Physella wrighti Te and Clarke, 1985 is an Endangered freshwater snail endemic to the Liard Hot Springs. The thermal characteristics of its environment suggest that water temperature (WT) is essential for the snail’s survival. Initially, Physella wrighti’s preferred WT was assessed, with 23 °C preferred. To determine if WT influenced the snail, the activity level, behaviour, survivability, number of egg masses (EMs) produced, number of eggs per mass (EPM), egg volume, EM viability, and incubation period (IP) were examined in 13 °C, 23 °C (preferred WT), and 33 °C water. No differences were found in activity level, but snails in 33 °C left the water more frequently, experienced total mortality, had the shortest survival length, produced the fewest EMs, however, had the shortest IP. Snails in 13 °C survived the longest, produced the most EPM, but had the lowest viability. Snails in 23 °C produced the most EMs and had the greatest viability, EPM did not differ from 33 °C, and IP was between 13 °C and 33 °C. These data indicate that Physella wrighti benefits more from WTs in the lower range of its habitat and has implications for its ecology and conservation.
COSEWIC first assessed Physella wrighti in 1998 (COSEWIC 2008). The species received the status of Endangered due to its extremely limited range, potentially limited population (estimated at fewer than 10000 snails), potentially large population fluctuations (at least one order of magnitude), potentially short lifespan, and the belief that it is a hot water specialist (COSEWIC 2008) (i.e., the species is adapted to survive only water temperatures (WTs) found in the LHS environment) thus has limited habitat availability (Lee and Ackerman 1999; DFO 2010). The status was re-evaluated in May 2000 and April 2008 (COSEWIC 2008). In 2003, Physella wrighti was listed as Endangered under Schedule 1 of SARA (Species at Risk Act 2002; COSEWIC 2008). At the provincial level, the conservation status for Physella wrighti is listed as S1 (critically endangered) (BC Conservation Data Centre 2008) and at the global level as G1 (critically imperilled) (NatureServe Explorer 2020).
In the LHS, Physella wrighti lives in WTs ranging from 23.5 °C to 36 °C (Te and Clarke 1985; Lee and Ackerman 1999; Salter 2003; COSEWIC 2008), but have been found in temperatures as high as 40 °C (COSEWIC 2008; DFO 2010), although it is not explicitly specified if this is above water and/or submerged in water. They have also been observed out of the water, crawling on various substrates even in January, with air temperatures ranging as low as −30 °C to −40 °C; substrates include mats of the algae Chara vulgaris Linnaeus (Characeae), grasses at the edge of the water, and other detritus and sediments (Te and Clarke 1985; Lee and Ackerman 1999; Salter 2001). Considering the variety of substrates on which Physella wrighti can be found, it is thought that the snail grazes on algal and microbial organisms (Lee and Ackerman 1999; Lauzier et al. 2011), not unlike other physid snails (El-Emam and Madsen 1982; Brown 2001; Dillon 2004). The currently reported WTs in which Physella wrighti exist and copulate do not appear to be specific to the areas where the snails are found within the hot springs. Instead, the temperatures appear to be applied to the overall water body (e.g., Alpha Pool and Alpha Stream) (Te and Clarke 1985; Lee and Ackerman 1999; British Columbia Ministry of Environment 2014). Although additional populations of Physella wrighti have been sought at other hot springs in British Columbia, including Grayling Springs (59°37′N 125°32′W and 59°37′N 125°38′W), Deer River Hot Springs (59°30′15.0″N 125°57′24.1″W), Toad River Springs (58°55′26.4″N 125°05′40.2″W), and Coal River Hot Springs (60°09′N 127°09′W), despite having similar temperature profiles, Physella wrighti have not been found (Te and Clarke 1985; Salter 2003).
At present, there is limited information available on the ability of snails to tolerate different temperatures and the impact of different temperatures on life history. Among the few studies available on pulmonate snails are van der Schalie et al. (1973), who studied the impact of WT on the life history of Physella gyrina, Lymnaea stagnalis Linnaeus, 1758 (Lymnaeidae), Stagnicola emarginata (Say, 1821) (Lymnaeidae), McMahon (1975) who studied the impact of heated water discharge on the life history of Physella virgata Gould, 1855, and Britton and McMahon (2004) who studied the impact of WT on the ability of Physella virgata to assimilate organic carbon. Studies on thermal tolerance are widely distributed across different clades (Russell-Hunter 1961a, b; van der Schalie et al. 1973; McMahon 1975, 1976; Britton and McMahon 2004; Muñoz et al. 2005; Seuffert et al. 2010), making it challenging to find comparative information on closely related taxa. To understand the thermal capabilities of Physella wrighti, one must study how the snail responds to different WTs. By acquiring more information on the snail’s biology, one can determine how to manage its habitat(s), mitigate threats, and identify other locations that may act as a habitat for this species.
While the LHS has been used by humans for over 180 years (Camsell 1956; Peepre et al. 1990; British Columbia Ministry of Environment 2009), Physella wrighti has persisted, surviving pool maintenance/structural modification, the introduction of cosmetic chemicals (i.e., soap and sunscreen), and trampling (Peepre et al. 1990; COSEWIC 2008; DFO 2010; B.C. Parks Facilities Information (personal communication, 2020)). Despite the species’ resilience, a single event, such as an abrupt change in water flow or the introduction of an exotic species, could have a negative impact on the species (COSEWIC 2008; DFO 2010; Fisheries and Oceans Canada 2017). Currently, there is a paucity of biological information for Physella wrighti. As a species at risk, it is necessary to examine this species’ life history with respect to a range of temperatures mirroring those seen in its native habitat. This will allow for the identification of other possible locations to survey for populations of the species and to anticipate and prevent incidents that may threaten the species.
To build knowledge of Physella wrighti, aspects of its life history were examined and considered with respect to different WTs. To ensure that observations remained unbiased, the activity level of Physella wrighti was assessed at 30 °C during the scotophase (night/dark period) and photophase (day/light period). Next, it was assessed if Physella wrighti had a preferred WT. Once the preferred temperature was established, it was used to set up experimental aquaria at the preferred WT and 10° above/below the preferred temperature. Under these conditions, the Physella wrighti’s period of activity, behaviour, survivability, number of egg masses (EMs) produced, number of eggs per mass (EPM), egg volume, EM viability, and incubation period (IP) were assessed.
2 Materials and methods
2.1 Specimen collection
Live specimens of Physella wrighti were collected from the LHS in September 2017, January 2018, and January 2019 (Fig. 1). Snails were collected from different locations along Alpha Stream and throughout Alpha Pool, Beta Pool, and the Epsilon/Delta/Gamma swamp complex. Collection sites consisted of 1 m2 plots from which no more than 10% of the total number of individuals was collected. Snails were collected based on availability (with no preference for size) as this was more conducive to random selection, maintaining collection efficiency, minimizing site damage, and minimizing unnecessary stress from the time and manipulation required to measure specimens during collection. Snails were not collected at the same spot for each collection due to the restricted collection conditions, as snail populations at some spots were too low to reasonably remove individuals from (e.g., could remove <10 snails). Snails were placed in 1 L plastic containers containing water from the collection site, and shipped in coolers to the Royal Saskatchewan Museum, Regina, Saskatchewan; the duration of transport did not exceed 48 h. Upon arrival, transport containers were submerged in 37.9 L holding aquaria at a temperature of 30 ± 2 °C for 2 h before releasing the snails into the holding aquaria. After establishing the temperature preference for Physella wrighti, newly arrived snails were acclimated at that temperature. Specimens were collected in accordance with applicable laws, guidelines, and regulations under SARA Permit No.: DFO-PPAC-00018.
2.2 Holding aquaria
Snails were cultured in 37.9 L aquaria at a WT of 30 ± 2 °C; this temperature corresponded to the transport temperature and is the middle of the range of field temperatures (23.5 °C–36 °C) reported by Lee and Ackerman (1999). The temperature of the aquaria was adjusted to 23 ± 2 °C to reflect the snail’s preferred temperature, as determined below. WT in the 37.9 L aquaria was maintained using Fluval® M100 fully submersible aquarium heaters.
Water changes were performed weekly using reverse osmosis (RO) water and mixed RO water/tap water treated with Seachem® Prime® as deemed necessary. Water changes consisted of a repeating schedule of a 10% water change on weeks 1 and 2 and a 20% water change on week 3, with larger water changes done as deemed necessary. Aquaria were supplemented with CaSO4 and NYOS® Alkalinity+ to reflect the water chemistry of the LHS; however, this did not appear to benefit the snails and supplementation was eventually ceased.
Solid waste materials were removed from the aquarium using a fine-mesh fishnet and a turkey baster. All waste was rinsed over silkscreen to prevent the loss of live snails. Dead snails were removed from the aquaria and preserved in ethanol.
To maintain a natural light spectrum, all holding aquaria were kept on a 12 h light/dark cycle using a single SunBlaster™ T5HO bulb per aquarium. Aquaria were partially covered with lids to discourage snails from crawling out of the aquaria and provide shelter from direct light.
2.3 Experimental aquaria set-up
Snails were kept in nine 9.5 L aquaria. Aquaria contained approximately 2.5 cm of filter sand and a sponge filter composed of a Top Fin® Filter Cartridge (for Top Fin® Multi-Stage Internal Filter 10). The activated carbon was removed and replaced by an air stone and airline filter to aerate the water. All aquaria were initially filled to a depth of 10.2 cm with 500 mL of stock aquarium water, 500 mL of tap water (treated with Seachem® Prime®), and the remaining amount with RO water containing CaSO4 supplement. No further supplementation was added.
Six aquaria were heated using one 50 W fully submersible aquarium heater per aquarium; three aquaria were heated to the preferred temperature of 23 °C (warm) and other three to 33 °C (hot). Three aquaria were placed in a Marathon Deluxe 8.5 cu.ft. All refrigerators were modified with a Penn A421ABC-02 temperature controller to maintain a WT of 13 °C (cold). Each aquarium was separated with a 2.5 cm thick piece of expanded polystyrene foam to aid in maintaining WTs and prevent snail travel between aquaria.
Aquaria were kept on a 12 h light/dark cycle with a single SunBlaster™ T5HO light suspended 12.5 cm above the aquaria. The aquaria were partially covered with lids to allow sufficient light to enter the aquarium and ensure snails had shelter from the light.
Aquaria were set up 6 days before adding snails; 10 snails were then acclimated and added to each aquarium. Acclimation involved placing snails in a container of water from the stock aquaria and floating the containers in the experimental aquaria for at least 1 h before releasing the snails into the experimental aquaria. Since Physella wrighti in the LHS demonstrates plasticity for a wide range of WTs, it was not deemed necessary to prolong acclimation time. Water was changed at least once per week using equal parts RO water and tap water treated with Seachem® Prime®. Water lost by evaporation was replaced using RO water.
Snails were fed a quarter to a half of Hagen® Nutrafin® Max Spirulina meal tablets and Omega One® Shrimp and Lobster Pellets. Portions were based on how much snails consumed; however, snails were fed at least once per week.
2.5 Experimental procedures
Prior to performing the temperature preference experiment, it was attempted to determine if there was a period of time where Physella wrighti was predominantly active to avoid conducting the preference trials during a time when the snails were inactive. To determine if Physella wrighti has a period of greater activity in response to photophase or scotophase, qualitative observations were made of the snails in the holding aquaria. Periods of observations were made at 1 h intervals in blocks from 01:00 to 07:00, 08:00 to 18:00, and 19:00 to 00:00; 07:00 to 18:59 was defined as the photophase and 19:00 to 06:59 as the scotophase. During the scotophase, aquaria were illuminated for 5 min or less per observation period because snails were too small to view under dim or red-light conditions. The WT was maintained at 30 ± 2 °C for these observations. Observations included whether Physella wrighti showed obvious location patterns within the aquaria, remained solitary or congregated, and whether snails were consistently active in the aquaria or appeared quiescent. The same observations were made for snails in the experimental aquaria. In consideration of the possible effects of different WTs on snail activity and time of day, additional observations on the snail’s location, direction of movement, and lack of movement were also made. Observations were made at 14 randomly generated times to reduce bias between scotophase and photophase observation times.
To determine the preferred temperature, snails were placed in a glass trough measuring 91.4 cm × 20.3 cm by 15.2 cm filled with 8.9 cm of water from the holding tanks (Fig. 2). A temperature gradient was established by heating one end of the trough and cooling the other (similar to the methods noted in Diaz et al. 2006, 2011). One end of the trough was heated to ∼45 °C using an immersion heater and a reptile heating lamp with a 120 W incandescent bulb, set to half power, and the opposite end was cooled to ∼5 °C using ice packs. Ice packs were replaced when approximately 80% thawed, and replacement was staggered to maintain WT. The trough was divided into six sections using 2.5 cm thick × 7.6 cm tall, rigid polystyrene foam with the top edges trimmed into a trapezoid shape to reduce sharp edges. The foam blocks were placed at 15.2 cm intervals; they helped generate a temperature gradient by reducing the flow of water along the length of the trough. It was judged that these divisions would not hinder the movement of the snails throughout the trough as they regularly have to navigate various obstacles in their natural habitat. The range of WTs was chosen to include the approximate temperatures in which Physella wrighti can be observed in the LHS (Te and Clarke 1985; Lee and Ackerman 1999; Salter 2003; COSEWIC 2008) and exposed the snails to WTs to which they may not normally be exposed. As Diaz et al. (2006, 2011) initially reported isotherms in their temperature gradients, a small aquarium air stone (Grreat Choice®) was added to each section, with an additional air stone to the heated section. Air was gently bubbled through each section using a Top Fin® AIR-8000 air pump to reduce the magnitude of the isotherms following Diaz et al. (2006, 2011). It was deemed that the slight current generated from the air stones would not hinder the movement of the snails throughout the trough as they are commonly located in areas with running water in their natural habitat. Top Fin® Digital Aquarium Thermometers were used to monitor the WT along the trough.
Snails used in the preferred temperature experiments were randomly selected from holding aquaria. Individual snails were marked on the shell for identification, using different colours of nail polish enamel (Clampitt 1970, 1974; Fenwick and Amin 1983). Snails were then placed in a medium-sized Marina® Hang On Holding and Breeding Box, receiving bubbled water from one of the holding aquaria, and left for ∼22 h to recover from tagging. Snails were then added to the trough where the WT was similar to the holding box. Snails were left in the trough for 2 h, then the WT at each snail’s location was noted. Since snails had been observed to traverse distances quickly in their natural habitat during specimen collection, a trial period of 2 h was a sufficient length of time for the trails. Snails were returned to holding aquaria, and the inner surface of the trough was cleaned and rinsed three times to remove residue from mucus trails to avoid any possible impact of the existing trails on the travel of snails in the subsequent trial. This process was performed at the same time, once a day, for 6 days, using a total of 30 snails. Five snails were used per trial, such that one snail from each of the same five holding aquaria was used per trial.
To determine if WT altered the behaviour of Physella wrighti, snails were observed for differences in emersion frequency and distance (how often the snails exited the water and the distance they crawled out of water), signs of irritation, congregation of snails, avoidance of areas within aquaria, if snails copulated, and the presence of EMs. The substrates upon which an EM was laid were also noted. Emersion distance was measured as vertical, linear distance from the water level to the snail. Snails were observed daily and during the 14 random times used to assess activity levels.
Snails in the experimental aquaria were observed daily from introduction to the date they were found dead. A snail was deemed deceased when it did not respond to external stimulus (i.e., gentle prodding) and was removed from the aquarium. Only data for snails that could be confirmed as dead within the previous 24 h were considered for analysis. New snails were added if the population in an individual aquarium dropped to seven out of ten snails; new snails were acclimated using the same procedure noted in the section “Experimental aquaria set-up”. Only the hot (33 °C) water treatments required replacement snails; a total of 18 snails were replaced. Individual snails in hot water aquaria were not initially marked. Thus, survivorship of snails could not be tracked beyond the first replacement of snails; analysis was performed on the available data. Survivorship data were unavailable for 10, 6, and 38 snails in the cold, warm, and hot water treatments, respectively. Snail survivorship was monitored daily for 108 days, after which survivorship was monitored twice per week for an additional 168 days, at which point the survivorship study was concluded. Deceased specimens were collected and stored at the Royal Saskatchewan Museum.
The number of EMs within each aquaria was recorded to determine the impact of WT on egg laying. Only EMs noticeable upon visual examination of the aquaria were considered. This prevented the disturbance and destruction of aquaria contents and inhabitants due to searching for EMs. Egg masses of all sizes were readily visible, and aquaria were thoroughly visually examined to ensure the maximum level of observations was reached; masses were counted daily for 108 days. Egg masses were examined for the number of EPM, size of eggs, viability of EMs, and embryo IP through two methods. The first method involved gently dislodging several EMs from the aquaria. Individual masses were placed in hinged plastic boxes measuring 2.2 cm × 2.9 cm × 2.9 cm with two holes covered with silkscreen to isolate the EM from the rest of the tank but allow gas and water exchange into/out of the box. Boxes were suspended at water level in each aquarium. Each box was removed from the aquaria and the EM within was examined using a stereomicroscope. For the second method, seven microscope slides were placed in each aquarium. Snails laid masses on the slides, and slides were removed from aquaria as necessary to examine EMs. This allowed for the examination of masses without dislodging masses from surfaces within the aquaria. These masses were examined using a compound light microscope. For both methods, masses were selected and examined based on availability and were immediately returned to their respective aquaria. All visible masses within the aquaria for which the number of eggs could be readily ascertained without disturbing the mass were included in the data for the EPM.
To assess potential differences in egg size between snails reared in the different WTs, the volume of each egg within an EM was calculated using the formula for a prolate ellipsoid. Using a calibrated ocular micrometre, the length and width of eggs were measured from the longest/widest portion of the inner membrane for each egg, as the outer membrane was not always visible. This allowed for a relative determination of egg size for all eggs by arbitrarily setting egg depth equal to egg width, as depth was not visible. Egg size was only considered for eggs in which embryos had not begun to revolve or eggs that contained no embryo to avoid measuring enlarged egg sizes due to an increase in embryo size. Egg viability was defined as snails developing fully without any visible malformations. Eggs were considered nonviable when an embryo started to degrade, as evident by a fractured appearance of the embryo, or if the embryo developed malformations followed by death within the egg.
The IP for embryos was defined from when an EM was laid to when the first snail hatched from an egg within the mass. Masses were included in the data only if it was clear that the mass had been laid within 12–24 h of being collected for observation.
2.6 Statistical analyses
Determining a model to show the relationship between a predictor(s) and a response can be accomplished using Frequentist or Bayesian methods of statistical testing (Ellison 2004). Bayesian statistical testing methods were used for data analysis due to their benefits compared to Frequentist methods of analysis (Ellison 2004; Stephens et al. 2005; Masson 2011). Frequentist testing applies null hypothesis significance testing (NHST) to data (Stephens et al. 2005). NHST generates a probability (p value) which indicates how likely or unlikely we are to observe the data if the null hypothesis (H0) is correct (e.g., the predictor variable does not affect the data) (Masson 2011). This does not provide the probability that the hypothesis is true, only how likely it is to observe the data under certain conditions. Bayesian methods address the probability that the hypothesis is true based on the data (Ellison 2004; Mason 2011). The analysis produces a credibility interval that provides the probability that the parameter(s) of interest (e.g., the mean) is found within a range of values that could result in the data (Ellison 2004). Bayesian methods are ultimately a more intuitive method of analysis.
Temperature preference was determined by fitting a Bayesian linear hierarchical model with holding aquarium and trial number as the predictors, a Gaussian distribution as the response (data appeared to follow a normal distribution and consisted of positive and negative continuous values), and uninformative priors for the model parameters using the packages BRMS (Bürkner 2017, 2018) and CODA (Plummer et al. 2006) in R3.6.1 (R Core Team 2019). Markov Chain Monte Carlo (MCMC) diagnostic methods were used to verify the reliability of the results: R-hat values, effective sample sizes (ESSs), and visual inspection of trace and posterior density kernel plots (Appendix A). MCMC diagnostics validated that the model had successfully converged; visual inspection of trace plots and posterior density kernel plots showed no trends, all R-hat values equalled one, and the ESSs were sufficiently large.
The effects of temperature on the number of days snails survived (survivorship), egg volume, and IP was assessed using a Bayesian generalized linear model with WT as the predictor variable and a gamma distribution with a log link for the response. A gamma distribution was chosen as the data are close to zero, continuous, and strictly positive. A preliminary examination of the data also supported using a gamma distribution. The effects of WT on the number of EMs and the number of EPM were also assessed using a Bayesian generalized linear model with WT as the predictor variable, and a negative binomial distribution (as the data are discrete data) with a log link function was applied. In the model for determining the effect of temperature on mass viability, a Bayesian generalized linear model with WT was the predictor variable and a binomial distribution was used as this distribution is appropriate in analyzing data that occur under a “yes” or “no” state (e.g., the egg is viable or nonviable). All model parameters were assigned default, uninformative priors. The MCMC diagnostics showed that the model had successfully converged since all R-hat values were equal to one, ESSs were sufficiently large (>1000 (Bürkner 2017)), and visual inspection of trace plots and posterior density kernel plots showed no trends (Appendix A). Analyses were performed using the packages BRMS (Bürkner 2017, 2018) and CODA (Plummer et al. 2006); figures were generated using ggplot2 (Wickham 2016) in R 3.6.1 (R Core Team 2019) with RStudio (RStudio Team 2018).
From a visual examination of Physella wrighti, snails remained active throughout all observation periods, and there was no visually definitive pattern of inactivity, suggesting individual snails rest only as required. There were no discernable patterns in the direction of snail movements in the aquaria; individuals appeared evenly distributed throughout each aquarium in the photo- and scotophase. Snails remained relatively scattered throughout the aquaria and did not appear to congregate on a particular substrate.
Physella wrighti showed the ability to tolerate a wide range of WTs; however, the preferred WT ranged from 15 °C to 30 °C (Fig. 3). Snails were observed crawling into the heated and cooled ends of the trough. The maximum WTs in which snails were observed were 38.1 °C and 39.1 °C. The minimum WT in which snails were observed was 5.8 °C, but snails were also seen crawling on the ice packs. One observed snail crawled into the hot water at 39.1 °C and then into the cold water at 5.8 °C during the 2 h observation period, indicating a capacity to tolerate large, rapid changes in WT. Snails also showed they were not hindered by the divisions in the trough or the air currents generated by the air stones by traversing across the entire length of the trough on multiple occasions. Analysis of the data showed Physella wrighti preferred a temperature of 23.9 °C (19.7 °C–29 °C, 95% credible interval); there were no clear differences between trials and holding aquaria. Knowing that WTs vary slightly, the resulting value was rounded down and used as the preferred temperature, as this was the desired level of accuracy to report to.
For the most part, Physella wrighti was not observed to show any changes in behaviour in response to the range of WTs examined. Snails were not visually observed to show a preference for a location or surface material (e.g., plastic, sand, glass) within aquaria. Snails were observed copulating in each temperature treatment and laid EMs even on the heated portion of the aquaria heaters. EMs were laid throughout the aquaria except in hot water aquaria, where snails laid very few EMs. Snails congregated when consuming food pellets and when copulating, which consisted of groups of two to four snails. No observable trends were seen for time of reproduction. Out of the total observations of emersion, snails in hot water were emersed for 72.4% of observations versus snails in cold water at 21.8% and snails in the preferred warm water at 5.7%. Snails in hot water crawled farther away from the water than snails in cold and warm water at distances of 7.5, 5, and 2.5 cm, respectively. Snails in hot water were also observed to crawl out of the water and onto the underside of the aquaria lids; this behaviour was not observed in the other temperature treatments.
Survival of field-collected snails varied among temperature treatments. The first snail death occurred in hot water aquaria within 24 h of exposure to hot water. Hot water aquaria were restocked with snails from the holding aquaria; however, rearing snails in hot water aquaria was unsuccessful. Snails in hot water aquaria did not survive for more than 60 days from the start of the study. There was a clear difference between the cold, warm, and hot water aquaria. Snails in hot water had a mean survival time of 6.9 days (3–18 days, 95% credible interval). In the cold water aquaria, few snails died early, and the majority survived much longer (Fig. 4). At 276 days after the start of the study, 57% of the cold water snails were still alive. The mean survival time for the cold water snails was 240 days (174–342 days; 95% credible interval). Snails in the preferred warm water survived longer than snails in hot water aquaria, with a mean survival time of 84 days (38–189 days; 95% credible interval). Several snails in the warm water survived throughout the main observation period but died shortly after the 108 day observation period. Snails in cold water had a survival length 2.9 times longer than snails in warm water and 34.5 times longer than snails in hot water. The length of survival of snails in warm water was 12.2 times longer than snails in hot water.
Physella wrighti first began laying EMs on different days in each WT. EMs were first observed in the preferred warm water 2 days after snails were introduced to the aquaria, 8 days after introduction to the hot water aquaria, and 13 days after introduction to the cold water aquaria. Snails laid a total of 348 EMs in the warm water, 39 in the cold water, and 5 in hot water (Fig. 5). Only the preferred warm water treatment resulted in snails laying EMs in all three aquaria. Snails in the cold water only produced masses in two aquaria, and those in hot water only produced masses in one aquarium. There is a clear difference in the number of masses that were laid between WTs. Snails in the warm water aquaria laid more masses than in the cold and hot water aquaria, and snails in the cold water aquaria laid more masses than in the hot water aquaria. On average, snails in the warm water aquaria laid 117 masses per aquarium (42–340 masses; 95% credible interval), whereas snails in the cold water aquaria produced 20 masses per aquarium (12–31 masses; 95% credible interval), and snails in the hot water aquarium produced 5 masses (0.88–23.81 masses; 95% credible interval).
While the number of EPM varied between masses (Fig. 6), the minimum number of eggs observed in a mass was 1, laid in warm water aquaria, and the maximum was 24, laid in a cold water aquaria. Snails in the cold water aquaria clearly laid more EPM, producing two times more EPM than snails in the warm and hot water aquaria. There was no clear difference in the number of EPM between the warm and hot water aquaria. On average, snails kept in cold water laid 11 EPM (9–13 eggs/mass; 95% credible interval), snails in the preferred warm water laid 5 EPM (3–8 eggs/mass; 95% credible interval), and snails in hot water laid 6 EPM (3–12 eggs/mass; 95% credible interval).
Most eggs laid by Physella wrighti were shaped roughly as a prolate ellipsoid. Estimated egg volume varied between eggs, with the minimum and maximum egg volumes of 0.055 and 0.521 mm3, respectively (Fig. 7), occurring in warm water aquaria. The average egg volumes were 0.16 mm3 (0.15–0.18 mm3; 95% credible interval) for cold water, 0.17 mm3 (0.14–0.20 mm3; 95% credible interval) for warm water, and 0.19 mm3 (0.14–0.27 mm3; 95% credible interval) for hot water. While the average volume of eggs produced by Physella wrighti in hot water was marginally larger than in cold and warm water, there was no clear difference in the average egg volume between temperatures (>95% certainty).
The viability of EMs ranged from 0% to 100% across temperature treatments, peaked at the preferred temperature (Fig. 8), and showed no obvious trend for the time it took an embryo to be nonviable. The average percent viability of EMs laid in cold water was less than half the viability of the preferred warm water at 31.2% (23%–40.1%; 95% credible interval) and 73.1% (52.7%–86.5%; 95% credible interval), respectively. EMs laid in hot water had greater viability than those in cold water but were still clearly lower than in warm water, with average viability of 56.7% (27.7%–81.1%; 95% credible interval) in hot water. EMs laid in warm water clearly had the greatest viability, and EMs laid in cold water clearly had the lowest (>95% certainty).
Incubation period differed between the WTs (Fig. 9). The minimum IP of 4 days occurred in hot water, whereas the maximum IP of over 45 days occurred in cold water. While IP between temperatures varied, IP within each temperature remained relatively clustered; as temperatures increased, the developmental period decreased across the three temperature treatments. On average, cold water prolonged the IP of Physella wrighti 6.8 times compared to hot water. The IP in hot water was 1.6 times shorter than in the preferred warm water. While incubation in warm water was not as fast as in hot water, IP in warm water was 4.1 times faster than in cold water. The average incubation time of embryos in cold, warm, and hot water was clearly different at 34.5 days (31.2–37.7 days; 95% credible interval), 8.4 days (6.8–10.3 days; 95% credible interval), and 5.1 days (3.3.7–7.0 days; 95% credible interval), respectively.
The ability of Physella wrighti to tolerate a range of WTs is surprising, considering the stenothermic hot water environment in which the snail is found. Thus, it is of interest to understand whether the conditions in the hot springs are required for this snail’s survival or if its isolation within the hot springs is due to other factors, such as increased fecundity or longer lifespan.
Some species of freshwater snails show a defined period of activity in response to photoperiod (Costil and Bailey 1998; Lombardo et al. 2010). Lombardo et al. (2010) examined six species of snails and found 70%–80% of snails observed were active during the day and that each species had a different time of day during which they were most active. In contrast, Costil and Bailey (1998) found no change in the activity level of Planorbarius corneus Linnaeus, 1758 (Planorbidae) at different times. Planorbarius corneus are arrhythmic, showing no activity patterns with respect to photoperiod; rather, the activity level depended on the individual snail. In both the constant and altered temperature experiments, Physella wrighti remained relatively evenly dispersed in each aquarium and did not congregate in any particular location. Snails did not appear to move within the aquaria in response to light or dark stimuli, and snails did not show any visually discernible patterns of movement. It was concluded that Physella wrighti shows arrhythmic activity, remaining equally active during dark and light periods.
A snail’s preferred WT plays a role in habitat selection (Vaidya and Nagabushanam 1978; Reynolds 1979) and can help determine a snail’s population distribution within and outside a surrounding geographical area. The preferred WT for Physella wrighti was approximately 23 °C, which is on the lower end of the commonly observed temperature range of 23 °C–40 °C (Te and Clarke 1985; Lee and Ackerman 1999; Salter 2003; COSEWIC 2008) where the snail has been found. It is plausible that Physella wrighti may only be tolerating the warmer WTs instead of requiring it for survival. The data suggest that Physella wrighti is capable of tolerating a wider range of WTs, between 5.8 °C and 39.1 °C. Further support comes from in situ observations of Physella wrighti crawling out of the water on rocks and metal hand railings in the Alpha Pool when the air temperature was below −22 °C in January 2018 and 2019 (personal observation). Clampitt (1970) found Physella gyrina and Physella integra Haldeman, 1841, can withstand a wide range of WTs, between 10 °C and 40 °C. Preferred WT may not always impact population distribution; selection could be influenced by thermal maximum/minimum, the requirement for surface air breathing (Clampitt 1970), access to food, environmental stability, or chemical composition of the water (von Oheimb et al. 2016). While Physella wrighti appears to show a preferred WT, its apparent ability to tolerate a wide range of WTs makes determining its population distribution more complex and indicates that other elements may influence its existence in the hot springs.
In response to temperature changes, organisms can show behavioural responses such as moving to find another temperature zone, entering dormancy until conditions are more favourable, or attempting to tolerate the current temperature (McClary 1964; Garrity 1984; Muñoz et al. 2005; Bae et al. 2015). The change in a snail’s behaviour with regard to WT is rather subtle. Physella wrighti exhibited mostly the same behaviours between the different temperature treatments, reproduced in all temperatures, explored all areas of the aquaria, and remained mobile in all temperatures, although snails in the cold water moved more slowly than snails in warm and hot water. In the cold and the preferred warm water aquaria, Physella wrighti did not demonstrate irritation through shell twitching, floating in the water column, or orientating their shells, as has been observed in other snails (McClary 1964; Garrity 1984; Muñoz et al. 2005; Bae et al. 2015). Snails in hot water left the water more frequently than in the warm and cold water and moved farther away from the surface of the water. This suggests that snails were experiencing irritation or stress in response to the hot water and were leaving the water to move to a more favourable location. Snails in the warm water rarely left the water, suggesting that the rare activity was exploratory.
Low oxygen levels are a common characteristic of hot spring water (Brues 1924). Snails absorb dissolved oxygen (DO) through their skin, and some species have additional respiratory structures that aid in facilitating the absorption of DO (Koopman et al. 2016). Bithynia tentaculata Linnaeus, 1758 (Bithyniidae) has a gill (Koopman et al. 2016), Planorbis planorbis Linnaeus, 1758 (Planorbidae) a lung (Koopman et al. 2016) (a modified portion of the mantle cavity (Brown 2001)), and Physa fontinalis Linnaeus, 1758 (Physidae) a lung and accessory gill (Koopman et al. 2016) (i.e., pseudobranch) (Strong et al. 2008). In water with low DO, snails that can breathe air may be able to maintain physiological functions by emersion to access atmospheric oxygen. Bae et al. (2015) and Koopman et al. (2016) demonstrated a possible link to snails emersion due to DO levels. Bae et al. (2015) noted Pomacea canaliculata Lamarch, 1822 (Ampullariidae) left the water as WT increased from 15 °C to 30 °C and noted that snails would breach the water to ventilate more often at 30 °C than 15 °C, suggesting that this was due to oxygen stress. Koopman et al. (2016) found that pulmonate snails were sensitive to hypoxia (25% saturation) and that snails surfaced more frequently to breathe compared to normoxia (100% saturation) and hyperoxia (300% saturation). Physella wrighti is a pulmonate snail (Turgeon et al. 1998) and may be sensitive to DO as shown in other species, but Koopman et al. (2016) also observed that, under normoxia in WTs between 10 °C and approximately 42 °C, pulmonate snails visited the surface more often at higher WTs indicating temperature related stress causing this behaviour. It is not known how reliant Physella wrighti is on DO versus atmospheric oxygen and how hypoxia could alter this snail’s behaviour without the effects of temperature stress. This study suggests that the behaviour is more related to temperature stress since snails did not just crawl out of the water but crawled further away from the water than snails in the other temperature treatments. Further research is required to determine any potential interactions of temperature and DO levels and how Physella wrighti would respond.
Aquatic snails seem to survive best in cooler to mid-range temperatures than at temperatures near or at their maximum survival temperature (van der Schalie et al. 1973; Vaidya and Nagabushanam 1978; El-Emam and Madsen 1982; Costil 1994). Since Physella wrighti is typically found in WTs 23.5 °C–36 °C (Te and Clarke 1985; Lee and Ackerman 1999; Salter 2003; COSEWIC 2008), it could suggest that mid to low WTs in this range would result in the greatest survival for this snail. The data indicated that Physella wrighti survives longer in cooler WTs (13 °C) than the snails are normally found in and that snails do not survive for as long in WTs (23 °C, 33 °C) present in the snail’s habitat. There is a clear relationship between the length of adult snail survival and WT; the length of survival decreases as WTs increase.
In cold conditions, ectotherms experience a reduced rate of biological functions due to the inability to maintain internal body temperature. Hubendick (1958) noted that while cold temperatures can significantly affect a snail’s life history, the effect is usually not fatal and only results in slowing a snail’s life history. This is due to reduced molecular kinetic energy slowing physiological processes (Clarke 2014). Hot temperatures stress organisms differently than cold temperatures. As temperatures become too high for an organism, it starts to experience thermal denaturation of its proteins, which may be unable to return to the functional conformation (Clarke 2014). This eventually leads to organ systems shutting down and the death of the organism. Physella wrighti snails in the cold water aquaria likely experienced a reduction in the rate of their life history, resulting in extended length of survival, whereas snails in hot water likely experienced irreparable damage to physiological systems resulting in shortened life history. Snails in the preferred warm water experienced a more optimal WT. The water was not so cold to hinder the rate of physiological processes but not so hot as to cause death.
Reproduction by snails in cold temperatures can be slowed or halted entirely. At low enough temperatures, snails have been shown to put more energy into growth than reproduction (e.g., Physella virgata (Britton and McMahon 2004)) as the rate of metabolic function is reduced to the point where organisms are simply surviving (Hubendick 1958). By exposing specimens of Physa fontinalis to three temperatures in the lab, Duncan (1959) demonstrated that in 18 °C water, Physa fontinalis produced EMs regularly. Still, EM production was reduced at 8 °C and almost ceased at 4 °C. Physella wrighti showed a similar pattern. Snails in the preferred warm water laid EMs consistently, and snails in cold water laid far fewer EMs. Further, EMs were only found in the two cold water aquaria located on the top shelf of a temperature-controlled refrigerator and had a WT of 12 °C–14 °C, whereas the third cold water tank was placed on a lower shelf and had a WT of 8 °C–11 °C. It is feasible that the lower temperature may have been too cold for snails to produce EMs but not to cause death. This indicates a possible reproductive threshold between 13 °C and 8 °C–11 °C for Physella wrighti. Further study is required at lower WTs to verify.
When WT is warm enough that snails are not expending energy solely on growth/survival, they begin to reproduce, with reproduction increasing as the biological function and temperature increase; when WT becomes too high, the reproductive output may be reduced due to thermal stress. van der Schalie et al. (1973) found EM production in Stagnicola emarginata increased as WT increased to the optimal survival temperature for the snail. Once the WT reached or exceeded the snail’s tolerance range, EM production ceased. Britton and McMahon (2004) found that when WT became too high for Physella virgata, energy was redirected from reproduction to survival. Physella wrighti seems to have produced EMs similarly; the number of EMs laid increased with WT until the tolerance range was reached, then egg laying almost stopped. Thermal stress in Physella wrighti can be further seen in a study that found snails kept in 28 °C water more regularly laid eggs above the water level, where the temperature was 25 °C (Lee and Ackerman 1999). The 28 °C water may have hindered reproduction, but because snails could find a suitable temperature, 25 °C, within their environment, reproduction still occurred. The lack of EM production at 28 °C indicates that the 33 °C water from this study may be too hot for Physella wrighti to produce EMs reliably.
The relationship between the numbers of EPM may be species-dependent among snails. McMahon (1975) studied two populations of Physella virgata, one living in artificially warmed water from heated discharge and the other living in unaltered water; snails in the warmer water produced fewer EPM than those in cooler water. van der Schalie et al. (1973) found the number of EPM for Lymnaea stagnalis increased as WT increased. Physella wrighti somewhat shows an inverse relationship between EPM and WT; the EPM decreased as temperature increased from 13 °C to 23 °C but did not change as WT increased from 23 °C to 33 °C. Snails kept in the cold water laid as many as two times more EPM than snails in the warm and hot water treatments. Snails in warm and hot water showed no difference in the EPM. As snails are approaching death, they may be laying as many eggs as physiologically possible (Britton and McMahon 2004; Emlen and Zimmer 2020). The lack of difference between warm and hot water treatments may be due to thermal stress causing snails in hot water to lay as many eggs as possible, resulting in no difference in the EPM between the warm and hot water. A greater range of temperatures would be required to more clearly demonstrate the relationship between WT and EPM.
There was no relationship found between egg volume and WT; while eggs laid in hot water were slightly larger, egg volume was not clearly different from the other temperatures. Lam and Calow (1989) studied egg volume in Radix peregra (Müller 1774) (Lymnaeidae) and found eggs produced in warmer water were significantly larger than those from one of the two unheated sites during 1 year but found no difference between all three sites the following year. They concluded that egg size was unlikely affected by temperature and that any discrepancies may have been due to other conditions or chance. The slightly larger egg volume observed for Physella wrighti may have been due to several factors, including the low number of eggs sampled.
Egg mass viability was impacted by WT, with the highest viability occurring in the preferred warm water aquaria at 73.1% and the lowest viability in the cold water aquaria at 31.2%. Lam and Calow (1989), Costil (1997), and Vaidya and Nagabhushanam (1978) examined the percentage of snails hatched from EMs with regard to temperature as a method of assessing viability. Considering the relationship between percent hatch and WT, it is apparent that the hatch rate may or may not be related to WT, depending on the snail species. For instance, Radix peregra, Planorbarius corneus, and Planorbis planorbis did not exhibit a relationship between WT and hatch rate (Lam and Calow 1989; Costil 1997). Indoplanorbis exustus Deshayes, 1833 (Bulinidae) showed an increased hatch rate with increasing WT, but when the temperature became too hot, the hatching rate decreased (Vaidya and Nagabushanam 1978). It is logical to observe a difference in EM viability with regard to temperature; if WT is too high or low, then the biological processes required for development become disrupted or altered (Hubendick 1958; Thomas and McClintock 1990; Britton and McMahon 2004). For Physella wrighti, there is a clear relationship between perecnt viability.
Based on observations from this study and that of Lee and Ackerman (1999), the IP of Physella wrighti in water 23 °C–28 °C is approximately 8 days. Examining WT and IP shows an inverse relationship between WT and IP for Physella wrighti, with the IP increasing as temperature decreases. Cold water is known to prolong the IP of snail embryos, whereas warm and hot water are known to reduce IP. The IP for Physella gyrina in room temperature water (20 °C–30 °C) is 7–8 days but drops to 5.5 days when the WT is 30 °C (DeWitt 1954). Thomas and McClintock (1990) found the same relationship for Physella cubensis Pfeiffer, 1839 where the longest IP took 31 days at 10 °C; the shortest took 9 days at 30 °C. Studies on Physella acuta Draparnaud, 1805 and Glyptophysa gibbosa Gould, 1847 (Planorbidae), Planorbarius corneus, and Planorbis planorbis show the same relationship between IP and WT (Costil 1997; Zukowski and Walker 2009). Duncan (1959) declared that, for Physa fontinalis, WT was the most crucial aspect affecting development speed. The relationship between WT and IP is due to increased temperature, causing an increased rate of biochemical processes during embryo development (Costil 1997), whereas decreased temperature reduces this rate (Hubendick 1958), thus altering Ips. It is more advantageous for most snails, Physella wrighti included, to produce eggs in warmer water than cooler water (excluding Ctmin and Ctmax) as decreased incubation time increases population numbers more quickly.
The data from this study indicate that hot water (33 °C) causes the fastest development but otherwise negatively impacts Physella wrighti’s life history. There seems to be a trade-off between cold (13 °C) water and the preferred warm (23 °C) water; snails live much longer in cold water but have the greatest reproductive capacity in warm water. This, along with the behaviours of Physella wrighti, indicates that this species can handle a wide range of WTs but is better suited to WTs lower than present in its current habitat. The currently reported WTs in which Physella wrighti exist and copulate do not appear to be specific to where the snails are found. Instead, the temperatures appear to be applied to the overall water body (e.g., Alpha Pool and Alpha Stream) (Te and Clarke 1985; Lee and Ackerman 1999; British Columbia Ministry of Environment 2014). This is relevant because water bodies are known to form thermoclines (Sundram and Rehm 1971). Thermoclines have been observed in Alpha pool, particularly during January; with the temperature differential between the incoming WT of −50 °C and an air temperature of −30 °C, there will be different thermal habitats throughout the pool (Wetzel 2001), resulting in microhabitats forming in the hot spring that may be slightly cooler in temperature. Thus, Physella wrighti may appear to reproduce in warm water when the actual WT is somewhat reduced.
The trends presented in this study have implications for the designation of Physella wrighti as a hot water specialist snail (COSEWIC 2008); they may not require hot water to survive. Physella wrighti may merely tolerate the hot WTs in response to other benefits in the LHS. Determining the presence of microhabitats may further alter our understanding of Physella wrighti and would be useful in understanding the distribution of the snail within its current environment. Due to Physella wrighti being listed as Endangered and endemic to the LHS, it is a fragile species and at risk of extinction (Fisheries and Oceans Canada 2017). Thus, studying and understanding how this snail operates within its environment is essential.
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The following describes the Markov Chain Monte Carlo (MCMC) diagnostics for the Bayesian analysis of the data presented in this paper. The MCMC diagnostics used consisted of R-hat values, effective sample sizes (ESSs), trace plots, and posterior density kernel plots. Kruschke (2015) explains that since there is a distribution of values that each parameter can occur within, trace plots (Fig. A1) can be used to verify that the predicted parameter values occur within as narrow an interval as possible (credible interval) while acknowledging the uncertainty of the true value for a given parameter. Multiple runs (known as a chain) of parameter predictions are performed for each parameter (to ensure the posterior distribution is represented properly) and are indicated by the different colours on a trace plot (e.g., see Fig. A1). Additionally, the posterior density is indicated in the plots in log form. The lack of vertical trends for each chain shows that each of the chains has converged around the same distribution and the model is said to have converged.
Kruschke (2015) further explains that the posterior density kernel plots (also known as density plots) (Fig. A2) show how often the values for a given parameter occurs for each of the chains noted above and can also be used to assess the model convergence.
Each chain is indicated by a different colour and should appear similar to the other runs (e.g., see Fig. A2). If each of the chains appears similar then the model is also said to have converged.
The Gelman–Rubin R̂ (or R-hat) statistic is used to examine the difference in the variance within and between the chains. The R-hat is also used to determine if chains have converged (Kruschke 2015). A value of one indicates that chains have converged (Kruschke 2015). All R-hat values for the analysis were equal to one.
Using the three different methods of diagnostics on chain convergence verified that were no errors made in assessing chain convergence for each statistical analysis.
The ESS is a measure of the actual sample size divided by autocorrelation in the chains of the posterior distribution (i.e., shows the number of independent values in the posterior distribution) (Kruschke 2015). Autocorrelation results from the sampling process for the posterior distribution and results in sampled posterior values that are not independent of each other (i.e., the values are correlated) (Kruschke 2015). To use this series of predictions, the result would be a biased posterior distribution; thus a biased model (Kruschke 2015). As long as the ESS is sufficiently large, there can be confidence that there is enough independent predictions to get information from the posterior distribution (Kruschke 2015). A desirable ESS is at least 1000 (Bürkner 2017). All of the ESS in this study were greater than 1000.
Figures A1 and A2 are examples of a diagnostic trace and posterior density kernel plot produced during statistical analysis for the survivorship of Physella wrighti. The remaining plots produced survivorship as well as for the other analysis performed were similar in appearance, so have not been presented here.
Data generated or analyzed during this study are available from the corresponding author upon reasonable request and has also appeared in Helmond (2020). Thermal tolerances of an endemic hot spring snail Physella wrighti Te and Clarke (Mollusca: Physidae). M.Sc. Thesis. pp. xiii + 136. Available from http://hdl.handle.net/10294/9343.
The authors declare there are no competing interests.
Funding was provided by Fisheries and Oceans Canada, the British Columbia Ministry of Environment and Climate Change Strategy, and the University of Regina.
Metrics & Citations
Erika Helmond, Kerri Finlay, Cory Sheffield, Mel Hart, and Jennifer Heron. 2023. Ascertaining the life history and thermal preferences and tolerances of the hot spring snail Physella wrighti Te and Clarke (Gastropoda: Physidae). Canadian Journal of Zoology.
101(8): 672-685. https://doi.org/10.1139/cjz-2022-0217
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