Fig 1.
Thermotaxis behaviors of Strongyloides free-living females near ambient temperatures are distinct from those of C. elegans adults.
For panels A-C, worms were placed at 23°C in a ~21–25°C gradient and allowed to migrate for 45 min. Cultivation temperature (TC) = 20°C. Starting temperature (Tstart) = 23°C (grey line). Black crosses show the starting positions of the worms. Randomly selected representative tracks of C. elegans adult hermaphrodites (A), S. stercoralis free-living females (FLFs) (B), and S. ratti FLFs (C). For all tracks, see S2 Fig. C. elegans hermaphrodites are seen engaging in negative thermotaxis toward their cultivation temperature, while S. stercoralis and S. ratti FLFs engage in positive thermotaxis. D) Quantification of the change in temperature experienced by C. elegans adults, S. stercoralis FLFs, and S. ratti FLFs. Values are the final temperature–starting temperature for each worm. Icons indicate responses of individual worms, boxes show medians and interquartile ranges, and whiskers show min and max values. n = 54 worms for C. elegans hermaphrodites (5 assays across 4 days), n = 76 worms for S. stercoralis free-living females (7 assays across 5 days), n = 59 worms for S. ratti FLFs (6 assays across 4 days). ns = not significant, ****p<0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test. E) Categorical distribution of thermotaxis behaviors in a ~21–25°C gradient across species. For each species, individual worms were considered to have engaged in positive or negative thermotaxis if their position at the end of the assay was outside of a 1 cm neutral exclusion zone centered on the starting position of each individual worm. Individuals that finished the assay within this zone were considered non-responding. n (negative/non-responding/positive) = C. elegans: 33/19/2; S. stercoralis: 3/25/48; S. ratti: 2/24/33. ***p<0.001, Fisher’s exact test with Bonferroni-Dunn correction for multiple comparisons.
Fig 2.
Strongyloides free-living females do not display noxious heat avoidance near human skin temperature.
Worms were placed at 30°C in a ~21–35°C gradient and allowed to migrate for 45 min. TC = 23°C. Colored tracks represent the paths of individual worms. Grey line represents Tstart = 30°C. Black crosses show the starting positions of the worms. Randomly selected representative tracks of C. elegans adult hermaphrodites (A), S. stercoralis free-living females (B), and S. ratti free-living females (C). For all tracks, see S2 Fig. C. elegans hermaphrodites are seen engaging in noxious heat escape behaviors, while S. stercoralis FLFs engage in positive thermotaxis and S. ratti FLFs engage in neither positive nor negative thermotaxis. D) Quantification of the change in temperature for C. elegans adults, S. stercoralis FLFs, and S. ratti FLFs. Values are the final temperature–starting temperature for each worm. Icons indicate responses of individual worms; boxes show medians and interquartile ranges; whiskers show min and max values. n = 50 worms for C. elegans hermaphrodites (5 assays across 3 days), n = 65 worms for S. stercoralis FLFs (5 assays across 3 days), n = 47 worms for S. ratti FLFs (6 assays across 4 days). ****p<0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test. E) Categorical distribution of thermotaxis behaviors in a ~21–35°C gradient across species. Individuals were considered to have engaged in positive or negative thermotaxis if their position at the end of the assay was outside of a 1 cm neutral exclusion zone centered on the starting position of each individual worm. Individuals that finished the assay within this zone were considered non-responding. n (negative/non-responding/positive) = C. elegans: 41/9/0; S. stercoralis: 2/7/56; S. ratti: 12/26/9. ***p<0.001, Fisher’s exact test with Bonferroni-Dunn correction for multiple comparisons.
Fig 3.
S. stercoralis FLF’s thermotaxis behavior is disrupted by the presence of an attractive odorant.
A) Diagram of the attractive odorant and temperature gradient experimental setup. Worms were placed at 23°C in a ~21–25°C temperature gradient. An attractive odorant (3m1b) was either not introduced, placed near the worm starting location (at 22.5°C), or placed far from the worm starting location (at 22°C). Assay duration: 45 minutes. Temperature-only data is reproduced from Fig 1. B) Heat map showing the temperature experienced by individual worms throughout the 45-minute assay. Cooler temperatures are represented by darker colors while warmer temperatures are represented by warmer colors. Heatmap rows represent the temperatures experienced by individual worms and are ordered by hierarchical cluster analysis such that worms with similar trajectories are grouped together. To view this data plotted as worm tracks, see S7A Fig. Triangles on the right of the heat maps indicate the individual worms chosen as representative tracks in Fig 3C. C) Example tracks of individual worms from each experimental condition. Tracks are color-coded by time in the assay as seen in the scale on the right. Gray line = 23°C (Tstart); black dots indicate worm starting position. For additional representative tracks selected randomly from the full track set, see S7B Fig. D) Quantification of the change in temperature experienced by worms (final temperature–starting temperature). Icons indicate responses of individual worms, boxes show medians and interquartile ranges, and whiskers show min and max values. n = 76 worms for temperature only, n = 65 worms for odorant near (6 assays over 3 days), and n = 66 worms for odorant far (6 assays over 3 days). ns = not significant, **p<0.01, ****p<0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test.
Fig 4.
Comparison of the sensory hierarchy across Strongyloides life stages.
Similar sensory cues result in different behaviors based on the sensory hierarchy of a specific life stage (indicated by direction of scale icon). At near-ambient temperatures, iL3s prioritize performing thermotaxis behaviors over responding to an attractive odorant [55]. At temperatures near host body heat, iL3 migration is influenced by attractive host odorants [55]. In contrast, free-living adults perform both thermotaxis and chemotaxis at near ambient temperatures, likely due to a sensory hierarchy that is less dominated by temperature cues.
Fig 5.
Exposure to high temperatures decreases the lifespan of Strongyloides spp. FLFs.
A) Diagram of survival assay. Individual adults were placed on NGM plates seeded with E. coli HB101. The plates were then incubated at either 23°C, 30°C, or 37°C. Plates were checked every 24 hours for survival. B-D) Probability of survival over time for C. elegans adult hermaphrodites (B), S. stercoralis FLFs (C), and S. ratti FLFs (D). Black circles represent assays run at 23°C, gold diamonds represent assays run at 30°C, and red triangles represent assays run at 37°C. n = C. elegans: 35 (23°C), 46 (30°C), 40 (37°C) worms; S. stercoralis: 48 (23°C), 58 (30°C), 48 (37°C) worms; S. ratti: 31 (23°C), 29 (30°C), 38 (37°C) worms. For all experiments, animals that were not found on the plate were censored. For C. elegans and one S. stercoralis experiment, the dates of censoring were not recorded; these animals (3 for C. elegans, 2 for S. ratti) have been excluded from survival analyses. Number of censored animals included in survival analyses = C. elegans: none; S. stercoralis: 5 (23°C), 0 (30°C), 0 (37°C); S. ratti: 4 (23°C), 5 (30°C), 6 (37°C). Survival curves reflect the combined survival of all worms across 5 (C. elegans, S. ratti) or 6 (S. stercoralis) independent experiments for each species. Survival curves for individual experiments are shown in S8 Fig. E) Table reporting average survival times for all species and temperature conditions. Values are mean ± standard error (number of animals). p-values are pairwise comparison of survival curves (23°C vs 30°C, 30°C vs 37°C, 23°C vs 37°C): p<0.001, Mantel-Cox test with Bonferroni-Dunn correction for multiple comparisons.
Fig 6.
Impact of near-tropical temperatures on S. stercoralis FLF brood size.
A) Diagram of the brood size assay. Individual adults were placed on NGM plates seeded with E. coli HB101. Plates were incubated at either 23°C, 30°C, or 37°C and checked every 24 hours for the number of eggs and larvae. B) Quantification of the impact of environmental temperature on brood size for C. elegans hermaphrodites (n = 36–47 adult worms), S. stercoralis free-living females (n = 32–54 adult worms), and S. ratti free-living females (n = 29–38 adult worms). Icons indicate responses of individual worms, boxes show medians and interquartile ranges, and whiskers show min and max values. ns = not significant, **p<0.01, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test. C) Diagram of the hatching assay. Individual adults were placed on NGM plates seeded with E. coli HB101. Plates were incubated for 4 hours at 20°C. After 4 hours, females were removed from plates and eggs were counted. Plates were then incubated at 23°C, 30°C, or 37°C. After 24–48 hours, unhatched eggs were counted. D) Quantification of hatching viability for C. elegans (n = 50–53 plates), S. stercoralis (n = 23–35 plates), and S. ratti (n = 22–27 plates). Icons indicate responses of individual worms, boxes show medians and interquartile ranges, and whiskers show min and max values. ns = not significant, **p<0.01, ****p<0.0001, two-way ANOVA with Tukey’s multiple comparisons test.
Fig 7.
Species- and life-stage-specific thermosensory behaviors and thermal physiology.
A) Summary of Strongyloides iL3 thermotaxis behaviors. Strongyloides iL3s display two modes of thermotaxis behavior: positive thermotaxis towards host body heat and negative thermotaxis towards below-ambient temperatures [55,56]. If a temperature gradient ends below ~30°C, Strongyloides iL3s are able to reverse an initial attraction to warmth, performing a “U-turn” behavior that triggers sustained negative thermotaxis towards below-ambient temperatures [56]. B) Summary of Strongyloides FLF thermotaxis behaviors and physiological response to increased temperatures. Strongyloides FLFs are attracted to temperatures above ambient. Sustained exposure to above-ambient temperatures drives reductions in adult lifespan; in contrast, brood size and hatching viability are enhanced (or stable) at near-tropical temperatures. C) Summary of C. elegans free-living hermaphrodite thermotaxis behaviors and physiological response to increased temperatures. Between 15–25°C, C. elegans adults show attraction to a “remembered” cultivation temperature and will undergo positive and negative thermotaxis towards that temperature [34–36,39,40]. At temperatures greater than 26°C, C. elegans adults display a noxious heat escape response and have a very decreased lifespan, brood size, and hatching viability [34,37–39,42,43,50,69].