Thermosensory behaviors of the free-living life stages of Strongyloides species support parasitism in tropical environments

Abstract

Soil-transmitted parasitic nematodes infect over 1 billion people worldwide and are a common source of neglected disease. Strongyloides stercoralis is a potentially fatal skin-penetrating human parasite that is endemic to tropical and subtropical regions around the world. The complex life cycle of Strongyloides species is unique among human-parasitic nematodes in that it includes a single free-living generation featuring soil-dwelling, bacterivorous adults whose progeny all develop into infective larvae. The sensory behaviors that enable free-living Strongyloides adults to navigate and survive soil environments are unknown. S. stercoralis infective larvae display parasite-specific sensory-driven behaviors, including robust attraction to mammalian body heat. In contrast, the free-living model nematode Caenorhabditis elegans displays thermosensory behaviors that guide adult worms to stay within a physiologically permissive range of environmental temperatures. Do S. stercoralis and C. elegans free-living adults, which experience similar environmental stressors, display common thermal preferences? Here, we characterize the thermosensory behaviors of the free-living adults of S. stercoralis as well as those of the closely related rat parasite, Strongyloides ratti. We find that Strongyloides free-living adults are exclusively attracted to near-tropical temperatures, despite their inability to infect mammalian hosts. We further show that lifespan is shorter at higher temperatures for free-living Strongyloides adults, similar to the effect of temperature on C. elegans lifespan. However, we also find that the reproductive potential of the free-living life stage is enhanced at warmer temperatures, particularly for S. stercoralis. Together, our results reveal a novel role for thermotaxis to maximize the infectious capacity of obligate parasites and provide insight into the biological adaptations that may contribute to their endemicity in tropical climates.

Author Summary

Soil-transmitted parasitic nematodes infect over 1 billion people and are a major source of neglected disease, particularly in the world’s most resource-limited communities. For most parasitic nematode species, reproductive adults exclusively reside within host animals. Species in the genus Strongyloides have a unique step in their life cycle that features soil-dwelling, non-parasitic adults. Previous studies of the free-living model nematode Caenorhabditis elegans have identified temperature as an important factor in ability of free-living nematodes to survive and reproduce in the environment. Our study investigates how the thermosensory behaviors of Strongyloides free-living adults contribute to their survival as well as their role in amplifying the quantity of infective larvae in the soil. We show that Strongyloides free-living adults display broad thermophilic preferences that are highly distinct from those of C. elegans adults. We also present the first evidence that thermotaxis acts as a robust mechanism for maximizing the infectious capacity of Strongyloides species located in tropical climates.

Introduction

Soil-transmitted parasitic nematodes are estimated to infect over 1 billion people worldwide and can cause a range of debilitating symptoms, including chronic gastrointestinal distress, anemia, malnutrition, and impaired growth in children [1–3]. The skin-penetrating human parasite Strongyloides stercoralis is estimated to infect at least 600 million people [4–7]. Chronic S. stercoralis infections, which arise due to the unique capacity of S. stercoralis to autoinfect hosts, can go undetected for many decades before evolving into a life-threatening systemic hyperinfection if an individual becomes immunocompromised [2,8,9]. Soil-transmitted parasitic worms primarily affect impoverished communities and places that lack adequate sewage management [6,7,10,11]. Current treatments only target ongoing infections and can be inadequate; no medical treatment exists to prevent infection or reinfection [12–14]. Additionally, there are currently a limited number of anthelminthic drugs, and anthelminthic resistance is already widespread among livestock parasites due to repeated dosage with anthelminthic drugs as part of regular deworming schedules [15,16]. Identifying novel therapeutic strategies, before drug resistance develops in humans, is therefore an important global health priority.

Many soil-transmitted parasitic nematodes have a life cycle that starts with developmentally arrested third-stage infective larvae (iL3s) finding and infiltrating a host animal [17]. Once inside a host, iL3s resume development and migrate through various host tissues to the small intestine, where they take up residence as parasitic adults. Parasitic adults produce offspring that are then voided from the host in feces. These offspring develop into iL3s and restart the cycle. Species in the genus Strongyloides have an additional, unique step in their life cycle, in which a fraction of the offspring of parasitic adults can alternatively develop into morphologically distinct free-living adults instead of infective larvae (Fig. S1) [8,18,19]. The offspring of these free-living adults exclusively develop into iL3s that then must find and invade host animals. The molecular mechanisms that drive the choice between homogonic development (post-parasitic larvae become iL3s) and heterogonic development (post-parasitic larvae become free-living adults) is poorly understood; however, the developmental decision depends on species identity, strain genetics, host immunocompetency, and environmental temperatures [20–25]. For example, exposure to temperatures above 34°C promotes direct development into iL3s for S. stercoralis, presumably to allow larvae to develop into iL3s while still inside the host; these iL3s re-penetrate host tissues to establish an autoinfection [2,9,21]. The free-living generation of Strongyloides spp. allows these species to sexually reproduce; unlike hookworms, Strongyloides parasitic adults are all genetically female and produce clonal progeny via parthenogenesis [8,20,26–29]. Furthermore, the Strongyloides free-living generation has been proposed to act as a means of amplifying the number of infective larvae that a single parasitic female can produce [30]. Despite its role in the Strongyloides life cycle, the Strongyloides free-living generation is generally understudied [31]. A better understanding of the sensory physiology and behavioral repertoires displayed by free-living Strongyloides adults could enable the development of novel strategies for predicting and controlling the infectious capacity of a neglected source of human disease.

The free-living adults of Strongyloides spp. live in the soil and experience similar environmental stressors to Caenorhabditis elegans, a nematode that is free-living throughout its entire life cycle. Furthermore, Strongyloides free-living adults share many morphological similarities with C. elegans adults [30,32,33]. Thus, to understand how sensory behaviors enable Strongyloides free-living adults to survive in the environment, we can compare their behaviors to those of C. elegans. One of the most ethologically significant and well-documented sensory behaviors exhibited by C. elegans is thermosensory navigation. C. elegans uses a complex set of thermosensory behaviors to stay within a physiologically and reproductively permissible temperature range (15-25°C) [34–41]. In brief, C. elegans will utilize both positive and negative thermotaxis to migrate towards a “remembered” cultivation temperature; at its cultivation temperature, C. elegans will engage in isothermal tracking [35,38]. At noxious temperatures ≥27°C and ≤5°C, C. elegans engage in noxious temperature responses, including reorientation behaviors and accelerated migration towards physiological temperatures [40,42]. These behavioral strategies are a critical mechanism for thermoregulation by a non-parasitic ectotherm: exposure to noxious heat stress causes dramatic drops in C. elegans fertility and longevity, in a manner partially dependent on strain and experience [34,43–50].

In comparison to C. elegans, the sensory behaviors and physiological requirements of soil-transmitted parasitic nematodes are far less documented. The few studies that have quantified the sensory behaviors of parasitic nematodes have demonstrated that the worms can detect a range of sensory cues, including chemosensory and thermosensory stimuli [51–54]. For thermosensation, recent work has shown that the iL3s of multiple mammalian-parasitic nematode species, including S. stercoralis, display highly robust parasite-specific thermosensory behaviors [55,56]. These include positive thermotaxis towards mammalian body heat and negative thermotaxis towards below-ambient temperatures; the switch between these two behavioral regimes is dictated by the ambient cultivation temperature, such that worms exposed to temperatures above ambient will engage in positive thermotaxis (and vice versa) [55]. As opposed to thermosensory behaviors being primarily used for physiological thermoregulation (as with C. elegans), the positive thermotaxis behaviors of iL3s are thought to reflect active host seeking; negative thermotaxis behaviors are potentially a mechanism enabling iL3s to either disperse from host feces or disengage from accidental heat seeking towards a non-host heat source [55,56]. Chemosensory behaviors also contribute to host-seeking and host-invasion behaviors by soil-transmitted parasites [52,53,57]. Indeed, life-stage-specific chemosensory preferences have previously been proposed to contribute to the restriction of host-seeking behaviors to iL3s. For example, although Strongyloides iL3s and free-living adults are both attracted to a variety of host odorants, only free-living larvae and adults are attracted to fecal odors [53,57]. These findings suggest that downregulation of attraction to fecal odors enables iL3s to disperse from feces in search of hosts [53,57]. Finally, although the impact of multisensory cues on parasite behavior has not been thoroughly investigated, initial work has indicated a sensory hierarchy in which strong thermal drive can override attraction to host-associated odorants in iL3s [55]. Importantly, this hierarchy depends on the specific thermal context in which an odorant is located. At temperatures below human skin temperature (~34°C), the infective larvae appear to ignore attractive odorants in favor of warmer temperatures [55]. In contrast, if an odorant is placed at temperatures closer to ~34°C, the infective larvae will decrease thermotaxis behaviors and increase local search behaviors [55]. Together, these findings suggest a sensory-dependent host-seeking strategy in which soil-transmitted iL3s first navigate towards a heat source, then determine whether that heat source is a potential host.

The thermosensory preferences of Strongyloides free-living adults are completely unknown. Do the physiological and behavioral responses of Strongyloides free-living adults to different environmental temperatures mirror those exhibited by C. elegans adults? To answer this question, we conducted a quantitative analysis of the thermal preferences and physiology of the free-living adults of two Strongyloides species: the human parasite S. stercoralis and the closely related rodent parasite Strongyloides ratti. We found that the thermosensory behaviors of Strongyloides free-living adults are distinct from both Strongyloides infective larvae and C. elegans. Across a broad range of thermal gradients, including those that trigger negative thermotaxis in iL3s and noxious heat escape in C. elegans, Strongyloides free-living adults display thermophilic migration. In addition, S. stercoralis free-living adults show a shifted multisensory hierarchy, such that chemosensory attractants influence behavior at temperatures below mammalian body heat. Finally, we discover that although attraction to warm temperatures shortens the lifespan of S. stercoralis free-living adults, their reproductive potential is enhanced. Our results suggest that Strongyloides free-living adults, whose progeny exclusively develop into infective larvae, use thermotaxis navigation as a mechanism to enhance the number of infective larvae in the soil. Together, these findings reveal biological adaptations that likely enhance the transmission of a potentially fatal human-parasitic nematode in tropical and subtropical climates.

METHODS

All protocols and procedures involving vertebrate animals were approved by the UCLA Office of Animal Research Oversight (Protocol ARC-2011-060) and by the UW Office of Animal Welfare (Protocol 4570-01), which adhere to the standards of the AAALAC and the Guide for the Care and Use of Laboratory Animals.

Maintenance of Strongyloides stercoralis

S. stercoralis UPD strain was serially passaged through male Mongolian gerbils (Charles River Laboratories) and maintained on fecal-charcoal plates as previously described [18]. Gerbils were inoculated with ~2,250 iL3s in 200 μL sterile phosphate buffered saline (PBS) via inguinal subcutaneous injection, under isoflurane anesthesia. Feces containing S. stercoralis were collected by placing infected gerbils on wire-bottomed cages overnight, with damp Techboard liners (Shepard Specialty Papers) on the cage bottoms. Fecal pellets were collected in the morning, mixed with autoclaved charcoal granules (Ebonex), and stored in 10-cm-diameter Petri dishes lined with Whatman filter paper. Fecal charcoal plates were stored at either 20°C for 48 hours or 25°C for 24 hours, then used in behavioral assays (free-living adults) or transferred to 23°C for 5-14 days until use (iL3s). S. stercoralis free-living adults and iL3s were collected from fecal-charcoal plates using a Baermann apparatus, as previously described [58]. For brood size, survival, and hatching assays, free-living adults were briefly suspended in BU worm saline [59] in a watch glass; individual worms were then pipetted onto assay plates using a dissecting microscope (Leica S9E).

Maintenance of Strongyloides ratti

S. ratti ED321 strain was serially passaged through female Sprague Dawley rats (Envigo) and maintained on fecal-charcoal plates as for S. stercoralis. Rats were inoculated with ~700 iL3s in 200 μL sterile PBS via inguinal subcutaneous injection, under isoflurane anesthesia. Collection and storage of feces containing S. ratti was performed as described for S. stercoralis.

Maintenance of C. elegans

C. elegans N2 strain (Caenorhabditis Genome Center, CGC) was maintained using standard methods [60]. In brief, young adult hermaphrodites used for maintenance and thermotaxis assays were raised on 2% Nematode Growth Media (NGM) plates seeded with a lawn of Escherichia coli OP50 bacteria (CGC; maintenance plates). To generate age-synchronized young adults for behavioral assays, adult hermaphrodites were allowed to lay eggs on maintenance plates for 4 hours; adults were then removed, and the plates were stored at either 20°C or 23°C until progeny reached the desired life stage.

Thermotaxis assays

Thermotaxis assays were performed on a custom thermoelectric behavioral arena, as previously described [49]. For some experiments, the thermoelectric control circuit included updated electrical components; a full parts list and wiring diagram are archived in a dedicated GitHub repository (https://github.com/BryantLabUW/Strongyloides-Free-Living-Thermosensory-Behaviors). Worm migrations were visualized using a 20 MP CMOS camera (Basler Ace acA5472-17um, Basler) at a frame rate of 0.2 frames per second for 45 minutes (adults) or 0.5 frames per second for 15 minutes (iL3s) using the pylon Viewer camera software suite (v7.4.0, Basler). Thermotaxis experiments were carried out on age-synchronized young adult hermaphrodites (C. elegans) and free-living adult males and females (S. stercoralis, S. ratti) that were incubated for at least 2 hours at the desired cultivation temperature (15°C, 20°C, or 23°C). For iL3 assays, worms were collected with a Baermann apparatus and suspended in ~1 mL BU saline in a watch glass, then incubated for at least 2 hours at the appropriate temperature. For multisensory assays, 5 μL of undiluted 3-methyl-1-butanol was placed on the thermotaxis plate immediately before worms were deposited. For post-hoc measurements of individual worm movements, x/y coordinates were generated using the Manual Tracking plugin for Fiji, then quantified and plotted using custom MATLAB scripts (MathWorks), as previously described [55,56,61]. All custom code and hardware specifications are archived on GitHub (https://github.com/BryantLabUW/Strongyloides-Free-Living-Thermosensory-Behaviors).

Brood size and survival assays

For C. elegans, age-synchronized adult hermaphrodites with 1-5 eggs were picked from maintenance plates onto treatment plates (2% NGM plates seeded with 50 μL HB101 bacteria) and allowed to grow for 1-3 days. For S. stercoralis and S. ratti, free-living adult females with fewer than five eggs and 3 free-living males were pipetted from a BU-filled watch glass onto treatment plates. Plates were randomly divided among the treatment temperatures (23°C, 30°C, or 37°C) and moved to their respective incubators. Experiments were performed in batches of at least two temperature conditions. Worms were checked for survival (movement or pharyngeal pumping) and brood size (number of eggs and larvae on plate) every 24 hours. If the worm was still alive, it was moved to a new treatment plate and returned to its treatment incubator for another 24 hours. This process was repeated until all worms had died. On the day each worm died, the number of eggs/larvae present when each plate was checked was recorded.

Hatching viability assays

Age-synchronized adult hermaphrodites (C. elegans) and free-living adult females (S. stercoralis, S. ratti) with greater than five eggs were transferred from maintenance plates (C. elegans) or a BU-filled watch glass (S. stercoralis, S. ratti) onto treatment plates. All treatment plates were moved to a 23°C incubator for four hours to allow worms to lay eggs. After four hours, adult worms were removed, and the number of eggs laid on each plate was recorded (E₀). Plates with fewer than eight eggs were excluded from the experiment. Plates were randomly divided among the treatment temperatures (23°C, 30°C, or 37°C) and moved to their respective incubators. Experiments were done in batches of at least two temperatures at a time. After 24 hours, the number of eggs remaining on treatment plates was recorded (E₁). Hatching viability was then calculated using the following formula:

$${(\text{E}}_0-{\text{E}}_1)/{\text{E}}_0\text{*100}$$

Statistical analysis

All statistical analyses except mean survival were conducted using GraphPad Prism 10 (Dotmatics). Mean survival values were calculate using OASIS 2 [62] Power analyses to determine appropriate sample sizes were performed using G*Power 3.1 [63]. Statistical details for experiments are provided in figure legends and Supplemental Data File 1.

Results

The free-living adults of Strongyloides species prefer temperatures warmer than their cultivation temperature.

To begin characterizing the temperature preferences of Strongyloides free-living adults, we first looked to see if they display behaviors similar to those of the constitutively free-living model nematode C. elegans. One well-documented behavior of C. elegans is that well-fed adult hermaphrodites migrate towards a remembered cultivation temperature when placed on a thermal gradient within their physiological temperature range (15-25°C) [34–36,39,40]. To test if Strongyloides free-living adults display similar thermal preferences, we generated post-parasitic free-living adults by cultivating feces containing post-parasitic larvae at 20°C for two days. Next, we placed isolated free-living females (FLFs) at 23°C in a large-format thermotaxis arena in which we established a ~21-25°C temperature gradient. Under these conditions, C. elegans adult hermaphrodites that were raised at 20°C displayed negative thermotaxis towards their cultivation temperature, as expected (Fig. 1 A, D). In contrast, we observed that S. stercoralis FLFs were more likely to engage in positive thermotaxis (Fig. 1B, D, E). S. ratti free-living females also did not migrate towards their cultivation temperature, instead displaying attraction to above-ambient temperatures, similar to S. stercoralis (Fig. 1C–E). We also tested the thermal preferences of Strongyloides free-living males (FLMs). S. stercoralis FLFs and FLMs displayed similar behaviors, while S. ratti FLFs traveled slightly further up the gradient than S. ratti FLMs (Fig. S2). The difference between S. ratti FLFs and FLMs was not due to systemic differences in velocity, as we observed no significant difference in mean speed (mm/s) between sexes (Fig. S2C).

Figure 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 (T_C) = 20°C. Starting temperature (T_start) = 23°C (grey line). Black crosses show the starting position of the worms. Representative tracks of C. elegans adult hermaphrodites (A), S. stercoralis free-living females (FLFs) (B), and S. ratti FLFs (C). 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 ~20-25°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. ***p<0.001, Fisher’s exact test with Bonferroni-Dunn correction for multiple comparisons.

S. stercoralis free-living females show positive thermotaxis in conditions that elicit noxious temperature escape behavior in C. elegans adults.

Another critical aspect of the C. elegans free-living thermosensory repertoire is the avoidance of noxious temperatures [37,64–66]. Experimentally, escape from noxious heat can be observed by placing adult hermaphrodites above 26°C, triggering migration down the thermal gradient towards physiologically permissive temperatures [37,38,40]. Notably, the behavioral strategies and cellular mechanisms underlying noxious temperature responses are partially distinct from those driving innocuous temperature responses [37,50,66–68]. To determine if thermal escape behaviors are present in Strongyloides FLFs, we compared the migration of C. elegans adult hermaphrodites and Strongyloides FLFs placed at 30°C in a ~21-35°C gradient (cultivation temperature, T_C = 23°C). As expected, C. elegans hermaphrodites showed robust migration towards cooler temperatures (Fig. 2A, D, E). Surprisingly, neither Strongyloides species displayed noxious temperature avoidance behaviors (Fig. 2B–E). S. stercoralis FLFs continued to show positive thermotaxis towards 34°C (Fig. 2B). In contrast, S. ratti FLFs did not significantly disperse from their starting temperature of 30°C (Fig. 2C–E). Finally, we tested whether S. stercoralis FLFs would display noxious temperature responses when exposed to mammalian core body temperatures. We found that S. stercoralis FLFs placed at 37°C in a ~32-38°C gradient did not migrate down the gradient (T_C = 23°C; Fig. S3). Together, these results indicate that the thermal preferences of Strongyloides free-living adults are distinct from those of C. elegans adult hermaphrodites, despite the similarities in their ecological niches (i.e., they are both non-parasitic bacterivores). Furthermore, it suggests that the cellular mechanisms that produce robust behavioral responses to noxious heat in C. elegans are either not present in Strongyloides spp., are tuned for temperatures much higher than mammalian body heat or have been co-opted for other functions.

Figure 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. T_C = 23°C. Colored tracks represent the path of individual worms. Grey line represents T_start = 30°C. Black crosses show the starting position of the worms. Representative tracks of C. elegans adult hermaphrodites (A), S. stercoralis free-living females (B), and S. ratti free-living females (C). 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. ***p<0.001, Fisher’s exact test with Bonferroni-Dunn correction for multiple comparisons.

Free-living adults display thermal preferences that are distinct from iL3s.

The iL3s of multiple mammalian-parasitic nematode species, including S. stercoralis and S. ratti, engage in negative thermotaxis towards cooler temperatures when placed near or below their ambient cultivation temperature [55,56]. This preference for cooler temperatures may act as a mechanism to disperse iL3s into cooler soil environments where discrimination between host-emitted heat and environmental temperatures is maximized. What are the behaviors of Strongyloides FLFs in cooler gradients? We first tested the preferences of S. stercoralis FLFs in a ~12-22°C gradient, conditions in which S. stercoralis iL3s engage in negative thermotaxis and accumulate at ~16°C (Fig. S5) [56]. Surprisingly, we found that S. stercoralis FLFs displayed relatively robust positive thermotaxis in these conditions (Fig. S5B, F). To test whether S. ratti display similar life-stage-specific differences in negative thermotaxis, we first tested the preferences of S. ratti iL3s in the ~12-22°C gradient. We found that S. ratti iL3s stayed closer to their starting temperature than S. stercoralis iL3s (Fig. S5C, E). Nevertheless, S. ratti FLFs also engaged in positive thermotaxis when placed at 20°C (Fig. SD, F). Together with our earlier experiments at above-ambient temperatures, these results indicate that Strongyloides FLFs are biased towards performing positive thermotaxis across a broad range of environmental temperatures.

Strongyloides spp. FLFs respond to host-associated odorants in near-ambient temperature gradients.

Like C. elegans, Strongyloides spp. can detect a range of non-thermosensory stimuli, including chemosensory cues [51,53,57,69]. In near-ambient temperature gradients, S. stercoralis iL3s prioritize thermosensory behaviors over chemosensory responses, such that exposure to temperature gradients below host body heat can override attraction to a host-associated odorant [49]. Do S. stercoralis FLFs display a similar sensory hierarchy? To test this question, we evaluated the impact of the attractive odorant 3-methyl-1-butanol (3m1b) on the behavior of S. stercoralis FLFs in a ~20-25°C gradient. In the absence of an odorant, S. stercoralis FLFs placed at 23°C displayed positive thermotaxis, traveling towards warmer temperatures (Fig. 1B–E; Fig. 3). In the presence of an odorant placed either near to the starting position of the worms (odorant location: 22.5°C, ~2.7 cm from T_start), or further away (odorant location: 22°C, ~5.5 cm from T_start), we observed a decrease in the distance individual worms traveled up the gradient when compared to worms in a pure thermotaxis (i.e., no odorant) gradient (Fig. 3; Fig. S6). The reduction partially reflects an increase in the number of worms that initially traveled down the temperature gradient towards the odorant before traveling back up the gradient (Fig. S6). This shifted migratory pattern was particularly noticeable when the odorant was placed in the “far” location (Fig. 3C; Fig. S6B). These results suggest a difference in the sensory hierarchy underlying the behaviors of S. stercoralis FLFs and iL3s, such that the FLF sensory hierarchy is less dominated by temperature cues (Fig. 4).

Figure 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. Temp 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 are ordered by hierarchical cluster analysis. 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 (T_start); black dots indicate worm starting position.

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.

Figure 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.

Increasing cultivation temperature decreases the lifespan of Strongyloides FLFs.

The multifaceted repertoire of thermosensory behaviors exhibited by C. elegans free-living adults (i.e., experience-dependent migration towards ambient cultivation temperature, isothermal tracking of cultivation temperature, and robust avoidance behaviors associated with noxious temperatures) are thought to be methods of thermoregulation in support of survival and reproduction [34–38,40,67,68]. Thus, our observation that the free-living adults of Strongyloides species almost exclusively engage in migration to above-ambient temperatures is highly intriguing. To begin searching for an explanation for the thermophilic preferences of Strongyloides free-living adults, we tested the impact of different constant environmental temperatures on the survival of free-living females (Fig. 5, Fig. S7). Individual S. stercoralis and S. ratti FLFs, as well as C. elegans hermaphrodites, were placed on NGM plates seeded with E. coli HB101 at either 23°C, 30°C, or 37°C and their survival was checked daily (Fig. 5A). As expected, increasing the cultivation temperature of C. elegans adults resulted in a dramatic decrease in lifespan (Fig. 5B, E). When the cultivation temperature was increased for S. stercoralis and S. ratti FLFs, the lifespan of both Strongyloides species also decreased (Fig. 5C, D, E). Notably, this decreased survival at warmer temperatures appears to conflict with the thermal preferences of the free-living adults. This raises the question of why Strongyloides FLFs are attracted to temperatures that decrease their longevity.

Figure 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. Individual worms were added to plates, then incubated at either 23°C, 30°C, or 37°C. Plates were checked every 24 hours for survival. 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. 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 Fig. S7.

B) Probability of survival over time for C. elegans adult hermaphrodites.

C) Probability of survival over time for S. stercoralis FLFs.

D) Probability of survival over time for S. ratti FLFs.

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.

S. stercoralis FLFs show increased reproductive success at near-tropical temperatures

One possibility is that the thermal preferences of Strongyloides FLFs support other critical biological processes – in particular, the reproductive capacity of this life stage. To test the hypothesis that exposure to warmer temperatures positively impacts Strongyloides reproduction, we counted the number of offspring produced by individual FLFs cultivated at different temperatures (Fig. 6A). Similar to previous studies, we found that exposure to warm temperatures rapidly and dramatically depresses C. elegans total brood size (Fig. 6B, Fig. S8) [45,46]. In contrast, S. stercoralis FLFs showed a significant increase in total brood size at 30°C (Fig. 6B). When the temperature was increased further to 37°C, we observed a decrease in total brood size such that the total numbers of eggs and larvae produced at 23°C and 37°C are not statistically different (Fig. 6B). This decrease in total brood size primarily reflects the decreased lifespan of S. stercoralis FLFs at 37°C. When we examined the number of eggs and larvae laid each day, we observed an increase in S. stercoralis daily brood size at 37°C compared to 23°C on the first experimental day (Fig. S8). Thus, the mechanisms that yield enhanced total brood sizes at 30°C are likely also active at 37°C; however, those increases are offset by decreased longevity of the FLFs. We also tested the impact of warm temperatures on S. ratti total brood sizes and found that they exhibited no change in total brood size between 23°C and 30°C and a decreased total brood size at 37°C (Fig. 6B). When we compared the S. ratti day-by-day brood sizes, we observed that exposure to 30°C did drive an initial increase in daily brood size compared to 23°C, similar to S. stercoralis (Fig. S8). These results suggest that S. stercoralis is physiologically adapted for warmer climates than S. ratti, which mirrors the geographical distributions of these two species: S. ratti is widely distributed throughout the world while S. stercoralis is most prevalent in tropical and subtropical climates [10,11,70].

Figure 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.

In C. elegans, exposure to noxious warmth not only decreases brood size but also decreases hatching viability [48,49,71,72]. The effect of increased temperature on Strongyloides FLF’s egg hatching viability is unknown and is particularly interesting because of our finding that Strongyloides brood sizes can be enhanced at warmer temperatures. Increased brood sizes could reflect compensatory mechanisms if the hatching viability of Strongyloides spp. is decreased at warmer temperatures, similar to C. elegans. Alternatively, hatching viabilities that are largely unaffected by warmer temperatures could indicate that parasites are generally adapted to warmer environmental temperatures. To determine hatching viability, we collected synchronized eggs by letting individual C. elegans adult hermaphrodites and Strongyloides adult FLFs lay eggs onto NGM plates seeded with E. coli HB101 for 4 hours at 23°C. After removing the adults, we counted the number of eggs on each plate, then incubated plates at either 23°C, 30°C, or 37°C for 24 hours (C. elegans) or 48 hours (Strongyloides spp.) before re-counting any unhatched eggs (Fig. 6C). As expected, we observed that C. elegans median hatching viability progressively decreased as a function of increased temperature (Fig. 6D). In contrast, S. stercoralis did not show any significant change in hatching viability as cultivation temperature increased up to 37°C (Fig. 6D). S. ratti also exhibited no significant change in hatching viability from 23°C to 30°C; however, we did observe a small, but significant, decrease in hatching viability when cultivated at 37°C (Fig. 6D). Notably, this decreased S. ratti hatching viability was still significantly higher than the C. elegans hatching viability at 37°C (p<0.0001, 2-way ANOVA with Tukey’s multiple comparisons test). Together, these experiments indicate that the range of reproductively and developmentally permissive temperatures is shifted towards warmer temperatures for Strongyloides spp. compared to C. elegans.

Discussion

Here, we present the first characterization of thermosensory behaviors by Strongyloides free-living adults and provide evidence to link life-stage-specific thermal preferences to physiological and reproductive consequences. We found that the free-living adults of S. stercoralis are attracted to above-ambient temperatures in a wide range of thermal conditions, including temperature gradients that trigger noxious heat escape behaviors in C. elegans, negative thermotaxis towards cultivation temperature in C. elegans, and negative thermotaxis towards below-ambient temperatures in S. stercoralis iL3s (Fig. 7). We showed that unlike iL3s, S. stercoralis free-living females can respond to chemosensory attractants in thermal gradients below human body temperature. Impressively, we saw that some worms can even migrate against their thermal preferences in favor of moving towards the odorant. We also performed the first systematic evaluation of the impact of environmental temperatures on the physiological and reproductive potential of S. stercoralis free-living adults. We found that exposure to 30°C drives an unexpected increase in the brood size of S. stercoralis free-living adults, even at the expense of their longevity. Finally, comparisons between S. stercoralis and S. ratti reveal that S. stercoralis is adapted to warmer temperatures than S. ratti, a difference that aligns with their narrower geographic distribution [10,11,70].

Figure 7. Species- and life-stage-specific thermosensory behaviors and thermal physiology.

A) Summary of Strongyloides iL3 thermotaxis behavior. 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 temperature. 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 [34,37–39,42,43,50,64].

Free-living nematodes can experience a range of environmental stressors; as ectotherms, environmental temperature is a critical source of potential stress that can significantly impact their individual fitness. The free-living model nematode C. elegans displays a complex thermoregulatory process that, in response to thermal stress, prioritizes individual survival and future fecundity over immediate production of offspring [73,74]. Major elements of the C. elegans thermoregulatory response include thermotaxis navigation towards physiologically permissive temperatures, inhibition of reproduction, and altering a range of other physiological processes [34,43,45,48,73,74]. Until now, it has been unclear whether this thermoregulatory strategy was a ubiquitous feature of ectothermic, bacterivorous nematodes. Our results demonstrate that the free-living adults of two nematode species, S. stercoralis and S. ratti, display an alternative thermoregulatory strategy, one that prioritizes immediate expansion of infective larval populations over individual adult survival. These findings demonstrate that despite their similarities to C. elegans, Strongyloides free-living adults display distinct thermosensory responses and physiological characteristics, underscoring the potential for diversity in the behaviors of ectotherms inhabiting ostensibly similar ecological niches.

Previous studies have found large differences in thermal preferences between C. elegans adults and host-seeking Strongyloides iL3s [55,56]. Our current results expand these differences to Strongyloides free-living adults. The specialized thermotaxis behaviors of S. stercoralis iL3s are thought to arise from adaptations to a neural circuit that is conserved with C. elegans. Our findings suggest that these adaptations are a persistent feature of the Strongyloides thermosensory circuit across life stages. However, our data also reveals plasticity in the thermosensory behaviors of Strongyloides as a function of life stage, specifically, the failure of Strongyloides FLFs to perform negative thermotaxis, a behavior that is robustly present in Strongyloides iL3s. These differences mirror the life-stage-specific differences in chemosensory preferences previously observed with Strongyloides species [52,53,57]. In C. elegans, changes in the behavioral valence of sensory stimuli across life stages can arise from plasticity in the functional architecture of sensory neural microcircuits [67,75,76]. Similar life-stage-specific changes in functional connectivity could account for the behavioral differences we see between Strongyloides iL3s and free-living adults. Taken together, our results emphasize the need for future studies that pinpoint the neural mechanisms that drive parasite-specific and life-stage-specific thermotaxis behaviors in Strongyloides species.

Our experiments demonstrate that soil-resident Strongyloides free-living adults cannot survive prolonged exposure to temperatures approximating mammalian body heat. This physiological inability contrasts strongly with the ability of host-resident Strongyloides parasitic adults to survive for many months within host bodies, as well as the ability of soil-resident iL3s to survive the thermal transition associated with host invasion [77]. Thus, ectothermic species that parasitize warm-blooded animals are not innately resistant to the negative impacts of thermal stress. What are the molecular and cellular mechanisms that not only enable Strongyloides parasitic adults to survive and reproduce within the host environment, but also produce a remarkable increase in maximum lifespan compared to free-living adults [30,77,78]? If these mechanisms are actively acquired or maintained following host infection, they may represent an exciting source of molecular targets for novel anthelmintic drugs. In the future, we hope that research leveraging our mechanistic understanding of C. elegans thermal physiology and the growing Strongyloides genetic toolkit will provide insight into the remarkable physiological abilities of parasitic adults [69,79].

Here, we have focused on understanding the thermotaxis behaviors and thermal physiology of Strongyloides free-living adults, a life stage observed only in Strongyloides and the closely related Parastrongyloides genera [80]. Although other medically important parasitic species, such as human hookworms, lack a free-living generation, they do deposit eggs and larvae into the environment that must also survive environmental temperatures long enough to mature into infective larvae that will locate and infect host animals. Is the ability of Strongyloides post-parasitic eggs to maintain high hatching viability in warm temperatures a common feature of parasitic nematodes that are endemic to tropical and sub-tropical regions? Recent experimental evidence is limited and potentially determined by experimental conditions: whereas one study reports that the eggs of Necator americanus, a human hookworm, display low rates of hatching at temperatures above 35°C, another reports that the eggs of both N. americanus and the human hookworm Ancylostoma duodenale are able to hatch (albeit with increasing mortality) at temperatures up to 40°C [81,82]. The eggs of other parasitic nematode species, including the pig large roundworm Ascaris suum, also display remarkable resistance to elevated temperatures [83–87]. For behavioral preferences, previous studies have found that S. stercoralis iL3s prefer temperatures warmer than the human hookworm Ancylostoma ceylanicum and other mammalian-parasitic nematodes [55]. Thus, the ability of S. stercoralis FLFs to maintain high hatching viability at 37°C may indicate a particularly high thermal tolerance, even among human-parasitic nematodes. Nevertheless, mammalian parasites must all survive and reproduce at body heat, suggesting the possibility of common molecular mechanisms for avoiding decreased longevity and diminished fecundity due to heat stress.

Our investigation of the impact of environmental temperatures on the effective fecundity of the Strongyloides free-living generation has intriguing implications for the role of this life stage in promoting infections across diverse climates. Our experiments show that the reproductive potential of Strongyloides free-living adults is relatively impervious to prolonged exposure to 30°C. Human infections with S. stercoralis predominantly occur in tropical and sub-tropical climates [10,11]. Our results suggest that the specialized physiology of S. stercoralis can contribute to the maintenance of infectious populations within these climates. Specifically, our findings deepen our understanding of the complex relationship between temperature, the choice between the heterogonic and homogonic developmental pathways, and the production of infective larvae. Previous work has shown that S. stercoralis post-parasitic L1 larvae that experience temperatures below 34°C are driven to develop into free-living adults [21]. Now, we show that after reaching adulthood, S. stercoralis adults are attracted to warm temperatures, potentially enhancing their reproductive output at the expense of individual longevity. Furthermore, even in cases where total reproductive output is only maintained, rather than enhanced (e.g., S. stercoralis at 37°C), exposure to warmer temperatures will shorten the time required to generate large numbers of iL3s, which may influence the functional infectivity of the soil-dwelling population [88–90].

Together, our results suggest that the thermotaxis behaviors and thermal physiology of S. stercoralis are particularly adapted for tropical climates. These findings are especially concerning in the context of anthropogenic climate change, where increases in global temperatures could shift the geographic ranges capable of supporting S. stercoralis transmission while also improving reproductive capacity and functional infectivity of these parasites [91,92]. Ultimately, our work emphasizes the importance of developing a deeper understanding of the complex behaviors and physiology of S. stercoralis, a highly neglected source of human disease.