate databases (GOA, KEGG, NCBI KOG), all the identified proteins were classified into nine functional classes: (1) general metabolic process, (2) energy metabolism, (3) nucleotide/Protein binding, (4) electron transport, (5) protein synthesis, (6) proteolysis, (7) response to heat, (8) stress response, and (9) signal transduction.

All chemicals were purchased from Sigma Aldrich, Oakville, Ontario, Canada, unless otherwise stated. Samples of Tasco^® powder were supplied by Acadian Seaplants Limited, Dartmouth, Nova Scotia, Canada. The wild type Caenorhabditis elegans strain N2 (var. Bristol), the transgenic strains CF1553 [muIs84[pAD76(sod-3::gfp)], CL2070 [dvIs70(hsp-16.2::gfp; rol-6(su1006))], CL2166 [dvIs19(pAF15(gst-4::gfp::NLS))], GR1307 [daf-16(mgDf50)], and TJ356 [zIs356 [daf-16::gfp rol-6(su1006)], and Escherichia coli strain OP50 were obtained from the Caenorhabditis Genetics Center, University of Minnesota, USA. Cultures of OP50 E. coli were grown overnight in LB broth and concentrated 10 times by centrifugation at 3500 × g for 10 min. The C. elegans strains were maintained at 20 °C on 1.2% solid nematode growth medium (NGM) [42] seeded with 50 μL of live OP50 E. coli as a food source.

Aqueous extract of Tasco^® (TWE) was prepared by dissolving 10 g of the product in 40 mL distilled water at 70 °C for 1 h. The mixture was centrifuged at 10,000 × g for 10 min at room temperature and the supernatant was pipetted out and put in a new tube. The pellet was redissolved in 40 mL water and the extraction procedure was repeated. The resulting supernatants were combined, dried under nitrogen (N₂) gas and stored at 4 °C until use. A stock solution equivalent to −40 mg/mL of Tasco^® was prepared in distilled water.

Synchronized N2 eggs were placed on NGM plates (~25 eggs/plate) containing TWE at various concentrations (i.e., 20, 100, 300, 600 or 1000 μg/mL) or vehicle (distilled water) with heat-killed OP50 as food source and maintained at 20 °C. Three day old adult worms, treated with TWE, were exposed to two thermal stress temperatures i.e., 30 °C and 35 °C in an incubator (Innova, New Brunswick, Canada). At 30 °C, the mortality of the worms was enumerated at 24 h intervals whereas at 35 °C, the number of individuals was counted every 2 h until all the worms had died.

To study the involvement of daf-16, a FOXO-family transcription factor that maneuvers the rate of ageing of C. elegans, in TWE-mediated stress tolerance, young adults of daf-16 loss-of-function mutant (GR1307) and wild type (N2) nematodes were incubated at 35 °C for 7 and 9 h, respectively, and the number of surviving individuals were recorded. C. elegans strains CL2070, CF1553 and CL2166 containing inducible GFP reporter for hsp-16.2, sod-3 and gst-4, respectively were cultivated on treatment plates from eggs for 3 days. For strains CF1553 and CF2166 the photographs were taken directly at day 3, whereas for strain CL2070, the worms were exposed to 35 °C for 2 h and allowed to recover at 20 °C for 4 h before photography. A total of 90 nematodes were used for each strain per condition. For daf-16::gfp localization, TJ356 C. elegans were used. Nematodes from normal or the heat stress conditions (i.e., 35°C for 2 h, followed by recovery at 20 °C for 4 h) were placed on 2% agar plates and pictures were taken using a fluorescence microscope. Epi-fluorescence images were acquired at constant exposure using the 20× objective lens. Fluorescence images were acquired at identical exposure times using 20× magnification for each strain. The fluorescence intensity for the head part of the worms was measured using the free Java image processing program ImageJ [43]

Synchronized C. elegans N2 strain eggs were placed on NGM plates (~25 eggs/plate) containing TWE or “vehicle” (distilled water) and maintained at 20 °C. The experimental plates were prepared by adding various concentrations (i.e., 20, 100, 300, 600, or 1000 μg/mL) of TWE stock solution to NGM (at 55 °C) having heat-killed the E. coli OP50 (at 65 °C for 30 min) to be used as food for the nematode. After 3 days, young adult worms were transferred to fresh treatment plates supplemented with 200 μM 5-fluorodeoxyuridine (FUdR) which was used to prevent development of progeny [44]. The nematodes were transferred to the new treatment plates every 2 days. Survival was evaluated daily and the animals were scored as dead if they failed to respond to gentle, repeated touches with a platinum pick. The first day of adulthood was considered as day 1. Individuals that crawled off the walls of the plates and died from desiccation were excluded from the analysis.

Synchronized C. elegans eggs were cultured on treatment plates at 20 °C for 6 days. Forty five individuals per treatment were mounted on 2% agar pads (in M9 buffer) and paralyzed with 25 mM sodium azide (in M9 buffer) and photographed under a fluorescence microscope. Length and width of the individuals were measured using ImageJ (NIH).

Synchronous eggs of the N2 strain were obtained by allowing the adults to lay eggs for 1 h and incubated at 20 °C on for 2 days. The larvae at L4 stage (30 per treatment) were transferred individually to treatment plates for egg laying. The worms were transferred to fresh plates daily until reproduction was completed. The mean number of offspring for each worm was counted beginning 48 h after egg-laying.

Synchronized eggs (N2 strain) were placed on treatment plates spotted with heat-killed OP50 E. coli, raised at 20 °C, and transferred to fresh plates with equal food/TWE treatments every 2 days after reaching adulthood. Thirty nematodes were selected randomly for each treatment and their feeding rate was scored by counting their pharyngeal bulb contractions over a 20 s period, using a microscope at room temperature (22 °C) (see [45]). The pharyngeal pumping rate was counted every 2 days for the first 9 days of adulthood.

Intracellular ROS in C. elegans were measured with H₂DCF-DA as the molecular probe. For ROS detection without any stress conditions, the L4 worms from seaweed treated or untreated conditions were used. The wild type N2 worms were treated the ROS detection under oxidative stress. The L4 worms from TWE 300 μg/mL treated plates along with untreated plates were incubated along with 300 μM juglone in the microplate reader during fluorescence detection. Briefly, the C. elegans were collected into 100 μL phosphate-buffered saline (PBS) with 1% Tween 20 in centrifuge tubes and pipetted into the black flat-bottom 96-wellmicrotiter plate (Costar) containing H₂DCF-DA (final concentration 50 μM in PBS). Samples were read every 2 h for 6 h in a Biotek microplate reader at 37 °C with excitation 485 nm and emission 530 nm.

C. elegans strain N2 was grown and maintained at 20 °C as described above. The N2 adults were allowed to lay eggs on NGM plates for 2 h at 20 °C. The eggs were transferred to control or TWE-supplemented NGM plates until the young adult stage was developed. The worms were heat-shocked (35 °C) for 2 h and 80–100 worms were picked directly into TRIzol Reagent (Invitrogen Life Technologies). The samples were flash frozen in liquid nitrogen. Total RNA was extracted based on the TRIzol method (Invitrogen) with minor modifications. Briefly, 100 μL of TRIzol with worms was added to 70 μL chloroform, mixed well and centrifuged at 10,000 × g for 10 min. The supernatant was mixed with 70 μL ethanol (70%) and loaded directly onto RNeasy spin columns (Qiagen, Canada) to precipitate RNA according to the manufacturer’s protocol. The quality and quantity of RNA obtained was assessed with Nanodrop ND-1000 (NanoDrop Technologies Wilmington, DE) and formaldehyde gel electrophoresis. Total RNA was reverse transcribed with 500 μg of DNA-ase digested total-RNA using Quantiscript reverse transcriptase (Qiagen, Canada) following the manufacturer’s instructions.

The induction of four genes: viz: hsp-16.2, daf-16, sod-3 and skn-1 was used to determine the heat stress response of C. elegans treated with TWE. The daf-16, sod-3, skn-1 and hsp-16.2 are known to be important in the life-span and stress tolerance in C. elegans. The primers used for qReal Time PCR were as follows: hsp-16.2, forward 5′-ACGCCAATTTGCTCCAGTCT-3′, Reverse 5′-GATGGCAAACTTTTGATCATTGTTA-3′; daf-16, forward 5′-TTTCCGTCCCCGAACTCAA-3′, reverse 5′-TTCGCCAACCCATGATGG-3′; sod-3, forward 5′-AGCATCATGCCACCTACGTGA-3′, reverse 5′-CACCACCATTGAATTTCAGCG-3′; skn-1, forward 5′-AGTGTCGGCGTTCCAGATTTC-3′ and reverse 5′-GTCGACGAATCTTGCGAATCA-3′; ama-1, forward 5′-CTGACCCAAAGAACA CGGTGA-3′, reverse 5′-TCCAATTCGATCCGAAGAAGC-3′. The C. elegans gene ama-1 was used as an internal control for quantification. Quantitative, Real-Time PCR was performed on StepOne™ Real-Time PCR System (Applied Biosystems) following the manufacturer’s instructions using SYBR green reagent (Roche Diagnostics, Mississauga, ON, Canada). Data were analyzed from two independent runs.

Each experiment was replicated at least three times with independent trials. Data were represented as mean ± SEM (standard error of the mean) and were analyzed by ANOVA and differences between groups were considered statistically significant at P < 0.05 using Tukeys HSD test with COSTAT^® statistical software. Results from the life-span experiment were processed using the Kaplan-Meier survival analysis of SPSS 15.0 and compared amongst the control and the TWE treated groups for significance by means of a log-rank, pairwise comparison test.