Hyposalinity reduces coordination and adhesion of sea urchin tube feet

ABSTRACT Climate change will increase the frequency and intensity of low-salinity (hyposalinity) events in coastal marine habitats. Sea urchins are dominant herbivores in these habitats and are generally intolerant of salinity fluctuations. Their adhesive tube feet are essential for survival, effecting secure attachment and locomotion in high wave energy habitats, yet little is known about how hyposalinity impacts their function. We exposed green sea urchins (Strongylocentrotus droebachiensis) to salinities ranging from ambient (32‰) to severe (14‰) and assessed tube feet coordination (righting response, locomotion) and adhesion [disc tenacity (force per unit area)]. Righting response, locomotion and disc tenacity decreased in response to hyposalinity. Severe reductions in coordinated tube foot activities occurred at higher salinities than those that affected adhesion. The results of this study suggest moderate hyposalinities (24–28‰) have little effect on S. droebachiensis dislodgement risk and survival post-dislodgment, while severe hyposalinity (below 24‰) likely reduces movement and prevents recovery from dislodgment.


Introduction
Climate change will increase the frequency and intensity of extreme climatic events (ECEs) such as heatwaves, droughts, storms, and intense precipitation (Coumou and Rahmstorf, 2012;Smith, 2011). Organisms may be particularly susceptible to their effects because they likely lack adaptations for these unprecedented events (Holt, 2008). In marine ecosystems, the biological consequences of thermal stress associated with heatwaves have received considerable attention (Smith et al., 2023), yet the effects of osmotic stress associated with low salinity (hyposalinity) following storm-related freshwater input remains poorly resolved (Cheng et al., 2016). Drastic changes in salinity are particularly problematic for stenohaline organisms, like sea urchins, that possess little to no capacity to osmoregulate (Burnett et al., 2002;Russell, 2013;Vidolin et al., 2007).
Sea urchins are dominant herbivores and ecosystem engineers in many benthic communities (Davidson and Grupe, 2015;Steneck, 2020). In temperate ecosystems, sea urchins are capable of reorganizing community structure by overgrazing macroalgae, decreasing primary productivity and habitat for many economically important species (Steneck, 2020).
Understanding how environmental stressors induced by ECEs, like periods of hyposalinity, will affect sea urchin populations is critical to predict the impact of climate change on the ecosystem services provided by temperate rocky reef systems. Sea urchin survival and success as herbivores is largely mediated by their tube feet, muscular hydrostatic extensions of the water vascular system involved in attachment, locomotion, feeding, respiration, and sensing (Flammang, 1996;Leddy and Johnson, 2000;Lesser et al., 2011;Rodriguez, 2003). Tube feet generally consist of an extensible stalk terminating in a disc containing a duo-gland adhesive system that secretes an adhesive and deadhesive to attach and detach to substrata, respectively (Flammang, 1996).

Journal of Experimental Biology • Accepted manuscript
The green sea urchin (Strongylocentrotus droebachiensis) is one of few euryhaline sea urchin species (Russell, 2013), occurring in the Northern Atlantic, Northern Pacific, and Arctic Oceans. It is a dominant grazer and an important fisheries species (Scheibling et al., 2020).
Here we assessed the impacts of hyposalinity on S. droebachiensis tube foot function. We quantified individual (disc tenacity [adhesive force per unit area]) and coordinated tube feet function (righting response [ability to right after inversion] and locomotion) along a salinity gradient. We hypothesized that both parameters would decline similarly in response to hyposalinity.

Sea urchin collection and experimental design
We collected sea urchins (44.95 ± 8.46 mm diameter, n = 30) from a depth of 9 m near Friday Harbor Laboratories on San Juan Island, Washington (48°32' 26.2392" N, 123°0' 40.3128" W) on October 31, 2022. Sea urchins were brought to the laboratory and divided into three size classes based on diameter (30-40, 40-50, and 50-60 mm). Sea urchins from each size class were equally divided into ten 18.4 x 13.3 x 13.3 cm containers (n=3 per container).
Containers were filled with ~3L of filtered natural seawater that was oxygenated with air diffusers and placed in a flow-through sea table at ambient temperature (11.3 ± 0.3 °C).
Each container was randomly assigned to an even integer treatment salinity ranging from 14 to 32 parts per thousand (‰). Containers were then lowered to their treatment salinity at a rate of 0.98 ± 0.17‰/10 minutes by adding chilled reverse osmosis (RO) water. Final treatment salinity was ± 0.5‰ of the target salinity. Tube foot function was assessed after sea urchins were kept in their target treatment salinity for 24 hours (Russell, 2013). To remove accumulated solid and nitrogenous waste, partial seawater changes were conducted after 6 and 12 hours with seawater matching the treatment salinity for each container. Sea urchins were not fed during the experiment.
Journal of Experimental Biology • Accepted manuscript

Tube foot function
Righting response, locomotion, and disc adhesive force were assessed for each sea urchin in seawater of their respective treatment salinity. Seawater of each treatment salinity was made by diluting filtered natural seawater with RO water. Disc area, used to calculate disc tenacity, was measured before the start of the experiment in filtered natural seawater at ambient salinity.

Righting response
Righting response is the time required for a sea urchin to completely flip from their inverted aboral body axis to their oral body axis (i.e., mouth facing the substrate). It is a common metric of neuromuscular coordination in sea urchins, requiring coordination of tube feet and spines (Collin et al., 2018). Sea urchins were placed, inverted, in a 10-gallon glass aquarium filled with ~8L of seawater and the time (sec) required for sea urchins to right themselves was recorded. Many sea urchins failed to right in 300 sec, suggesting they could not right themselves within a biologically relevant timeframe. In these instances, we recorded righting time as 300 sec. We converted righting response (sec) to an activity coefficient (AC, sec -1 ) using the formula = 1000 ℎ − 3.33 ̅ such that faster righting times corresponded to higher AC, slower righting times correspond to lower AC, and failure to right in 300 sec corresponded to an AC of 0 (Barker and Russell, 2008).

Locomotion
Locomotor performance was assessed by placing sea urchins at one end of a 10-gallon glass aquarium, dispensing 0.5ml of 2% bleach in seawater at the ambulacral column opposite to the desired direction of movement. Preliminary trials indicated sea urchins actively move away from a dilute bleach solution. We recorded sea urchin movement with a DSLR camera (Nikon D5600; Nikon USA, Melville, NY USA) positioned beneath the aquarium with a 1 cm 2 scale.
One frame per second for the first 30 seconds of movement was isolated (resulting in 30 frames) and analyzed in ImageJ using the MTrackJ plugin (Meijering et al., 2012). We tracked the center of the sea urchin's exposed jaw across the 30 frames and calculated instantaneous speed using the distance covered and time taken from one frame to the next. Maximum locomotor speed (cm sec -1 ) was calculated as the maximum instantaneous frame-to-frame speed recorded in each trial. If no tube feet attached within five minutes, the trial was discontinued, and the sea urchin was omitted from data analysis. For each sea urchin, the highest value of each of three trials was used to calculate disc tenacity by dividing the maximum adhesive force by the mean disc area of that sea urchin.

Statistical analysis
All analyses were performed in R (https://www.r-project.org/). We used general additive models (GAMs -package mgcv; Wood, 2011) to compare the relationship between salinity and tube foot functionality among the three performance metrics (activity coefficient, maximum speed, disc tenacity). Salinity was modeled as a thin plate smoothing spline and smoothing parameters selected using generalized cross validation criteria. The degree of non-linearity of the models are provided as effective degrees of freedom (edf), whereby higher edf corresponds to a more non-linear relationship. A linear regression showed no significant relationship between sea urchin diameter and any of the tube foot functionality metrics (activity coefficient: t Normal distribution of residuals was assessed with a histogram and quantile-quantile plot.
Homogeneity of variances was verified by plotting the model residuals versus the fitted values.
( Figure S2). Activity coefficient data violated assumptions; natural log transformation improved assumptions, but the results were qualitatively similar to those with non-transformed data ( Figure   S2, S3), we reported the latter for ease of interpretation and visualization.
The mean value of each performance metric was calculated for each salinity and the greatest mean was used as maximum performance. Performance of each sea urchin was then graphed as a percentage of maximum performance to facilitate comparisons between performance metrics.

Results and Discussion
Our results show that sea urchin tube foot adhesion and coordination decline with decreasing salinity in distinct linear and non-linear relationships. Activity coefficient had the least linear relationship of the three metrics (edf = 3.522), resembling a cubic or quartic function ( Figure 1A, Table 1). Maximum locomotor speed had the most linear relationship, with an edf of 1, suggesting a positive linear relationship between salinity and maximum locomotor speed ( Figure 1B, Table 1). Disc tenacity had an edf of 2.516, resembling a quadratic or cubic function ( Figure 1C, Table 1). One sea urchin did not extend tube feet during adhesive force measurements at 14‰ and was omitted from data analysis. Furthermore, sea urchins experienced severe reductions in each performance metric at different salinities. Relative to maximum performance, activity coefficient was severely reduced at  24 ‰ (Figure 2A), locomotion was severely reduced at  20 ‰ ( Figure 2B), and disc tenacity was severely reduced at  16 ‰ ( Figure 2C).
Reduced tube foot functionality under hyposalinity may be the result of the dilution of ions in seawater important for tube feet neuromuscular activity and adhesion. Calcium, sodium, and potassium, for example, are essential for many physiological and cellular activities, including neuromuscular function (Carafoli, 2005;Hill, 2012). At lower salinities, these ions may be below the concentrations required for normal neuromuscular function and thus Journal of Experimental Biology • Accepted manuscript responsible for reduced tube foot coordination. A decrease in concentration of ions important for neuromuscular function may therefore explain the declines of highly coordinated activities (locomotion, righting response) at higher salinities than disc tenacity. However, calcium is hypothesized to induce secretion of the adhesive in tube feet, and many marine adhesives use non-covalent interactions such as calcium and magnesium bridging to achieve adhesion to the substratum (Hennebert et al., 2015;Lebesgue et al., 2016). Thus, changes in various ion concentrations from hyposalinity may impact the formation, release, and composition of the adhesive. Future work is needed to better understand the physiological and cellular mechanisms responsible for reduced tube foot functionality in hyposaline conditions.
The inability to regain attachment and proper orientation after dislodgement from hydrodynamic forces and predators can be fatal to sea urchins (Dayton, 1973;Dayton, 1975).
The observed decline in activity coefficient suggests hyposalinity will aggravate the negative impacts of dislodgment for sea urchins experiencing severe hyposalinity (below 24‰) by reducing the ability to recover proper orientation. Ability to evade high hydrodynamic forces and predators may also decrease under hyposalinity as sea urchins become slower in response to a negative stimulus ( Figure 1B). The effect of severe hyposalinity on sea urchin survival may be partially alleviated by relatively little change in disc tenacity between 18-28‰ ( Figure 1C, 2C).
Individual tube feet attached readily to glass capillary tubes in 18‰ seawater and thus may retain sufficient functionality at low salinities to allow sea urchins to remain attached and compensate for reduced adhesion by recruiting more tube feet to the substratum, preventing dislodgment (Narvaez et al., 2022).
Hyposalinity may have a pronounced effect on tube foot functionality that persists after returning to average conditions, thus future experiments should investigate the potential for recovery in tube foot functionality once sea urchins are returned to average salinity conditions. Sea urchins likely experience multiple hyposalinity events within the same year. Repeated exposures may have a detrimental effect on performance or may result in acclimation where performance increases over time. Indeed, Strongylocentrotus droebachiensis growth rate acclimates to hyposalinity and populations exposed to hyposalinity exhibit greater ability to right themselves under hyposalinity than unexposed populations, suggesting local acclimation (Drouin et al., 1985;Himmelman et al., 1984;Russell, 2013). However, tube feet exhibit considerable phenotypic plasticity in performance and morphology (Cohen-Rengifo et al., 2017;Narvaez et Journal of Experimental Biology • Accepted manuscript al., 2020;Narvaez et al., 2022;Santos and Flammang, 2007;Stark et al., 2020;Toubarro et al., 2016). Given that plastic traits are often energetically expensive (Dewitt and Scheiner, 2004), maintenance of homeostasis and tube foot performance may present competing energetic demands, resulting in lower tube foot performance under stressors, such as hyposalinity. The sea urchins used in this study were collected from a depth of 9 m and although they were likely protected from hyposalinity, we lack long-term salinity measurements along a bathymetric gradient at our collection sites. Therefore, future studies should measure salinity levels within different sea urchin microhabitats and test the capacity and extent that sea urchins acclimate to hyposalinity within and between populations with differential exposure to hyposalinity.
Future ECEs associated with global climate change will likely expose sea urchins to more intense and frequent variations in salinity (Nyhagen et al., 2018). Our results suggest moderate hyposalinity (24-28‰) will have limited effects on S. droebachiensis performance. Severe hyposalinity (below 24‰) will likely increase dislodgment risk, reduce movement, and prevent recovery from dislodgment. However, S. droebachiensis is one of few euryhaline sea urchin species and likely represents a "best case scenario" for sea urchin performance under hyposalinity when compared to stenohaline species. Therefore, understanding the performance of stenohaline sea urchin species under hyposalinity and the capacity for performance to acclimate to hyposalinity will strengthen predictions about the consequences of ECEs on these critical members of marine communities.

Acknowledgements:
We thank the two anonymous reviewers for their insightful comments and John McCormack for help with data collection.

Competing interests
We declare we have no competing interests.   Normality assumption shown as a histogram and QQplot of the model residuals.

Authors' contributions
Homoscedasticity shown as a scatterplot of residuals versus fitted values.