Durotaxis in Nematode Caenorhabditis elegans

Durotaxis in Nematode Caenorhabditis elegans

Article Durotaxis in Nematode Caenorhabditis elegans Lipika Parida1 and Venkat Padmanabhan1,* 1 Department of Chemical Engineering, Indian Institute...

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Durotaxis in Nematode Caenorhabditis elegans Lipika Parida1 and Venkat Padmanabhan1,* 1

Department of Chemical Engineering, Indian Institute of Technology, Kharagpur, West Bengal, India

ABSTRACT Durotaxis is a process where cells are able to sense the stiffness of substrates and preferentially migrate toward stiffer regions. Here, we show that the 1-mm-long nematode, Caenorhabditis elegans are also able to detect the rigidity of underlying substrates and always migrate to regions of higher stiffness. Our results indicate that C. elegans are able to judiciously make a decision to stay on stiffer regions. We found that the, undulation frequency, and wavelength of worms, crawling on surfaces show nonmonotonic behavior with increasing stiffness. A number of control experiments were also conducted to verify whether C. elegans are really able to detect the rigidity of substrates or whether the migration to stiffer regions is due to other factors already reported in the literature. As it is known that bacteria and other single-celled organisms exhibit durotaxis toward stiffer surfaces, we conjecture that durotaxis in C. elegans may be one of the strategies developed to improve their chances of locating food.

INTRODUCTION Caenorhabditis elegans are model organisms for various studies in genetics and developmental biology primarily because of their fully mapped genome (1–3), relatively simple anatomy (4), short life span and ease of genetic manipulation (5). With just over 300 neurons, the nematode exhibits a remarkable ability to navigate through complex environments in search of food and/or to avoid danger (6,7). Locomotion is considered to be one of the most fundamental and simplest among the varied behavioral facets of C. elegans. It has been established that these animals move forward by propagating undulatory waves along their body (8–10). The understanding of neuromuscular processes and biomechanics of C. elegans locomotion will help in establishing an intrinsic relationship between the neural and muscular activities within the animal. In addition, it is also important to study the contact mechanics between the worm and its environment, particularly during crawling, where the substrate properties could have a significant impact on its behavior. The ability of C. elegans to promptly sense the environmental cues and modify their behavior appropriately is of utmost importance for their survival. The nematode typically moves forward by propagating undulatory waves in a dorsal-ventral plane, generated by alternating contraction and relaxation of dorsal and ventral

Submitted January 26, 2016, and accepted for publication June 27, 2016. *Correspondence: [email protected] Editor: Sean Sun. http://dx.doi.org/10.1016/j.bpj.2016.06.030 Ó 2016 Biophysical Society.

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muscle groups running along its length (11,12). It has been reported that both shape and speed of these undulations change in response to the surrounding environment (11,13,14). In low viscous liquids, C. elegans exhibit a swimming gait characterized by C-shaped conformations with high frequency and long wavelengths (13,15–18), whereas on moist surfaces like agar gels, the worms exhibit a crawling gait characterized by S-shaped undulations with low frequency and short wavelengths (13,19). On these substrates, a thin film of water results in strong capillary forces causing the worm to carve a groove on the surface. The worm then pushes itself against the sides of this groove, which gives rise to anisotropy in the transverse and lateral friction between its body and the substrate, causing it to move forward (17,19–22). It has been observed that C. elegans adapt very quickly to the mechanical load induced by the surrounding environment by modulating their gait (17,19,23). Their locomotory patterns change from gaits that resemble swimming to the crawling gait with varying viscosity of the solution. Previous studies have also revealed that C. elegans are able to detect the moisture content or humidity of the surrounding environment and are shown to actively avoid regions of higher humidity by performing reversals (24–26). In this work, we report that C. elegans respond to substrate stiffness not only by modifying its gait, but also by migrating to stiffer regions of the substrate. The animals were found to exhibit durotaxis and always migrate to the stiffest region, irrespective of their crawling velocity. A series of control experiments, including chemotaxis, changes

Durotaxis in Nematode C. elegans

in humidity of the air, water content on the surface, and effect of height difference at the interface were performed to conclude that the animals were indeed able to detect the rigidity of underlying substrates and exhibit durotaxis toward stiffer surfaces. A closer look into the behavior of animals at the interface between regions of different stiffnesses reveals that C. elegans are aware of the change in substrate stiffness and make a judicious decision to migrate to and stay on regions with higher rigidity. Based on our analysis of the behavior of worms on surfaces with varying rigidity, we conjecture that this unique behavior in C. elegans could possibly be one of their strategies for improving the chances of locating bacteria, which is their primary source of food (27).

measured at successive frames for a given period of time and the wavelength as the spatial period of the nematode’s wave motion.

Substrate preparation Substrates with different stiffness were prepared by dispersing various concentrations of SiNP (12 nm diameter; purchased and used as received from Sigma Aldrich, St. Louis, MO) and gelatin (purchased and used as received from Loba Chemie, Colaba, Mumbai) in 50 mL of water using a high-intensity ultrasonic processor for 20 min. The agar powder was added to the solution and was heated to a temperature of 95oC until a clear solution was obtained. The amount of agar added was such that its concentration in the solution was 1 wt %. The solution was then poured into a petri dish and allowed to cool down to room temperature for gelation.

Rheology of agar gel MATERIALS AND METHODS Worm strains and culture We used wild-type N2 C. elegans grown on Escherichia coli OP50 lawn in a 6015 mm petri dish containing 1 wt % agar in an incubator maintained at 20 C. All experiments were conducted using a synchronously staged worm population prepared using the hypochlorite digestive treatment (28). Wellfed young adults (3 days old) were selected for all experiments to exclude the possible effects on animal physiology from starvation (29). To conduct experiments, 1 ml of M9 buffer solution was added to a plate of synchronized worms and the petri dish was gently swirled for 10 s to wash the worms off the plate. The plate was tilted and the liquid containing worms was transferred to a 15 mL centrifuge tube. The worms were then washed with M9 solution three to four times to remove bacteria and other food sources. Approximately 100–150 worms were transferred to a fresh agar plate and allowed to crawl freely by absorbing the excess amount of water with tissue paper, trying to avoid direct contact between the tissue paper and the substrate.

Worm acquisition The behavior of C. elegans on substrates with varying stiffness was captured using a Zeiss (Oberkochen, Germany) Stemi 2000-C stereo microscope mounted with a video camera (640  480 pixels and an eight-bit monosensor). The microscope was outfitted for bright-field illumination from a light source reflected from a mirror positioned at an angle of ~45 such that the agar plates were illuminated from below. Eight-bit gray-scale images and black-and-white videos of the worms crawling were captured for analysis. To estimate the kinematic properties, out of many worms in the frame, we considered only those that moved in the forward direction. From these videos, the frequency was measured directly by counting the number of cycles made by the worm’s head in a specific period of time. The wavelength and velocity of worms were estimated from the ‘‘skeletonized’’ data and the centroid, respectively. The following procedure was used to skeletonize the worm. The worm was first separated from the background by applying a greyscale threshold to the images. Small nonworm objects were removed from the images based on their size, and ‘‘holes’’ in the thresholded worms’ bodies were filled. Using the MATLAB (The Mathworks, Natick, MA) Image Processing toolbox routines, including bwareaopen.m, imfill.m, and bwmorph.m, a morphological thinning operation was performed that resulted in a skeleton running down the center of the worm’s body. The skeleton data were then obtained from individual points collected from the binary skeleton image by determining which pixels had a value equal to 1 and utilizing their row and column positions as Cartesian coordinates. The centroid is taken as the midpoint of this data set. The velocity was then calculated by estimating the displacement of the centroid

The elastic modulus of agar gel was estimated by means of a stress-andstrain-controlled Anton Paar (Ashland, VA) MCR-301 rheometer equipped with double-plate geometry (diameter 50 mm) operated in oscillatory mode. Agar gel samples with different concentrations of nanoparticles were prepared in 60  15 mm petri dishes, and after the gels were formed, samples were carefully cut and placed in the holder. All measurements were controlled and analyzed with Physica RheoPlus software. An amplitude sweep from 0.01% to 100% strain was performed to identify the linear viscoelastic region. The temperature was kept constant at 20 C. Frequency sweeps at 0.3% strain over the range from 0.1 to 10 Hz were then used to extract the storage modulus of each sample. All data reported in this work were recorded at a frequency of 10 rad/s.

Preparation of assay plates To prepare the durotaxis assays, we first poured pure agar solution into a petri dish and allowed it to cool down and form the gel. As soon as the solution stopped flowing, one-half of the gel was carefully removed from the plate and the solution containing the additive was poured in its place (Fig. 1 c). Extreme care was taken to maintain a smooth interface between the two sides. The petri dish was then allowed to cool down to room temperature and maintained at 20 C in an incubator for ~1 h before using the worms for the experiments. In all the experiments, the worms were initially placed at the center of the dish, on the interface, and allowed to crawl freely for 1 h before images were captured for analysis. To ensure that there is no height difference at the interface between the two sides that might have an effect on the preference of worms for one of the sides, the two sides were prepared separately but with identical concentrations of agar (1 wt %) and no additives. We observe that there is no significant difference in the concentration of worms on either side of the assay plate, as shown in Fig. S1 in the Supporting Material.

Chemotaxis experiment For the chemotaxis experiments, 0.5% SiNP and 2% gelatin solutions were used as test compounds. From each of these solutions, 5 mL was put on the agar plate 16–17 h before the experiment. Shortly before the chemotaxis assay, 1 mL of 1 M sodium azide was spotted onto the same position to anesthetize the animals at the center of the gradient. As a control, sodium azide was also spotted at a position ~4 cm away from the center of the test-compound gradient. To calculate the chemotaxis index (CI), ~100 animals were then placed equidistant (~3 cm) from these two spots and the excess water was removed using a tissue paper. Extreme care was taken to avoid direct contact between the tissue paper and the substrate. When the animals began to crawl on the agar surface, the assay plates were left undisturbed for 30 min at room temperature. The number of worms paralyzed near the

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Methods. All experiments, except those that were used to study the effect of water content on the agar surface, were performed after 1 h of preparation of the assay in a controlled environment. Staged worms were placed at the middle of assay plates and allowed to crawl freely for 1 h before images were captured for analysis. a

b

Substrates with a step change in stiffness In the first set of experiments, we used pure 1 wt % agar on one side and agar-SiNP blend with varying concentrations of SiNP on the other. The images of assays after the worms were allowed to crawl freely for 1 h on agar/agar-SiNP substrates are shown in Fig. 2, a–d. The region to the left of the interface (shown with a dark line) corresponds to pure agar (lower stiffness) and the region to the right corresponds to agar with SiNP (higher stiffness). We observe that in all cases, the majority of C. elegans have migrated to the agar-SiNP side, which is the stiffer region. The fractional

c FIGURE 1 Elastic modulus of (a) agar-SiNP and (b) agar-gelatin composite gels as a function of SiNP and gelatin concentrations, respectively. The x axes in both plots are broken for clarity. (c) Schematic of the durotaxis assay. The worms were initially placed at the middle of the plate and allowed to crawl freely for 1 h before image capturing for estimating the distribution of worms.

spotted regions was counted, and the CI was then calculated by CI ¼ ðNA  NB Þ=ðNA þ NB Þ; where NA and NB are the numbers of worms in the regions with and without additives, respectively. The procedure was repeated three times with both additives to obtain the average value.

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RESULTS AND DISCUSSION Although the elasticity of agar gels can be varied by changing the concentration of agar (30), we keep it constant in all our experiments, because the animals were grown on agar plates and may show chemotactic behavior toward higher concentrations of agar. We vary the stiffness of 1 wt % agar gel by adding various concentrations of silica nanoparticles (SiNP) and gelatin. The elastic modulus (E) of the gel increased with the inclusion of SiNP (Fig. 1 a), whereas it decreased with the addition of gelatin, as shown in Fig. 1 b. SiNPs used in this study were insoluble in water, but they reinforce the gel network formed by the double helices of agar molecules, resulting in an increase in the stiffness. Although gelatin has been known to increase the thickness of agar solution (31), the gel formed by mixing agar and gelatin shows a decrease in its stiffness with increasing concentrations of gelatin (31–33). The assays for durotaxis experiments were prepared such that the plates contained pure agar on one side and the agar-additive blend on the other (Fig. 1 c). Care was also taken to maintain a smooth surface at the interface between the two regions (Fig. S2). A detailed description of the procedure is provided in Materials and

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e

FIGURE 2 C. elegans durotaxis on agar/agar-SiNP substrates with pure 1 wt % agar on the left and 1 wt % agar containing (a) 0.008 wt %, (b) 0.01 wt %, (c) 0.05 wt %, and (d) 0.5 wt % SiNP on the right. The scale bars in all images are 1 mm long, and the vertical line at the bottom of each image represents the interface between the two regions. (e) Distribution of worms on soft (pure agar; light) and stiff (agar-SiNP; dark) regions.

Durotaxis in Nematode C. elegans

distribution of worms (f) on the two regions, calculated by counting the number of worms on each side and dividing it by the total number of worms, is shown as a function of ratio of stiffnesses (Estiff/Esoft) in Fig. 2 e. In all figures, the error bar represents 1 standard deviation in the data obtained from four independent experiments. The distribution is greatly influenced by the ratio Estiff/Esoft. For a small concentration of SiNP, Estiff/Esoft ~ 1, indicating that the difference in stiffness between the two regions is very small. As a result, the worms were almost equally distributed on both regions. However, as the ratio increases, the probability of locating the animals on the stiffer side also increases. For the highest concentration of SiNP, we observed that >95% of the worms migrated to the stiff region. The second set of experiments were performed with the pure 1 wt % agar on one side and agar-gelatin blend with varying concentrations of gelatin on the other. Fig. 3, a–d, shows the images of assay plates after the worms were allowed to crawl freely for 1 h on agar/agar-gelatin substrates.

In this case, the region to the left of the interface, which corresponds to pure agar, is the stiffer side, and the region to the right, which corresponds to agar with gelatin, is the softer side. It is clear that the majority of C. elegans have again migrated to the stiffer region, which is the side corresponding to pure agar. The fractional distribution of worms (f) on each side of this assay, shown in Fig. 3 e, also indicates that as the concentration of gelatin increases, the ratio Estiff/Esoft deviates from 1 and the probability of locating the worms on the stiffer side (pure agar) increases. A comparison between the distribution plots in Figs. 2 e and 3 e shows that for a given ratio Estiff/Esoft, the probability of locating the worms on the stiffer region with SiNP is consistently higher than that in the gelatin case. This indicates that C. elegans are not only able to detect the relative difference between the soft and stiff regions, but also show a stronger preference to stiffer surfaces; meaning that the higher the stiffness of the substrate, the greater is the tendency of C. elegans to migrate to it. In other words, the worms appear to be more sensitive to the difference in stiffness when the stiffnesses of both the regions are higher. In both these cases, although the worms were seen to cluster up on the stiffer side, they moved independently until they came close enough to form clusters. Substrate with a series of step changes in stiffness

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FIGURE 3 C. elegans durotaxis on agar/agar-gelatin substrates with pure 1 wt % agar on the left and 1 wt % agar containing (a) 0.25 wt %, (b) 0.5 wt %, (c) 1 wt %, and (d) 2 wt % gelatin on the right. The scale bars in all images are 1 mm long, and the vertical line at the bottom of each image represents the interface between the two regions. (e) Distribution of worms on soft (agar-gelatin; light) and stiff (pure agar; dark) regions.

To analyze the behavior of C. elegans on substrates with varying degrees of stiffness, we prepared plates that contained strips of agar with various concentrations of gelatin and SiNP on either side of a pure 1 wt % agar strip such that the stiffness increased gradually as the worms moved from left to right on the plate. The animals were placed at the center of the plate on the strip corresponding to pure agar and allowed to crawl freely for 1 h before image capturing for analysis, as shown in Fig. 4 a. We note that the number density of worms on the extreme right, which corresponds to the stiffest region, is significantly higher than that on other regions. The fractional distribution of worms on the plate was calculated by counting the number of worms on each strip and dividing it with the total number of worms, as shown in Fig. 4 b. We observed that the majority of the worms migrated to the stiffest region of the plate, corresponding to the highest concentration of SiNP (0.5 wt %). The probability of locating the worms increased gradually as a function of substrate stiffness as a result of a small but gradual change in the stiffness of consecutive sections on the assay plate. As the velocity of worms was significantly affected by the stiffness of the underlying substrate, the worms that were placed at the center of the assay in the above experiment moved quickly to other regions where the velocity was drastically reduced. To verify whether the concentration of worms on the stiffer region is not a statistical error due to

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FIGURE 4 C. elegans behavior on a substrate with varying degrees of stiffness. Images of assay plates containing strips of agar with increasing stiffness from left to right. The worms were initially placed on (a) the middle strip that has no nanoparticles and (c) the extreme left strip with 2% gelatin and were allowed to crawl freely for 1 h. The scale bars in both images are 1 mm long. (b and d) Distribution of worms on various regions of the assay plate, corresponding to (a) and (c), respectively.

low velocity, we performed a similar experiment where the worms were now placed on the far left strip of agar with a high concentration of gelatin (softest region in the series), where the velocity is comparable to that on the stiffest side (strip on the extreme right). We observed that after 1 h, the majority of the worms still migrated to the stiffest region, confirming that the observed distribution of worms, with the majority on the stiffest side, was in fact due to the difference in the stiffness of the substrate. Kinematics of C. elegans To understand the reason behind migration of C. elegans to stiffer regions, we estimated the velocity of animals (defined as the average distance traveled by the centroid of worms in 1 s) and plotted it as a function of elastic modulus of the substrate, as shown in Fig. 5 a. The locomotion assays consisted of a significantly lower number of worms. It is known that in the absence of food, the worm exhibits back-and-forth motion. Thus, to estimate the kinematic properties, we considered worms that moved only in the forward direction during a specific period of time. We note that the average velocity shows a nonmonotonic behavior with increasing stiffness. As the concentration of gelatin was increased, the substrates became softer and the velocity with which the worms trav-

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eled also decreased monotonically, whereas with addition of SiNP, the velocity increased to a concentration of up to 0.01 wt % and then showed a steep drop in its value. However, irrespective of the worm’s crawling velocity, C. elegans always migrated to the stiffest region. We also quantified the shapes of C. elegans by estimating their undulation frequency, which is defined as the average number of undulation cycles the worm performs in 1 s, and wavelength, as shown in Fig. 5, b and c, respectively. The error bars in these results were obtained from four independent experiments with five worms from each, giving us an average of >20 worms. We observed that both frequency and wavelength of undulations showed nonmonotonic behavior with increasing stiffness of the substrate. The maximal values for both frequency and wavelength occurred at the same stiffness where the velocity was found to be the highest. This suggests that the undulation frequency and wavelength of crawling worms are directly related to the velocity at which they move forward on wet surfaces. These results agree well with previous work by Karbowski et al. (19). To fully eliminate the possibility of velocity of crawling motion being the reason behind the migration of C. elegans toward stiffer surfaces, we performed two additional sets of experiments, one in which the soft and stiff regions were prepared by carefully selecting the concentrations of gelatin and SiNP (0.25% and 0.02%, respectively) such that the velocity, frequency, and wavelength of worms were similar on both sides, and the other in which the velocity is maximal on the softer side (corresponding to a concentration of 0.01 wt % SiNP) and lowest on the stiffer side (corresponding to a concentration of 0.5 wt % SiNP). In both cases, we observed that C. elegans migrated to the stiffer region, even though the kinematic properties were similar on the two sides in the first case and the worms moved with a higher velocity on the softer side in the second (Fig. 5, d and e). These observations led us to conclude that none of the kinematic properties were responsible for this peculiar behavior of C. elegans. To further understand the nature of C. elegans migration to stiffer surfaces, we calculated the probability of worms making a U-turn and getting back to their initial location when crossing the interface from either direction (stiff to soft and soft to stiff), as shown in Fig. 5 f. When the worms crossed the interface from the stiffer to the softer region, a majority of them spontaneously made U-turns (by means of U turns and loops) and got back to the stiffer side, whereas the worms that crossed the interface from the softer to the stiffer region made no such attempts to get back; rather, they continued to go in the direction of increasing stiffness. This suggests that C. elegans are not only able to detect the rigidity of underlying substrates but can also make a judicious decision to stay on stiffer regions. We now use the U-turn probability data to estimate the number of worms on a given side at steady state as

Durotaxis in Nematode C. elegans

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FIGURE 5 (a) Velocity, (b) undulation frequency, and (c) wavelength of crawling worms on substrates with varying stiffness. The solid line separating the two regions corresponds to pure agar with no additives. All three parameters show a maximum at E ¼ 5.37 kPa, corresponding to an SiNP concentration of 0.01 wt %. (d) Assay with 0.25 wt % gelatin on the left and 0.02 wt % SiNP on the right, where the velocity, frequency, and wavelength of worms are equal. (e) Assay with 0.01 wt % SiNP, where the velocity of worms is highest, on the left, and 0.5 wt % SiNP, where the velocity is lowest, on the right. The scale bars in (d) and (e) are 1 mm long and the vertical line at the bottom of each image represents the interface between the two regions. The distribution of worms on soft (light) and stiff (dark) regions are shown next to the experimental images. (f) Probability of U-turns made by C. elegans when they cross the interface from stiff side to soft side (squares) and from soft side to stiff side (circles) as a function of the ratio of elastic moduli.

Nstiff ¼ Nstiff/soft  Pstiff/soft þ Nsoft/stiff  1  Psoft/stiff



(1)  Nsoft ¼ Nstiff/soft  1  Pstiff/soft þ Nsoft/stiff  Psoft/stiff ; (2) where Nstiff/soft is the number of worms crossing the interface from the stiff to the soft side and Nsoft/stiff is the number of worms crossing the interface from the soft to the stiff side during the time frame of our observation, for all the different stiffness combinations studied in this work. We then calculate the fraction of worms on the stiffer side as fstiff ¼

Nstiff  Nstiff þ Nsoft

(3)

and compare this fraction to that reported in Figs. 2 and 3, as shown in Table 1. Control experiments Addition of certain additives to agar may have an effect on both worms and the substrate. To account for all the possible

effects of additives used in this study on C. elegans and agar, we conducted several control experiments to verify whether the observed behavior is due to substrate stiffness or whether some other factor is playing a significant role in dictating that the worms prefer the agar with SiNP and avoid the agar with gelatin. We first note that acidity of the substrate does not change significantly upon addition of gelatin and SiNP (Table S1). Chemotaxis toward additives

C. elegans are widely studied on gelatin substrates in laboratories, so we conducted several experiments to check whether the worms show any specific affinity toward or avoidance of silica and gelatin. In the first set of experiments, the plates were prepared as for a regular chemotaxis assay (see Materials and Methods for details), with a few drops of either silica or gelatin solution along with the anesthetic spotted at one location and only the anesthetic spotted at a control location (Fig. S3). The worms were then placed at a location that is equidistant from the two locations and allowed to crawl freely. The CI for SiNP and gelatin, given in Table S2, indicates that the worms show no specific preference toward either of the additives used in this study.

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Parida and Padmanabhan TABLE 1 Assays Estiff =Esoft 1.10 1.40 1.57 1.96 1.15 1.68 3.29 5.08

Fraction of Worms on the Stiffer Side of Durotaxis fstiff from Eq. 3

fstiff from Fig. 2

0.57 5 0.02 0.68 5 0.01 0.77 5 0.01 0.91 5 0.03 0.59 5 0.03 0.66 5 0.01 0.78 5 0.02 0.89 5 0.03

0.55 5 0.01 0.65 5 0.03 0.76 5 0.02 0.90 5 0.02

fstiff from Fig. 3

Effect of additives on the humidity 0.53 5 0.02 0.64 5 0.01 0.83 5 0.02 0.91 5 0.02

To maintain consistency with the setup used in durotaxis experiments, we studied the behavior of worms on a similar assay with pure agar on one side of the plate and agar with the corresponding additive on the other. The difference between assays here and the durotaxis experiments is that here, the additive solutions were spotted on top of the agar substrate after the formation of gel so that they are superficially present on the substrate and do not modify its stiffness (see Table S3), whereas in the latter case, the additives were mixed with the agar solution and were embedded in the gel. An equal amount of water was spread on the pure agar side to maintain the same moisture content on both sides. Fig. 6 shows the experimental images of assays with SiNP (Fig. 6 a) and gelatin (Fig. 6 b) solutions placed on the right side of

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the plate. We note that the distribution of the worms is not affected by the presence of either SiNP or gelatin on the plate. This confirms that the worms do not show any preference for or avoidance of these additives unless they modify the stiffness of the substrate.

FIGURE 6 Chemotaxis experiment with (a) 0.5% SiNP solution and (b) 2% gelatin solution spotted on the righthand side of the assay. (c) Relative humidity of the air above the substrates with various concentrations of the additives. The prefixes G and S represent the gelatin and SiNP additives, respectively, and the number represents the concentration. For instance, G0.25 means substrate containing 0.25 wt % gelatin. A stands for pure agar substrate. (d) Experimental image of an assay with a linear gradient in the humidity and a step change in the stiffness. The stiffer region (right) is more humid. The scale bars in all experimental images are 1 mm long.

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Although there is no reported work in the literature, to our knowledge, which provides evidence that SiNP and/or gelatin, the additives used in this study, modifies the humidity of the substrate as well as the air above the surface, we measured the humidity levels and also designed some experiments to verify whether the migration of C. elegans toward regions of higher stiffness is due to differences in the moisture levels on and/or above specific regions of agar. The moisture content of the air above the agar substrates, measured using the colorimetric cobalt (II) chloride indicator paper strips, with varying concentrations of SiNP and gelatin, is shown in Fig. 6 e. We note that there is no significant difference in the humidity due to the presence of additives in the substrate. To confirm that neither humidity nor the water content of the substrate is playing a role in this migration of C. elegans to specific regions, we designed a few experiments where the assays were prepared with regions differing in moisture level. To study the effect of water content on the agar surface on the behavior of C. elegans, we prepared the two sides of the assay at different times before doing the actual experiments. The softer side, with the pure agar solution, was prepared 24 h before the start of experiments and the side with agar containing SiNP (the stiffer side) was prepared 1 h before the experiments. Due to the difference in the time allowed for evaporation of water from the agar substrates, the softer side (pure agar) had less water content than the stiffer side (agarþSiNP). Fig. S4 shows the amount of water surrounding the worm’s body on both sides of the assay. The average width of the water layer surrounding the worm, as shown in Table S4, indicates that the worm is surrounded by more water on the stiffer side. We observed that for high concentrations of SiNP, even though the moisture content on the SiNP side of the assay was significantly higher than that on the pure agar side, the worms preferred to migrate to the SiNP side. However, for low concentrations, as the difference in stiffness between the two sides decreased, the effect of humidity dominated and the worms migrated to the pure agar side (Fig. S5). A similar observation was made when the experiments were performed with gelatin, where for low concentrations of gelatin, the water content became the dominating factor and the worms migrated to the less moist region (softer side), but as the concentration of gelatin was increased, the difference in stiffness dominated over humidity and the majority of the worms migrated to the stiffer region, which was the more moist side. Next, to delineate the effect of humidity in the air above the surface, we prepared both sides of the assay (soft with

Durotaxis in Nematode C. elegans

pure agar and stiff with various concentrations of SiNP) 1 h before conducting the experiments and placed ~50 mg of silica gel on the softer side. As silica gel is hygroscopic, it absorbs moisture from the air and creates a gradient of moisture content in the air above the substrate. We note that the difference in humidity has a stronger effect on the behavior of the worms than the moisture content on the surface. As a result, when the difference in stiffness between the two regions was moderate, majority of the worms migrated to the softer side (Fig. S6). However, when the concentration of the SiNP was increased to 0.5%, stiffness dominated and the worms migrated to the stiffer side, which was more humid, as shown in Fig. 6 f. These results show that humidity and stiffness are two distinct factors affecting the behavior of C. elegans. In summary, we studied the behavior of C. elegans on surfaces with different stiffnesses and observed that the animals were able to detect the rigidity of the underlying substrate and make a judicious decision to migrate to and stay on regions with higher stiffness. The animals were observed to migrate always to the stiffest region, even though their velocity was not the highest. A closer look into the behavior of C. elegans at the interface revealed that majority of the worms spontaneously responded to the change in stiffness of the substrate by making U-turns when they crossed the interface from the stiffer side. This surprising behavior was also observed in cases where the conditions were such that the velocity was highest on the softer side. Based on our results, we conjecture that this behavior of C. elegans may be one of their strategies to improve their chances of finding food. We hope that our study will be a starting point for more thorough investigations in this area, which not only will help in decoding the language of nematode motion but also will enable a fundamental understanding of such behavior in small organisms.

SUPPORTING MATERIAL

This work is supported by a grant from the Department of Science and Technology, New Delhi, India, awarded to V.P. (DST SR/S3/CE/072/2012).

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AUTHOR CONTRIBUTIONS

18. Vidal-Gadea, A., S. Topper, ., J. T. Pierce-Shimomura. 2011. Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin. Proc. Natl. Acad. Sci. USA. 108:17504– 17509.

V.P. conceived the project, V.P. and L.P. designed the experiments, L.P. carried out the experiments, and V.P. and L.P. wrote the article.

ACKNOWLEDGMENTS We thank Jayanta Chakraborty (IIT Kharagpur) for providing access to the microscope and Swati Neogi (IIT Kharagpur) for helping us with the estimation of modulus. We also thank Jerzy Blawzdziewicz (Texas Tech University), Siva Vanapalli (Texas Tech University), Sudarsan Neogi (IIT Kharagpur), and Rabibrata Mukherjee (IIT Kharagpur) for useful discussions. LP is supported by the Institute Scholarship at IIT Kharagpur.

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