Swimming is more energetically demanding than crawling for C. elegans

C. elegans locomotor patterns are modulated by their physical environment, with crawling the behavioral response on the surface of firm substrates like agar, and swimming the behavior adopted in liquids. The increased activity of C. elegans in a liquid environment, revealed by a higher frequency undulatory behavior [12–14], might suggest that this locomotion mode requires more energy than crawling. However, the mechanical resistance an animal encounters when crawling on an agar surface can be up to 10,000-fold larger than that encountered when swimming in a low viscosity liquid [15, 16]. Given the considerable differences in swim versus crawl behavior, we sought to resolve the question of which locomotor behavior is more energetically demanding by directly measuring the metabolic rate of C. elegans under both conditions and calculating the energy cost of the swim and the crawl.

Microcalorimetry directly assays the heat produced by live C. elegans and measures the total metabolic rate (the heat generated includes that produced by both aerobic and anaerobic metabolism) [17] (Fig. 1a). We first measured the standard metabolic rate (SMR) at 20 °C, which corresponds to the energy consumed by C. elegans when there is no locomotion. We immobilized animals either by levamisole-induced paralysis of wild-type N2 animals or by the genetic paralysis of unc-54 mutants deficient in a muscle myosin heavy chain. Microcalorimetry measures for both chemical and genetic immobilization showed that stationary animals spent significantly less energy in a liquid environment than on a solid environment (Fig. 1b), possibly due to differences in oxygen availability on agar versus liquid. We next measured the active metabolic rate (AMR), which corresponds to the total energy consumed during a unit time of locomotion. We found that the AMR was similar for N2 crawling on an agar surface and swimming in M9 buffer (Fig. 1c). We then calculated the energy cost of both locomotion forms by subtracting the measured SMR from the AMR (Fig. 1d). Our energy cost calculations revealed that swimming was a more energetically demanding locomotion mode for C. elegans than crawling, requiring enhanced energy as compared to the cognate immobilized baseline (Fig. 1e). The C. elegans body wall muscle that mediates locomotion is likely the focus for most energy expenditure during swimming. Given the mammalian exercise definition as “any planned, structured, and repetitive bodily movement produced by skeletal muscles that results in energy expenditure [18]”, our findings suggest that swimming can be considered as exercise for the nematode C. elegans.

Fig. 1 Swimming is more energetically demanding than crawling for C. elegans. a A microcalorimeter measuring unit composed of a reference ampoule (without animals) and a test ampoule (with C. elegans) within a precisely temperature-regulated water bath. Heat flows are monitored by super-sensitive heat detectors. b Microcalorimetry measurements of standard metabolic rate (SMR, metabolic rate at rest) at 20 °C on a solid (nematode growth medium (NGM) agar) or in a liquid (M9 buffer) environment. We immobilized animals for SMR measurements by levamisole-induced paralysis for N2 animals (n = 4 independent trials) or by genetic paralysis of unc-54 mutants deficient in a major muscle myosin (n = 5 independent trials), and measured heat output normalized to the total amount of protein in each sample. c Microcalorimetry measurements of active metabolic rate (AMR) at 20 °C on a solid (NGM agar) or in a liquid (M9 buffer) environment by N2 animals (n = 7 independent trials). Heat output was normalized to the total amount of protein in each sample. Because unc-54 mutants cannot move, their AMR cannot be measured. d Calculating the energy cost of locomotion. The energy cost of crawling or swimming equals the difference between the AMR and the SMR on a solid or in a liquid environment, respectively. e Energy cost of both locomotion forms calculated based on the microcalorimetry measures presented in (b) and (c). Note that when using unc-54 SMR to calculate the energy cost of locomotion, we compared it to N2 AMR, and the genetic background of these two strains might differ slightly. Statistical significance was determined by paired two-tailed Student’s t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. ns non-significant Full size image

C. elegans get tired after acute swim exercise

Young adult C. elegans swim continuously in M9 buffer for just over 90 min before entering an episodic phase during which periods of active swimming alternate with periods of quiescence [19]. For that reason, we decided to adopt an acute exercise protocol in which young adult C. elegans continuously swam in M9 buffer up to the 90 min transition point and were then returned to a bacterially seeded nematode growth medium (NGM) agar plate. We transferred crawling control animals for the same 90 min to an unseeded NGM agar plate (Fig. 2a). This experimental design guaranteed that any differences observed between exercised and control animals were not due to differences in food availability or to the rough handling associated with pick-mediated transfer of animals. For all the experiments presented here, samples were collected either immediately before exercise or at different time points post-exercise.

Fig. 2 C. elegans get tired after acute swim exercise. a The acute swim exercise (M9 buffer) and control (unseeded plate crawl) protocol for C. elegans. b Crawling distance traveled by N2 animals at different time points after a 90 min swim exercise. Each point represents a single animal (n = 49–50 animals). c Crawling distance ratio of exercise to control N2 animals 5 min after a swim exercise of displayed durations (n = 40–50 animals). Unpaired two-tailed Student’s t test was used in (b) and (c) to compare crawling distances of control versus exercise animals. *P < 0.05; ****P < 0.0001. NGM nematode growth medium, ns non-significant Full size image

We observed the crawling behavior of animals upon return to food-containing plates after a 90 min swim. We measured the locomotion on plates by the track length left on the bacterial lawns to show that animals that had swum for 90 min initially moved less than their non-swim siblings (Fig. 2b). Our quantification 5 min immediately after the end of the swim exercise revealed that exercised animals crawled less than half the distance covered by the control animals when returned to the seeded NGM agar plates. Notably, however, exercised animals recovered to normal movement levels within 1 hour, as we found no difference in crawling distance between exercised and non-exercised controls at 1 and 2 hours post-exercise (Fig. 2b).

To determine if the reduction in locomotion distance might be attributed to a change in the physical environment (liquid to solid), we exercised C. elegans for different periods of time. When animals swam in M9 buffer for 5 min or 30 min, we found no reduction in the crawling distance after animals were returned to seeded plates (Fig. 2c), showing that this temporary slow-down phenotype is not a consequence of the transition from a liquid to a solid locomotory mode. In fact, we observed the first signs of tiring and reduced crawling ability only after a 60 min swim exercise, which became more pronounced with the 90 min exercise period (Fig. 2c). Our results reveal that a long swim exercise can induce locomotory fatigue as measured shortly after exercise completion. The fact that C. elegans get tired after an acute swim period further supports that swimming is more energetically demanding than crawling.

C. elegans swim exercise is associated with an increase in mitochondrial oxidative stress

It is well known that physical exercise is associated with oxidative stress in human and mammalian muscle [20, 21]. To address whether the generation of reactive oxygen species (ROS) and an oxidative stress response in muscle are features of C. elegans swim exercise, we first took advantage of the reduction-oxidation-sensitive green fluorescent protein (roGFP), which allows for ratiometric quantification of redox changes in live cells [22]. Using a C. elegans strain that expresses roGFP specifically in the body wall muscle mitochondria [23], we determined that immediately after the swim exercise, the muscle mitochondrial matrix of swimmers was significantly more oxidized than the mitochondrial matrix in matched non-exercise controls (Fig. 3a). The increased oxidation level was maintained 1 hour after the end of exercise, but by 4 hours post-exercise the mitochondrial oxidative environment again matched control levels (Fig. 3a). Thus, as is true in mammals, acute physical exercise in C. elegans increases mitochondrial oxidation in muscle, and nematodes are able to adapt/clear elevated oxidation levels within a few hours to re-establish the mitochondrial oxidative environment to basal levels.

Fig. 3 C. elegans swim exercise increases muscle mitochondrial oxidation and induces a specific transcriptional oxidative stress response. a Relative mitochondrial oxidation level of P myo-3 mito-roGFP-expressing animals at different time points post-exercise (higher 405/488 ratios indicate increased oxidation levels). We took confocal images of body wall muscle at both 405 nm and 488 nm excitation and used the mean fluorescence intensities of mitochondrial regions (40–50 regions per animal) to calculate the 405/488 ratios. Each point represents the 405/488 ratio average from a single animal (n = 60–63 animals); statistical significance was determined by unpaired two-tailed Student’s t test. b Heat map summarizing quantitative polymerase chain reaction results in N2 animals for commonly used stress reporter genes at different time points post-exercise (n = 5 independent trials). Expression data are presented as the log 2 fold change of exercise samples relative to control samples in a color gradient from red (downregulation) to dark green (upregulation). White represents no change in expression levels. Paired two-tailed Student’s t tests were used to compare relative expressions of control versus exercise samples at each time point. See Additional file 1 and Additional file 4A–F for detailed results for each gene. c Percentage of surviving N2 animals during treatment with 3 mM juglone 4 hours post-exercise (n = 60 animals). d Average increase in percent survival during treatment with 3 mM juglone 4 hours post-exercise of N2, sod-3(tm760), sod-4(gk101), and sod-5(tm1146) exercised animals relative to control counterparts (n = 60 animals). The average increase in percent survival was calculated from all the time points between 15 and 45 min of treatment duration. Note that the reduced survival benefit of exercised sod-4 mutants was not due to a reduced swimming capacity relative to N2 and the other sod mutants. See Additional file 3B–D for detailed survival curves of sod mutants. Statistical significance in (c) and (d) determined by a log-rank test comparing control versus exercise survival curves. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ER-UPR endoplasmic reticulum unfolded protein response, HSR heat shock response, ns, non-significant, mito-UPR mitochondrial unfolded protein response Full size image

Changes in specific oxidative stress response transcripts accompany swim exercise

Having documented evidence of a transient oxidative increase in C. elegans body wall muscle mitochondria after acute exercise, we used quantitative polymerase chain reaction (qPCR) to quantitate transcript levels of selected cellular oxidative stress-responsive genes immediately after the swim and during recovery. We first focused on key antioxidant defense genes encoding superoxide dismutase (SOD, the sole detoxifying enzyme for superoxide) and catalase (detoxifies H 2 O 2 ). The C. elegans genome encodes five SODs: SOD-1 (constitutive) and SOD-5 (inducible) are cytoplasmic, SOD-2 (constitutive) and SOD-3 (inducible) are mitochondrial, and SOD-4 is extracellular [24]. There are three C. elegans catalases, CTL-1 (cytoplasmic), CTL-2 (peroxisomal), and CTL-3 [25, 26]. Our quantitation revealed that sod-4 and sod-5 are significantly upregulated immediately after the swim exercise, but rapidly return to control levels in later time points; sod-3, ctl-1, and ctl-2 are significantly downregulated following exercise; and sod-1, sod-2, and ctl-3 transcripts showed little or no change between control and exercised animals (Fig. 3b and Additional file 1A–H). We also analyzed the expression of gst-4 and gcs-1, which encode phase II detoxification enzymes involved in glutathione antioxidant defense [27–29]. While the transcript levels of gst-4 (mainly expressed in body wall muscle [30]) changed significantly during the swim/recovery period, the levels of gcs-1 (mainly expressed in the pharynx and intestine [29]) did not (Fig. 3b; Additional file 1I, J). Finally, we also included the heat shock protein genes hsp-16.2 and hsp-16.41 (genes divergently transcribed from a shared promoter [31]) in our qPCR analysis, given their previous documentation as oxidative stress-responsive reporters in C. elegans (in addition to heat shock inducibility) [32–34]. Both hsp-16.2 and hsp-16.41 showed a marked upregulation in swim-exercised animals, with peak expression occurring at 2 hours post-exercise (Fig. 3b; Additional file 1K, L).

Importantly, the expression changes we documented were exercise-dependent rather than attributable to liquid exposure, given that paralyzed unc-54 mutants placed in M9 buffer for 90 min did not show similar expression patterns for the most strongly affected transcripts in wild-type animals (Additional file 2). Our sampling of oxidative stress response machinery thus shows that an acute swim exercise in C. elegans induces a “mixed” oxidative stress transcriptional response in the hours post-exercise, featuring upregulation of some oxidative stress genes (e.g., sod-4, sod-5, gst-4, hsp-16.2, hsp-16.41) and concomitant downregulation of others (e.g., sod-3, ctl-1, ctl-2).

Mild stresses are thought to induce protective defenses via a process called hormesis, and we wondered if a swim session and its associated physiological changes might enhance robustness. To address whether acute swim bouts might enhance overall oxidative defenses in exercised animals, we treated nematodes at 4 hours post-exercise with juglone, a xenobiotic compound that generates high levels of ROS [35]. We found that a significantly higher proportion of swim-exercised animals survived the juglone treatment as compared to their non-exercised control counterparts (Fig. 3c). This exercise benefit was mostly gone by 24 hours post-exercise, even though exercised animals still exhibited a trend for increased juglone resistance (Additional file 3A).

Given the crucial role of SODs in addressing high levels of superoxide, we investigated whether the SOD genes modulated by swim exercise in C. elegans (i.e., sod-3, sod-4, and sod-5) had any role in the increased survival to juglone treatment. We found that exercised sod-3 and sod-5 mutants exhibited increased survival relative to their control counterparts, identical to N2 animals (30–35%). However, exercised sod-4 mutants exhibited a diminished response of only a 15% survival increase (Fig. 3d; Additional file 3B–D). We conclude that even a single swim exercise bout is associated with physiological changes, partially dependent on the extracellular SOD-4, that have a role in protection against a subsequent lethal oxidative stress.

Swim exercise does not induce a generalized stress response in C. elegans

To address how generally stress responses might be activated during the swim, we determined expression levels by qPCR of commonly studied reporter genes that are activated by multiple types of stress: hsp-1 and hsp-70 for heat shock response (HSR) [36, 37]; hsp-6 and hsp-60 for mitochondrial unfolded protein response (mito-UPR) [38]; hsp-4 for endoplasmic reticulum unfolded protein response (ER-UPR) [39]; nhr-57 for hypoxia response [40]; and gpdh-1 and nlp-29 for osmotic response [41, 42].

Our qPCR data suggest that swimming in M9 buffer for 90 min does not induce short-term HSR, mito-UPR, or ER-UPR because the reporter genes tested exhibited no induction in exercised animals (some of them were even slightly downregulated at specific time points) (Fig. 3b; Additional file 4A–E). Interestingly, we found that hypoxia-induced reporter nhr-57 was significantly upregulated immediately after swim exercise and then returned to control levels in the following time points (Fig. 3b; Additional file 4F). Because nhr-57 upregulation did not occur when unc-54 mutants were exposed to M9 buffer (Additional file 4G), the expression changes can be attributed to exercise rather than to transfer into liquid. Data suggest that although hypoxia-associated signaling and responses occur during the swim, hypoxia signaling may be rapidly reversed upon return to a solid environment. Regarding osmotic stress reporter genes, both nlp-29 and gpdh-1 transcripts exhibited a highly dynamic expression pattern even in non-exercised control animals (Additional file 4H, I), suggesting that under our experimental setup, physical manipulations, rather than osmotic stress, drove their expression.

Overall, our data establish that a 90 min swim in M9 buffer does not lead to full-blown transcriptional activation of multiple stress pathways. Rather, specific and distributed exercise-dependent transcriptional changes accompany the acute swim bout. Moreover, the exercise experience is associated with enhanced oxidative stress defenses, suggesting that specific modulation of oxidative stress pathways may suffice to provide significant physiological benefit at least during the hours following the exercise bout.

Transcriptional changes are consistent with reduced glucose metabolism after swim exercise

The two main sources of energy during physical exercise are carbohydrates and fats. Therefore, we analyzed how acute swim exercise might affect gene expression of key members of both C. elegans carbohydrate and fat metabolism. To evaluate potential carbohydrate utilization, we focused our analysis on glucose metabolism: glucose transport, glycolysis, and gluconeogenesis (Fig. 4a). We found persistent downregulation of transcript levels of the main C. elegans glucose transporter, encoded by fgt-1, between 1 and 4 hours post-exercise (Fig. 4b; Additional file 5A). Expression of three essential glycolytic enzymes was also significantly downregulated in exercised animals at different time points: hxk-2, hexokinase (first enzyme of glycolysis); pfk-1.1, phosphofructokinase (rate-limiting enzyme of glycolysis); and pyk-1 and pyk-2, pyruvate kinases (last enzymes of glycolysis) (Fig. 4b; Additional file 5B–E). ldh-1, encoding the B subunit of lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate in a post-glycolysis process, was also downregulated between 1 and 4 hours after swim exercise (Fig. 4b; Additional file 5F). Finally, a similar temporal pattern of downregulation was observed for pck-1, a phosphoenolpyruvate carboxykinase that is the rate-limiting enzyme of gluconeogenesis (Fig. 4b; Additional file 5G; magnitude of downregulation greater for pck-1). The shared theme of expression downregulation of seven out of seven selected genes indicates that glucose metabolism as a whole may be reduced in the hours following acute swim exercise.

Fig. 4 Transcriptional changes in C. elegans are consistent with reduced glucose metabolism after swim exercise. a Diagram of glucose metabolism highlighting the steps that we chose for our expression analysis. C. elegans proteins are shown in red. For simplicity, several metabolic steps are omitted. b Heat map summarizing quantitative polymerase chain reaction results in N2 animals at different time points post-exercise for glucose metabolic genes (n = 5 independent trials). Expression data is presented as log 2 fold change of exercise samples relative to control samples in a color gradient from red (downregulation) to white (no change). Paired two-tailed Student’s t tests were used to compare relative expressions of control versus exercise samples at each time point. See Additional file 5 for detailed results for each gene. **P < 0.01; ***P < 0.001; ****P < 0.0001 Full size image

Transcriptional changes suggest increased fat metabolism during exercise

Fat metabolism is a complex network of enzymatic reactions that can lead to outcomes as diverse as lipid storage, fatty acid breakdown, lipid incorporation into cell membranes, or cell signaling, depending on the metabolic state and requirements of the organism. A large proportion of lipids in C. elegans, such as triglycerides, are stored in lipid droplets across different tissues. For fatty acids to be used as a source of energy, lipases have to break down triglycerides into glycerol and fatty acids. Fatty acids are then available for activation (conversion into acyl-coenzyme A(CoA)) followed by beta-oxidation, which generates acetyl-CoA that can enter the tricarboxylic acid cycle. We focused our analysis of lipid metabolism gene expression on fatty acid breakdown and lipid storage (Fig. 5a).

Fig. 5 Transcriptional changes in C. elegans suggest increased fat metabolism during swim exercise. a Diagram of fat metabolism highlighting the steps that we chose for our expression analysis. C. elegans proteins are shown in red. For simplicity, several metabolic steps are omitted. b Heat map summarizing quantitative polymerase chain reaction results in N2 animals at different time points post-exercise for fat metabolic genes (n = 5 independent trials). Expression data is presented as log 2 fold change of exercise samples relative to control samples in a color gradient from red (downregulation) to dark green (upregulation). White represents no change in expression levels. Paired two-tailed Student’s t tests were used to compare relative expressions of control versus exercise samples at each time point. See Additional file 6 for detailed results for each gene. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 Full size image

Our qPCR analyses revealed that transcripts of three C. elegans lipases, C07E3.9, C03H5.4, and atgl-1 (the latter of which is documented to be expressed in muscle [43]), were significantly upregulated immediately after swim exercise. C07E3.9 maintained a higher expression level in exercised animals even after 4 hours, whereas C03H5.4 and atgl-1 returned to control levels quickly after exercise completion. Other C. elegans lipases, known to be primarily expressed outside of muscle [43, 44], either did not change their expression (D1054.1) or were downregulated (hosl-1 and lipl-4) after exercise (Fig. 5b; Additional file 6A–F). The enzyme responsible for fatty acid activation (acyl-CoA synthetase), encoded by acs-2, and the alpha subunit of the mitochondrial trifunctional protein responsible for the last three steps of fatty acid beta-oxidation, encoded by ech-1.1, were also upregulated at the end of swim exercise, followed by a post-exercise downregulation (Fig. 5b; Additional file 6G, H). Among the tested genes involved in fatty acid breakdown, an expression pattern emerged in which upregulation was observed specifically at the time point immediately after swim exercise. The transcriptional changes we quantitated suggest that C. elegans adapt gene expression in a manner expected to increase fat metabolism during physical exercise.

Consistent with the hypothesis of increased lipid breakdown during swim exercise, we also observed a strong downregulation of C. elegans delta-9 fatty acid desaturases, encoded by fat-5, fat-6, and fat-7, at the 0 hour time point (Fig. 5b; Additional file 6I–K). Downregulation of these desaturases as occurs during swim exercise should promote fat breakdown instead of fat synthesis/storage [45, 46]. Finally, we analyzed the transcriptional levels of genes encoding proteins involved in intracellular transport of lipids. Interestingly, two out of three fatty acid binding proteins, lbp-7 and lbp-8, and one acyl-CoA binding protein, acbp-3, showed strong downregulation between 0 and 2 hours post-exercise (Fig. 5b; Additional file 6L–O). It has been proposed that LBP-7, LBP-8, and ACBP-3 can inhibit beta-oxidation by sequestering fatty acids [47, 48]. Furthermore, acbp-3 mutants have been shown to contain lower levels of triglycerides accompanied by an increase in fatty acid beta-oxidation [49], suggesting that post-exercise downregulation of lipid binding proteins may promote fat breakdown.

Lipid storage is depleted specifically in C. elegans body wall muscle after swim exercise

Our expression analyses strongly suggest that lipid catabolism increases in C. elegans during swim exercise, which led us to explore whether lipid stores in different C. elegans tissues are affected by physical exercise. To evaluate tissue-specific fat use, we used C. elegans strains in which lipid droplets were fluorescently labeled by Perilipin 1-GFP in a tissue-specific manner [50]. We measured lipid droplets in these strains via quantitative image analysis immediately after the swim, comparing exercised to non-exercise animals. While the number of lipid droplets in the intestine (Fig. 6a, b) or hypodermis (Fig. 6c, d) was not changed during exercise, we measured a significant decrease in the lipid droplets of body wall muscle immediately after swim exercise (Fig. 6e, f). Muscle lipid droplet number returned to control levels just 1 hour later (Fig. 6e), revealing the highly dynamic regulation of fat metabolism in exercised muscle. These results confirm that C. elegans increase fat breakdown during swim exercise in a tissue-specific manner, with intramuscular lipid reserves being used to fuel the high-energy swimming locomotion. Moreover, our data underscore that even a single swim bout suffices to induce physiological change in exercised muscle.