THERMAL TOLERANCES OF AN ENDEMIC HOT SPRING SNAIL

PHYSELLA WRIGHTI TE AND CLARKE (MOLLUSCA: PHYSIDAE)

A Thesis

Submitted to the Faculty of Graduate Studies and Research

In Partial Fulfillment of the Requirements

For the Degree of

Master of Science

in

Biology

University of Regina

By

Erika Kirsten Helmond

Regina, Saskatchewan

July 2020

Copyright 2020: Erika Helmond

UNIVERSITY OF REGINA

FACULTY OF GRADUATE STUDIES AND RESEARCH

SUPERVISORY AND EXAMINING COMMITTEE

Erika Kirsten Helmond, candidate for the degree of Master of Science in Biology, has presented a thesis titled, Thermal Tolerances of an Endemic Hot Spring Snail Physella wrighti Te and Clarke (Mollusca: Physidae), in an oral examination held on July 28, 2020. The following committee members have found the thesis acceptable in form and content, and that the candidate demonstrated satisfactory knowledge of the subject material.

External Examiner: *Prof. Bruce Leighton, Simon Fraser University

Co-Supervisor: *Dr. Kerri FInlay, Department of Biology

Co-Supervisor: *Dr. Cory Sheffield, Adjunct

Committee Member: *Dr. Mel Hart, Department of BIology

Committee Member: *Jennifer Heron, Adjunct

Chair of Defense: *Dr. Maria Velez, Department of Geology

*All present via Zoom ABSTRACT

Physella wrighti (Te and Clarke, 1985) is an Endangered freshwater snail endemic to the

Liard Hot Springs in northern British Columbia. It inhabits water temperatures from

23.5oC to 36oC and is active year-round. Despite its conservation status, little else is known about this species. To advance our understanding of P. wrighti in its environment, I investigated how water temperature affects aspects of its life history in a lab setting. I first investigated if P. wrighti would be more active in the scotophase versus the photophase at 30oC and observed no differences in activity level. I tested if P. wrighti had a preferred water temperature by allowing snails to explore a gradient of temperatures, and determined that the snails preferred 23oC. I reared snails in 13oC

(cold), 23oC (warm), and 33oC (hot) water to examine if water temperature would affect the snail’s period of greatest activity, behaviour, survivability, number of egg masses produced, number of eggs per mass, egg volume, egg mass viability, and incubation period. I found no differences in activity level and no difference in behaviour except snails in hot water left the water more often and crawled farther away. Snails in the hot water experienced complete mortality with an average survival of 7 days; snails in warm and cold water survived an average of 84 and 240 days, respectively. Snails in warm water produced the most egg masses, with an average 348 masses compared to 39 and 5 masses in cold and warm water, respectively. The number of eggs per mass was greatest in the cold water, with an average 11 eggs compared to 5 and 6 eggs in warm and hot water, respectively. I found no difference in egg volume between water temperatures, but mass viability was highest in warm water. The incubation period was shortest in hot water and longest in cold water. These data suggest P. wrighti grows and survives better i in water temperatures at the low end of the range observed in its current habitat and may only be tolerating the warmer water. This has implications for the ecology and conservation of this species.

ii ACKNOWLEDGEMENTS

I want to acknowledge my supervisors Dr. Kerri Finlay and Dr. Cory Sheffield, and my committee members Dr. Mel Hart and Jennifer Heron, for whom their patience, guidance, and support, through this project have been invaluable to me and helped me greatly in my professional development. I thank Dr. Mark Vanderwel for his willingness and unending patience and support in helping me to develop an understanding of statistics. I want to thank Xingzi Zhou and Ann King for their help during my project. Xingzi’s work in helping me monitor aquarium conditions and providing me with additional aquarium observations and Ann’s aid with data entry are greatly appreciated. I thank Dawn Marks and Corey McCowan for their help during fieldwork. I want to note that Dawn’s ability to keep field notes is inspirational and provided me with excellent examples of how to improve my field note skills. I also thank the Greater Vancouver Zoo and Andrea

Gieliens, Lead Biologist, for providing me with some perspective on aquarium operations. Furthermore, I want to thank the Leavitt Lab for their help during my preliminary work, as this aided me in determining where to focus my time and resources.

Likewise, I want to thank my fellow students. There is a great community of students at the University of Regina. I thoroughly enjoyed spending time with them and always appreciated our brainstorming sessions.

Finally, I would like to thank my funding partners: the University of Regina, the British

Columbia Ministry of Environment and Climate Change Strategy, the Royal

Saskatchewan Museum, the Friends of the Royal Saskatchewan Museum, and Fisheries and Ocean Canada. Without their support, none of this would have been possible.

iii DEDICATION

I dedicate this thesis to my family: Dan Helmond, Wendy Helmond, Myckala Helmond, and Trevor Helmond. I cannot thank you enough for your constant support throughout all the challenges and fun times that come with working on a project like this. All of your interest in this tiny little snail has made working on this project even more fun than I could have imagined. I always enjoy spending time discussing my project with you and cannot say enough how much I appreciate that you never got bored listening to my many stories and reflections.

iv TABLE OF CONTENTS

ABSTRACT ...... i

ACKNOWLEDGEMENTS ...... iii

DEDICATION...... iv

TABLE OF CONTENTS ...... v

LIST OF FIGURES ...... x

CHAPTER 1: INTRODUCTION TO PHYSELLA WRIGHTI AND THE LIARD

HOT SPRINGS, AND RESEARCH OBJECTIVES ...... 1

1.1 History of the Liard Hot Springs ...... 5

1.2 Physical description of Physella wrighti ...... 9

1.3 Life history of Physella wrighti ...... 13

1.4 Conservation status of Physella wrighti ...... 14

1.5 Population Threats of Physella wrighti ...... 15

1.6 Research objectives ...... 17

CHAPTER 2: EFFECTS OF WATER TEMPERATURE ON THE ACTIVITY,

BEHAVIOUR, AND SURVIVORSHIP OF PHYSELLA WRIGHTI ...... 19

2.1 Introduction...... 19

v 2.2 Experimental Methods ...... 23

2.2.1 Field Collection of Physella wrighti ...... 23

2.2.2 Feeding Physella wrighti ...... 25

2.2.3 Observed Period of Greatest Activity – Constant Temperature ...... 25

2.2.4 Determining the Temperature Preference for Physella wrighti ...... 26

2.2.5 Observed Period of Greatest Activity – Varying Temperatures ...... 30

2.2.6 Behavioural Observations of Physella wrighti ...... 31

2.2.7 Determining Survivorship of Mature Physella wrighti ...... 31

2.3 Results ...... 33

2.3.1 Observed Period of Greatest Activity – Constant Temperature ...... 33

2.3.2 Determining the Temperature Preference for Physella wrighti ...... 33

2.3.3 Observed Period of Greatest Activity – Varying Temperatures ...... 35

2.3.4 Behavioural Observations of Physella wrighti ...... 35

2.3.5 Determining Survivorship of Mature Physella wrighti ...... 37

2.4 Discussion ...... 42

vi CHAPTER 3: THE EFFECTS OF TEMPERATURE ON ASPECTS OF

REPRODUCTION OF PHYSELLA WRIGHTI ...... 51

3.1 Introduction...... 51

3.2 Experimental Methods ...... 52

3.2.1 The Number of Egg Masses Produced by Physella wrighti ...... 53

3.2.2 The Number of Eggs per Mass Produced by Physella wrighti ...... 53

3.2.3 Determining Differences in the Size of Eggs Laid by Physella wrighti.. 54

3.2.4 Viability of Eggs per Egg Mass Produced by Physella wrighti ...... 56

3.2.5 Physella wrighti Embryo Incubation Period ...... 56

3.2.6 Statistical Analysis ...... 59

3.3 Results ...... 60

3.3.1 The Number of Egg Masses Produced by Physella wrighti ...... 60

3.3.2 The Number of Eggs per Mass Produced by Physella wrighti ...... 61

3.3.3 Determining Differences in the Size of Eggs Laid by Physella wrighti.. 64

3.3.4 Viability of Eggs per Egg Mass Produced by Physella wrighti ...... 64

3.3.5 Physella wrighti Embryo Incubation Period ...... 67

vii 3.4 Discussion ...... 67

CHAPTER 4: GENERAL DISCUSSION AND CONCLUSION ...... 78

4.1 Discussion ...... 78

4.1.2 Challenges of Studying Aspects of Life History in Gastropods ...... 78

4.1.3 Current Opinions on Taxonomy and Physella wrighti as a Distinct

Species ...... 81

4.1.3 Ecological Considerations for the snail Physella wrighti ...... 83

4.1.3.1The Potential Ecological Roles of Physella wrighti in the Liard Hot

Springs Ecosystem ...... 83

4.1.3.2 Invasive Species – Potential Threats to the Population of Physella

wrighti with a note on the Potential of Physella wrighti as the invader ..... 86

4.1.4 Summary of Findings ...... 88

4.1.5 Potential Impact of Temperature on Physella wrighti Reproductive

Potential ...... 92

4.1.6 Considerations for Future Research on Physella wrighti ...... 97

4.2 Final Conclusion ...... 100

REFERENCES ...... 102

viii APPENDIX A –AQUARIUM SET-UP AND MAINTENANCE ...... 118

A.1.1 Stock Aquarium Set-Up ...... 118

A.1.2 Experimental Aquarium Set-up ...... 122

APPENDIX B: CONVERGENCE PLOTS ...... 128

APPENDIX C: UNIDENTIFIED SNAIL SPECIMENS FROM LIARD HOT

SPRINGS ALPHA STREAM ...... 132

APPENDIX D: LARVAL STAGE FOR THE HOT SPRING MITE

THERMACARUS NEVADENSIS ...... 134

ix LIST OF FIGURES

Figure 1. Live specimen of Physella wrighti taken September 2017 along Alpha Stream

in the Liard Hot Springs, Liard River Hot Springs Provincial Park, northern British

Columbia, Canada (59o25’35”N 126o06’35”W)...... 2

Figure 2. Location of Liard River Hot Springs Provincial Park noted by the arrow at

59o25’35”N 126o06’35”W ...... 3

Figure 3. Location of Liard River Hot Spring Provincial Park along the Alaska Highway

as noted by the asterisk ...... 4

Figure 4. Map of Liard Hot Springs and Campground ...... 7

Figure 5. Ventral view of a dead specimen of Physella wrighti ...... 10

Figure 6. Dorsal view of a dead specimen of Physella wrighti ...... 10

Figure 7. Rendering of a right coiled (dextral) gastropod shell ...... 12

Figure 8. Physella wrighti copulating in a warm water aquarium; mantle digits visible 12

Figure 9. Temperature Preference Trough ...... 27

Figure 10. Frequency of water temperatures chosen by Physella wrighti ...... 34

Figure 11. View of some of the male genitalia of Physella wrighti during copulation ... 36

Figure 12. Physella wrighti egg mass laid on an aquarium heater in a warm water

aquarium ...... 38 x Figure 13. Three Physella wrighti copulating...... 39

Figure 14. Three copulating Physella wrighti, with penial complexes visible ...... 39

Figure 15. Physella wrighti on aquarium lid in a hot water aquarium (33oC) ...... 40

Figure 16. Survival of field-collected Physella wrighti snails in each of the tested water

temperatures ...... 41

Figure 17. One of four eggs in an egg mass laid by Physella wrighti in a warm water

aquarium (23oC) ...... 55

Figure 18. Examples of viable embryos of Physella wrighti in egg masses laid in warm

(23oC) and cold water (13oC) aquariums ...... 57

Figure 19. Various non-developed and non-viable embryos of Physella wrighti in egg

masses laid in warm water (23oC) and cold water (13oC) aquariums ...... 58

Figure 20. The total number of recorded egg masses laid by Physella wrighti in each

water temperature...... 62

Figure 21. The number of eggs per mass produced by specimens of Physella wrighti

cultured at three different water temperatures ...... 62

Figure 22. An egg mass laid by Physella wrighti containing one egg, the minimum

number contained within an egg mass ...... 63

Figure 23. An egg mass laid by Physella wrighti containing 24 eggs, the maximum

number contained within one egg mass ...... 63 xi Figure 24. A comparison of egg volume (mm3) for eggs produced by specimens of

Physella wrighti cultured at three different water temperatures ...... 65

Figure 25. Comparison of the percent viability of egg masses laid by specimens of

Physella wrighti in three different water temperatures ...... 65

Figure 26. Comparison of the incubation period for embryos of Physella wrighti cultured

in three different water temperatures ...... 66

Figure A.1 Various setups of the stock aquariums used to keep Physella wrighti ...... 119

Figure A.2. The heated aquariums used in the hot and warm water experiments ...... 123

Figure A.3. The aquariums used for the 13oC (cold water) experiments ...... 123

Figure A.4. A sponge filter used for aeration in one of the experimental aquariums .... 124

Figure A.5. One of the microscope slides present within an experimental tank ...... 126

Figure A.6. The Plastic aquarium covers used on the experimental aquariums ...... 126

Figure B.1. Markov chain Monte Carlo diagnostic trace plot for the effect of hot water

produced during the assessment of the difference in the length of survival for wild

snails with regards to temperature ...... 129

Figure B.2. MCMC diagnostic posterior density kernel plot for the effect of hot water

produced during the assessment of the difference in the length of survival for wild

snails with regards to temperature ...... 129

xii Figure C.1. Several Specimens of the unidentified Snail in Alpha Stream, Liard Hot

Springs, Liard River Hot Spring Provincial Park ...... 132

Figure C.2. One of the unidentified snails from Liard Hot Springs, Liard River Hot

Spring Provincial Park ...... 132

Figure C.3. Various angles of the unidentified snail from Liard Hot Springs, Liard River

Hot Spring Provincial Park ...... 133

Figure D.1. One specimen of presumed Thermacarus nevadensis during its larval stage

present under the shell of its presumed host Physella wrighti ...... 134

Figure D.2. The inferred larval stage for Thermacarus nevadensis, indicated by the

arrows, present under the shell of its presumed host Physella wrighti ...... 134

Figure D.3. The inferred larval stage for Thermacarus nevadensis present under the shell

of its presumed host Physella wrighti ...... 135

Figure D.4. The larval stage for Thermacarus nevadensis present on presumed host

Physella wrighti ...... 135

Figure D.5. The larval stage for Thermacarus nevadensis present on the presumed host

Physella wrighti ...... 136

Figure D.6. The larval stage for Thermacarus nevadensis present on its presumed host

Physella wrighti ...... 136

xiii CHAPTER 1: INTRODUCTION TO PHYSELLA WRIGHTI AND THE LIARD

HOT SPRINGS, AND RESEARCH OBJECTIVES

The earth is an amazingly biodiverse planet, home to over 1.8 million known species

(Roskov et al., 2019), with new species discovered regularly. At present, many species are at risk of extinction. In Canada, the Species at Risk Act (S.C. 2002 c.29) defines a

Threatened species as one that will likely become Endangered if there are no actions taken to mitigate the reasons leading to the species’ extirpation/extinction, and an

Endangered species as a species for which extirpation or extinction is imminent. For species with these listings, it is important to understand their function within their ecosystem to provide us with the best chance at being able to prevent their extinction. In one way extinction is a natural phenomenon of evolution. For example, extinction can occur due to natural disasters or through natural selection; populations that do not evolve to survive in a changing environment cannot be expected to exist within that environment. In a different way, extinction can be driven by human influences that result in the loss of a species to unnatural factors, e.g. poaching or poor management practices.

It is a primary concern to prevent unnatural extinctions; thus, taking the time to understand what is driving a species towards extinction and the impact of its loss is very important. However, it is challenging to achieve such goals without first understanding the organism’s life history. Once the life history is established, we can prioritize what actions should be taken to protect a species.

Physella wrighti (Physidae) (Figure 1) is a freshwater pulmonate snail found only in the

Liard Hot Springs in the Liard River Hot Springs Provincial Park (59o25’35”N

1

Figure 1. Live specimen of Physella wrighti taken September 2017 along Alpha Stream in the Liard Hot

Springs, Liard River Hot Springs Provincial Park, northern British Columbia, Canada (59o25’35”N

126o06’35”W). Photo credit: Erika Helmond.

2

Figure 2. Location of Liard River Hot Springs Provincial Park noted by the arrow at 59o25’35”N 126o06’35”W. Image from COSEWIC (2008b).

3

Figure 3. Location of Liard River Hot Spring Provincial Park along the Alaska Highway as noted by the asterisk. Image modified from Peepre et al. (1990).

4 126o06’35”W) in northern British Columbia, Canada (Figures 2, 3) (Te and Clarke, 1985;

Salter, 2001; COSEWIC, 2008). The first specimens of P. wrighti were originally collected between 1972 and 1973 (Clarke, 1974; Te and Clarke, 1985), but were not recognized as a distinct species until 1978 when Te (1978) discussed the snail (termed

OTU-82) as possibly being an unclassified species of snail. Later, Te and Clarke (1985) officially named and described the snail as Physella wrighti. The Committee on the

Status of Endangered Wildlife in Canada (COSEWIC) first assessed P. wrighti in 1998, resulting in the species receiving the status of endangered (COSEWIC, 2008b). This status was re-examined and confirmed in May 2000 and April 2008 (COSEWIC, 2008b).

Physella wrighti was listed on Schedule 1 of the federal Species at Risk Act (SARA)

(S.C. 2002 c.29) in 2003. Although P. wrighti has been sought out at other hot springs, including Grayling Springs (59o37’N 125o32’W and 59o37’N 125o38’W), Deer River Hot

Springs (59o30’15.0”N 125o57’24.1”W), Toad River Springs (58o55’26.4”N

125o05’40.2”W), and Coal River Hot Springs (60o09’N 127o09’W), it has not been found

(Te and Clarke, 1985; Salter, 2003; Heron, 2017, personal communication). Currently, there is a paucity of information on P. wrighti’s life history; by acquiring more information on its biology, we can determine how to manage its habitat, mitigate threats, and identify other locations that may act as habitat for these snails.

1.1 History of the Liard Hot Springs

Hot Springs are generally defined as having water that discharges from a ground source at ≥5oC above the average annual air temperature for the locality (White, 1975). The

Liard Hot Springs (LHS) is located in northern British Columbia at mile 496 of the

5 Alaska Highway (Figures 2, 3). The hot springs are thought to have been in use since before 1835 by First Nations present in the area, including the Beaver, Cree, Saulteau,

Slavey, and TseK’hene cultures from the Blueberry River First Nation, Doig River First

Nation, Fort Nelson First Nation, Halfway River First Nation, McLeod Lake Indian

Band, Prophet River First Nation, Saulteau First Nations, and West Moberly First

Nations (British Columbia Ministry of Environment, 2009); however, the earliest documented use is from 1835 by people exploring the area (Camsell, 1956). The hot springs were a popular rest and bathing spot for construction workers during the development of the Alaska Highway (Peepre et al., 1990). It is now visited by tourists from around the world, with over 135,000 visitors recorded in some years (Nicole

Tattam, 2020, personal communication). The hot springs were listed as a Class A provincial park in April of 1957 (Peepre et al., 1990), to ensure the land within the park would be preserved in as natural a state as possible and still be enjoyed by visitors (B.C.

Parks, 2020a).

The hot springs complex consist of Alpha Pool, Alpha Stream, the hot water swamp that

Alpha stream empties into, Beta Pool and its outlet stream, and the Delta/Epsilon/

Gamma hot water swamp complex (Figure 4). The park has additional unique features including the Hanging Gardens, composed of tufa deposits (a porous rock formed by the precipitation of calcium carbonate (Ford, 1989)), and a couple of smaller, cool water creeks that empty into the hot water pools (Peepre et al., 1990; B.C. Parks, 2020b).

Workers built the first boardwalk to the Alpha and Beta Pools in 1942; it is likely that

Alpha Pool was dammed at this time to deepen it (Peepre et al., 1990; B.C. Parks,

6

Figure 4. Map of Liard Hot Springs and Campground. The map shows the Alpha and Beta Pools,

Delta/Epsilon/Gamma hot water swamp complex, and Alpha Stream. Image adapted from B.C. Parks

(2010).

7 2020b). Subsequently, stairways were added to both Alpha and Beta Pools for easier access, and change rooms and compost toilets were built (dates unknown) (Peepre et al.,

1990; COSEWIC, 2008b). There have been renovations to the hot springs infrastructure, due to the degradation of the pre-existing structure/facilities, that replaced the boardwalk to Alpha Pool, as well as upgraded the change rooms, deck, and stairs at Alpha Pool that took place in 2012 (Formline Architecture, 2012). In addition to these upgrades, the boardwalk to Beta Pool and the structures around Beta Pool have been removed, with the intent that the pool is renaturalized (work completed 2011) (Jennifer Heron, 2020, personal communication), and the existing composting toilets at Alpha Pool were renovated in 2014 (B.C. Parks Facilities Information, 2020, personal communication). A weir was built prior to 1973 to separate Alpha Pool into the warmer upper pool (average water temperature 40oC) and the cooler lower pool (average water temperatures 26-38oC created via diversion of cool water stream into the pool) (Peepre et al., 1990; DFO,

2010). Despite the potential impacts from development, the LHS remains relatively unaltered (Peepre et al., 1990), and P. wrighti is still present throughout the Alpha Pool and Stream, Beta Pool and Stream, and Delta/Epsilon/Gamma (personal observation).

The chemical composition of the water in Liard is predominantly calcite-sulphite

(COSEWIC, 2008b), which differs from other hot springs in western Canada, namely

Fairmont Hot Springs (50o19’41.0”N 115o50’35.1”W) and Radium Hot Springs

(50o38’06.0”N 116o02’18.8”W), which have a calcite water signature. The LHS is also

- - characterized as having a pH of 6.5-7.5, alkalinity (HCO3 ) of 180 mg/L, Nitrate (NO3 ) level of < 1 mg/L, and Calcium (Ca2+) of 210-226 mg/L (Te and Clarke, 1985; Lee and

Ackerman, 1999; Grasby et al., 2000). The water signature of the LHS is thought to be 8 from meteoric water (i.e. from precipitation) flowing to a depth of around 3.4 km below the crust where the water temperature reaches 120oC; these are the only conditions that would account for this geochemical signature (GW Solutions, 2010). The exact source location and flow path the source water takes are currently unknown (GW Solutions,

2010).

Adding to the uniqueness of the LHS is the high volume flow rate of Alpha Stream from

Alpha Pool, which measures 81 litres per second (Peepre et al., 1990; COSEWIC,

2008b), compared to Radium Hot Springs at 28 litres per second and Banff Hot Springs

(51o09’03.4”N 115o33’38.5”W) at 8.3-15 litres per second (Parks Canada, 2018). The

LHS are considered the second-largest hot springs in Canada, with an overall flow rate of about 120-135 litres per second; the largest spring is the Grayling River Hot Springs with a flow rate of 144 litres per second (St. Pierre, 1980, as cited in Peepre et al., 1990).

1.2 Physical description of Physella wrighti

Physella wrighti belongs to the family Physidae, subfamily Physinae, genus Physella, subgenus Physella (Turgeon et al., 1998). It has a sinistrally coiled shell (i.e. coils to the left) with no operculum (i.e. the plate that closes over the aperture) as is typical for physids (Figure 5) (Clarke, 1981). The official name and description of P. wrighti come from Te and Clarke (1985) who described P. wrighti as having a somewhat shiny shell of a light horn/yellowish colour (Figures 5, 6). The shell is narrowly elongate-ovate with an average length of 5.5 mm, 3.5-4 whorls in adult specimens (Figures 5, 6). The penultimate whorl may be exerted; and, in some specimens, the pre-penultimate whorl may also be exerted (Figures 5, 6). Physella wrighti’s protoconch (i.e. the smallest whorl 9

Figure 5. Ventral view of a dead specimen of Physella wrighti. The shell is sinistrally Figure 6. Dorsal view of a dead specimen of Physella wrighti. The shell is sinistrally coiled and horn/yellow in colour. This specimen’s shell length measured approximately coiled and horn/yellow in colour. This specimen’s shell length measured approximately

5.93 mm from the top of the spire to the end of the body whorl. A) body whorl; B) 6.43 mm from the top of the spire to the end of the body whorl. A) body whorl; B) penultimate whorl; C) pre-penultimate whorl; D) protoconch; E) Spire; F) sutures; G) penultimate whorl; C) pre-penultimate whorl; D) protoconch; E) Spire; F) sutures. Photo columellar fold; H) aperture. Photo Credit: Erika Helmond. credit: Erika Helmond. 10 of the shell) is rounded, conical, and varies slightly in colour from light to slightly darker horn/yellow. The spire shape is long and acute with deep to very deep sutures (i.e. grooves between whorls) in most individuals; the shell does not show any thickenings from periods of growth-rest, and has few to no spiral striations (slight grooves that follow the angle of the whorls (Getz et al., 2017)) (Figures 5-7). The main whorl/body whorl may be minimally shouldered in some individuals (i.e. whorls may be slightly more angular (Arnold, 1965)). Te and Clarke (1985) describe the aperture as acute above and narrow but round at the base, with the lateral portion being somewhat flat (Figure 6). The columella fold (i.e. the outer facing layer of the central axis (columella) of the whorls

(Arnold, 1965)) varies in thickness between specimens, with some individuals having only a slightly thickened fold, whereas others possess a definitively thickened fold

(Figure 6). Typically, the columella fold is oblique and not twisted. Physella wrighti usually does not have a palatal callus (i.e. a thickening of the outer edge of the aperture that is furthest from the columella (Perez and Cordeiro, 2008)) but can have a whitish wash although no thickening can be observed. Most individuals of P. wrighti have a parietal callus (a thickening of the shell on the inner lip of the aperture (Arnold, 1965)) that is obviously layered near the columellar fold, where it also broadens from being rather narrow (Figures 6, 7).

The body of P. wrighti is dark grey-black with a black mantle that is mostly devoid of any circular patterning (Figures 1, 8). Physella wrighti generally has six, pointed mantle digits only on the columellar side (Figure 8). The digestive tract mostly lacks pigmentation and is laterally lobed, while the renal complex is curved, and can be entirely

11

Figure 8. Physella wrighti copulating in a warm water aquarium;

mantle digits visible. Mantle digits are visible on the right side of the

Figure 7. Rendering of a right coiled (dextral) gastropod shell. Labels top (acting male) specimen. Photo credit: Erika Helmond. a) and b) note the types of striations that can occur on gastropod shells. a) transverse/longitudinal striae; b) spiral striae. Image adapted from

Getz et al. (2017).

12 tubular or lamellar internally. The bursa copulatrix (i.e. a storage sac for sperm in the female reproductive system (Roth, 1960)) of P. wrighti is small, rectangular, positioned horizontally, and has a duct attached at the side, whereas the penis is described as a round, loose-packed preputial gland that can range in size from small to medium. The gland has a two-part penial sheath, one part being glandular and one non-glandular, with the transition between the sections not very constricted. The glandular portion is closer to the preputium, short, and somewhat swollen in appearance; the non-glandular is a transparent, flaccid membrane that contains the tube-like penis. The penial sheath is approximately 1.5 times the length of the preputium. A more detailed description can be found in Te and Clarke (1985).

1.3 Life history of Physella wrighti

The life history, diet, and habits of P. wrighti are not well described. In the LHS, P. wrighti can generally be found living in water temperatures ranging from 23.5-36oC (Te and Clarke, 1985; Lee and Ackerman, 1999; Salter, 2003; COSEWIC, 2008) but can be found in water up to 40oC (personal observation; COSEWIC 2008), and crawling out of the water onto various materials in January when the air temperature was between -30oC and -40oC. Within these water temperatures, P. wrighti can be seen crawling through mats of the algae Chara vulgaris, leaves off of Betula papyrifera (Paper birch) trees, sediments, wood, and grasses at the edge of the water (personal observation; Te and

Clarke, 1985; Lee and Ackerman, 1999; Salter 2001). Considering the variety of substrates on which P. wrighti can be found, it is thought that the snail grazes on algal and microbial organisms (Lee and Ackermn, 1999; Laurzier et al., 2011), not unlike other

13 physid snails (Brown, 2001; Dillon, 2004); however, there have been no studies on the diet of P. wrighti.

Physella wrighti is hermaphroditic, possessing both male and female reproductive organs

Te and Clarke, 1985), and lays eggs in clear, crescent-shaped jelly masses, similar to those commonly observed in most physids (Lee and Ackerman, 1999). Egg masses contain 6-18 eggs, with the average size of a freshly laid egg approximately 0.135 mm, and an approximate incubation period of nine days (Lee and Ackerman, 1999).

Hatchlings, with an average shell length of 0.69 mm, are visible to the naked eye (Lee and Ackerman, 1999).

1.4 Conservation status of Physella wrighti

The conservation status of P. wrighti is assessed at the provincial, national, and global levels. At the provincial level, P. wrighti is listed as S1 (critically endangered) (B.C.

Conservation Data Center, 2008) and at the global level, is listed as G1 (critically imperilled) (NatureServe Explorer, 2020). The national, legal status of P. wrighti in

Canada as recommended by COSEWIC, and listed under SARA, is Endangered under

Schedule 1 of the Species at Risk Act (COSEWIC, 2008b). Physella wrighti was assessed as Endangered by COSEWIC due to its extremely limited range (i.e. endemic to the

LHS), potentially limited population (estimated at fewer than 10,000 snails), potentially large population fluctuations (at least one order of magnitude), potentially short life span, and the belief that it is a hot water specialist (Lee and Ackerman, 1999; COSEWIC,

2008b).

14 1.5 Population Threats of Physella wrighti

Humans pose different threats to P. wrighti, such as habitat alteration for recreational activities (DFO, 2010; Laurzier et al., 2011). As noted earlier, there have been many renovations to the hot springs, many of which occurred before the discovery of P. wrighti; thus, it cannot be determined what impact the renovations (e.g. the installation of the Alpha Pool dam) may have had on the population (DFO, 2010). Since we have become aware of P. wrighti, renovations to the surrounding structures have been undertaken with great care to minimize impact (e.g. Elevate Environmental Inc., 2012).

While maintenance of the present structures can help prevent destructive renovation activities (DFO, 2010; Laurzier et al., 2011), there could be future renovations to the pools due to address accessibility concerns (Trumpener, 2019), and deal with unavoidable structural deterioration.

The presence of harmful chemicals in the hot springs poses a threat to the population of

P. wrighti (Peepre et al., 1990; DFO, 2010; Fisheries and Oceans Canada, 2017).

Chemicals used for personal care (e.g. cosmetics and insect repellants) could have a negative impact on P. wrighti’s life history and biological functions (DFO, 2010;

Laurzier et al., 2011), considering that such products are known to have deleterious effects on other invertebrates (Schmitt et al., 2008; Neuparth et al., 2014; Bal et al.,

2016; Gibson et al., 2016; Campos et al., 2017). There has been an increase in the number of visitors to the hot springs over time. Peepre et al. (1990) stated an average of

40 000 visitors, Lee and Ackerman (1999) suggested an average of 100 000 visitors, and the average count from 2015 to 2018 was just over 132, 000 visitors (Nicole Tattam,

15 2020, personal communication). If visitor numbers continue to grow, there will be an increased chance of chemicals impacting the snail population.

Mortality from visitors trampling snails also poses a threat to P. wrighti (DFO, 2010).

Visitors have been known to explore the areas surrounding the hot springs (COSEWIC,

2008b), and the continuation of such actions remains a concern (DFO, 2010). It appears that, despite signage requesting visitors to stay on the constructed paths, there is still exploration of the edges of Alpha Stream and Pool (personal observations, September

2017). While trampling of snails does not appear as a primary concern (Fisheries and

Oceans Canada, 2017), I suggest that the risk not be discounted since trampling could cause a reduction in the snail’s population. A More notable concern is the trampling and destruction of habitat from visitor exploration since this can result in the loss of habitat for that is already extremely limited (Fisheries and Ocean Canada, 2017).

Another threat to P. wrighti is the potential for the dam and/or weir in Alpha Pool to fail structurally (DFO, 2010, Fisheries and Oceans Canada, 2017). While the consequences of this happening are unknown, it could alter the flow of water into the Alpha Stream, creating a loss of habitat in Alpha Pool (DFO, 2010; Fisheries and Oceans Canada,

2017), and desiccation of snails in Alpha Pool (Fisheries and Oceans Canada, 2017).

There is a high amount of drilling for oil and gas that occurs in northern B. C. (Heron,

2007; DFO, 2010; GW Solutions, 2010). Drilling is considered to be a threat to P. wrighti due to possible interruption of the path that the source water takes to get to the

LHS. Since the exact path the water takes remains unknown, companies could easily

16 drill in a crucial location, causing the water to be diverted from the hot springs (DFO,

2010; GW Solutions, 2010). If this were to happen, there could be a loss of the warm water or the hot springs could completely dry up, eliminating any chance of survival by

P. wrighti (COSEWIC, 2008b; GW Solutions, 2010).

1.6 Research objectives

There is limited information available regarding the life history of P. wrighti. Thus, it is necessary to establish as much about its life history as possible to anticipate and prevent incidents that might threaten this endangered snail species. To build knowledge of P. wrighti, I investigated several aspects of its life history and how these might be affected by its habitat. I first examined if there was a difference in the activity level of P. wrighti during the photophase (light period) or scotophase (dark period), then investigated if P. wrighti had a preferred water temperature. Using that preferred water temperature, I established a series of aquariums at a range of three water temperatures, 13oC (cold),

23oC (warm), and 33oC (hot), to see if the range of water temperatures had a measurable difference in various aspects of P. wrighti’s life history. Using these water temperatures,

I assessed the response of P. wrighti’s period of activity and behaviour to determine if the snails showed any stress responses to different water temperatures. I examined how water temperature altered this snail’s survivability, the number of egg masses it produced, the number of eggs per egg mass, the egg volume, and the viability of egg masses.

As one of the only four documented species of snails for which live specimens were recorded from in the LHS (Salter, 2001; Salter 2003), P. wrighti could provide valuable services to its unique ecosystem as has been suggested for the Banff Springs Snail,

17 Physella johnsoni (Physidae) (also referred to as Physa johnsoni) (Lepitzki, 2002).

Snails, in general, provide ecosystem services by breaking down and recycling waste from flora and fauna, and maintaining microbial balance (Brown, 2001). Thus, by working towards being able to protect P. wrighti, we are also working towards preventing the destruction of an incredible ecosystem.

18 CHAPTER 2: EFFECTS OF WATER TEMPERATURE ON THE ACTIVITY,

BEHAVIOUR, AND SURVIVORSHIP OF PHYSELLA WRIGHTI

2.1 Introduction

Ectotherms are organisms that are reliant on the temperature of their surroundings to modulate their body temperature as they are incapable of internal thermoregulation

(Reynolds, 1979). As such, ectotherms must move towards or away from a heat source to reach a suitable temperature to carry out their life cycle (Reynolds, 1979; Kavaliers,

1980). Depending on a species’ life history requirements, the ideal temperature conditions can be defined as the range of temperatures in which individuals tend to congregate or spend most of their time (Vaidya and Nagabhushanam, 1978; Reynolds and Casterlin, 1979). Increases and decreases in temperature norms in a species’ habitat can alter an individual’s internal chemical and physical processes (Hubendick, 1958;

Reynolds, 1979), which can result in an altered life history (van der Schalie et al., 1973).

Snails (Mollusca, Gastropoda) are ectotherms that depend on the temperature of the surrounding environment to thermoregulate (Vaidya and Nagabhushanam, 1978).

Aquatic snails must, therefore, rely on water temperature to maintain body function. At present, there is little information available on the ability of snails to tolerate different temperatures and the impacts of varying temperatures on survival. The available information is widely distributed between different clades (Russell-Hunter, 1961a,

1961b; McMahon, 1975; Muñoz et al., 2005; Seuffert et al., 2010), making it challenging to find comparative information on closely related snail taxa. Thus, current information is only generally comparative between species and not representative between all species. 19 Temperature preference has been observed to affect habitat selection of some species of snails (Vaidya and Nagabhushanam, 1978; Reynolds 1979), whereas other species tolerate and move through such a wide range in temperatures that the preferred temperature does not seem to impact their distribution within an environment (Clampitt,

1970). Alternatively, a snail species’ distribution could be affected by aspects such as its minimum or maximum thermal tolerance, the requirement for surface breathing

(Clampitt, 1970), access to food, environmental stability, or the chemical composition of the water (von Oheimb et al., 2016). Thus, knowing a snail’s preferred temperature or temperature range can provide important information for its distribution.

Before determining an aquatic snail’s preferred water temperature range, or even how it responds to different temperatures, it is desirable to determine its diel activity patterns to ensure observations and experiments are made at the appropriate time. For example,

Heiler et al. (2008) found Pomacea canaliculata (Ampullariidae) was most active in the dark. As such, it may not be appropriate to make observations or perform experiments during daylight hours. It is not uncommon to observe diel activity patterns in snails

(Heiler et al., 2008; Lombardo et al., 2010); thus, it is important first to understand a snail’s activity patterns to ensure data is collected at an appropriate time.

Snails can exhibit a variety of behaviours in response to stress from hot and cold; Muñoz et al. (2005) noted that, when faced with extreme conditions, an animal’s response can be

“fight or flight” depending on its mobility. For snails, this could mean either leaving the area in search of more suitable conditions, or staying in the area and tolerating the temperature for as long as possible. For example, Littorina aspera (Littorinidae) (now

20 known as Echinolittorina aspera), Littorina modesta (now known as Echinolittorina modesta), and Echinolittorina peruviana have been observed tolerating extreme heat from the mid-day sun by pointing the apex of the shell towards the sun to minimize exposure of the shell to solar heat (Garrity, 1984; Muñoz et al., 2005). Alternatively, the apple snail, Pomacea canaliculata (Ampullariidae), exhibits temperature-dependent behaviours, digging in the substrate only at cooler temperatures, and swimming in the water column at mid-range temperatures (Bae et al., 2015). McClary (1964) observed specimens of Pomacea paludosa twitching their shells in response to increased temperature. Thus, the wide range of behaviours must be considered when examining a snail’s preferred temperature, and how they respond to temperature fluctuations.

In addition to behaviour, survival/mortality is a crucial consequence of temperature on snail life history. Some species survive better at colder than warmer temperatures (e.g.,

Lymnaea emarginata (Lymnaeidae) (now known as Ladislavella emarginata) (van der

Schalie et al., 1973)), whereas other species show the opposite trend (e.g. Indoplanorbis exustus (Bulinidae) (Vaidya and Nagabhushanam, 1978)); regardless, the optimal survival is almost always somewhere between the tested extremes (El-Emam and

Madsen, 1982; Costil, 1994). Thus, it is necessary to expose snails to a range of water temperatures to determine the temperature at which survival is optimal.

The hot spring snail Physella wrighti is an Endangered snail endemic to the extreme environment of the Liard Hot Springs (LHS) (COSEWIC, 2008b) and typically found in ambient water temperatures ranging from 23.5-36oC (Te and Clarke, 1985; Lee and

Ackerman, 1999; Salter, 2003; COSEWIC, 2008). Only one previous captive study on P.

21 wrighti has been published (Lee and Ackerman, 1999), which addressed some aspects of

P. wrighti’s life history, including the average number of eggs laid per egg mass; however, the study did not investigate P. wrighti’s temperature preferences or tolerances, changes in behaviour, or survivorship in response to different water temperatures.

While P. wrighti seems to survive well in its hot spring environment, it is unknown if P. wrighti is present in the hot springs because it prefers warmer water and is incapable of surviving in cooler conditions, or if this species is only tolerating its environment for other reasons (e.g. access to food). Additionally, we do not know how P. wrighti responds to temperatures outside the ranges of its natural habitat. For instance, do water temperatures outside its normal range alter its behaviour and/or survivability?

In this chapter, I provide insight into P. wrighti’s thermal tolerances and provide additional comparative information on its life history in response to different water temperatures. To achieve this, I observed P. wrighti in aquariums maintained at 30oC for a 24 hour period to determine if the snails have an observable period of greatest activity in response to photophase and scotophase. Next, I determined if P. wrighti has a preferred temperature range by creating a temperature gradient using the modified methods of Diaz et al. (2006, 2011), and a modified version of methods for determining acute temperature preference from Reiser et al. (2014) and Reynolds and Casterlin

(1979). I assessed different aspects of P. wrighti’s life history, including its observed period of greatest activity during the photophase and scotophase, behaviour, and survivability in different water temperatures. By assessing said aspects, I provide insight

22 into whether P. wrighti is truly physiologically restricted to its hot spring habitat or if it is only tolerating this habitat.

2.2 Experimental Methods

2.2.1 Field Collection of Physella wrighti

Live specimens of P. wrighti used in these experiments were collected from the Liard

Hot Springs, Liard River Hot Springs Provincial Park, British Columbia, Canada in

September 2017, January 2018, and January 2019 to ensure there were relatively freshly collected snails available for experimentation and observation.

Upon locating a group of snails, we used two 1 m sticks to create a 1 m2 plot from which to sample, and performed an abundance count of the snails present in the plot. The number of snails collected at each plot never exceeded 10% of the total number of snails in the plot. We collected snails from the Alpha stream, Alpha Pool, and the

Epsilon/Delta/Gamma complex (Figure 4). We also collected snails from the Beta pool/stream complex (Figure 4); however, Beta pool/stream had a lower abundance of snails with only two locations containing snails. We gently removed snails from the collection site by hand and placed them in one-litre plastic transport containers. Before collecting the snails, we rinsed containers three times with water downstream of our collection site to rid the plastic of inorganic residue. Containers were then filled half to three-quarters full with water from the collection site. To prevent overcrowding snails, only 100 snails were placed within each collection container.

We packed samples into coolers and travelled with them to the facilities at the Royal 23 Saskatchewan Museum, Regina, Saskatchewan. The total time of transport, from collection to destination, was a maximum of 48 hours.

The snails used in the experiment Observed Period of Greatest Activity - Constant

Temperature were collected from Alpha Pool, Alpha Stream, Beta Pool, and the

Epsilon/Delta/Gamma complex in September 2017, three months before observations.

Snails used in the Determining Temperature Preference for Physella wrighti experiment were collected from Alpha Pool in January 2018, four months before observations. In both cases, the duration of time between snail collection and the timing of experimentation was greater than I desired; however, the snail’s remote habitat, the costs and effort required to collect specimens, as well as the timing in which the experiments were prepared made this unavoidable. To mitigate differences between the field and lab conditions, I kept the snails under conditions as similar as possible to their natural habitat. The snails I used for the experiments Observed Period of Greatest

Activity - Varying Temperatures, Behavioural Observations of Physella wrighti, and

Determining Survivorship of Mature Physella wrighti were collected from Alpha Pool in January 2019, 10 days before experimentation. I had no size preference for which snails were collected, opting instead for the availability of snails.

Once specimens Regina, I acclimated snails to a series of 10 US gallon aquariums with the water temperatures set to 30 ± 2oC (for the snails used in the experiments Observed

Period of Greatest Activity - Constant Temperature and Determining the

Temperature Preference for Physella wrighti) and 23 ± 2oC (for the snails used in the

Observed Period of Greatest Activity - Varying Temperatures, Behavioural

24 Observations of Physella wrighti, and Determining Survivorship of Mature Physella wrighti experiments) (See Appendix A for a detailed stock aquarium setup and maintenance). I used 30oC initially as this corresponded to the middle of the range of inhabited temperatures in the field noted by Lee and Ackerman (1999), whereas 23oC corresponded to the preferred temperature that I later determined so I adjusted the stock tank water temperatures accordingly (see Determining the Temperature Preference for

Physella wrighti). I acclimated all snails to the stock aquariums by floating the transport containers, with the snails and the transported water, in the stock aquariums for at least two hours before releasing snails into the aquariums.

2.2.2 Feeding Physella wrighti

I fed captive snails using Hagen® Nutrafin® Max– spirulina meal tablets in ¼ portions of tablets for smaller populations and ½ to full tablets for larger populations. I provided snails in the Observed Period of Greatest Activity - Varying Temperatures and

Determining the Temperature Preference for Physella wrighti, Behavioural

Observations of Physella wrighti, and Determining Survivorship of Mature Physella wrighti experiments with Omega One® Shrimp and Lobster Pellets, to provide snails with additional calcium to aid in maintaining shell strength. I fed snails once per week; however, if I saw that the food disappeared abnormally fast, I increased the feeding as I deemed necessary.

2.2.3 Observed Period of Greatest Activity – Constant Temperature

To determine if Physella wrighti has a period of greater activity in response to

25 photophase and scotophase, I made qualitative observations of the snails in their stock tanks (see Appendix A for detailed aquarium set up and maintenance) during three periods of observation, occurring from 01:00 to 07:00, 08:00 to 18:00, and 19:00 to 00:00 at one-hour intervals. The period 07:00 to 18:59 was the photophase and 19:00 to 06:59 was the scotophase. Since snails were too small to view under red light conditions, I turned the lights on in the room for no more time than necessary to make observations

(<5 minutes/observational period). The aquarium temperatures were maintained at 30oC

± 2oC. My objective for this experiment was to determine if P. wrighti showed any obvious patterns of location within the aquariums, remained solitary or congregated, and if snails consistently active in the aquariums or appeared quiescent.

2.2.4 Determining the Temperature Preference for Physella wrighti

To determine the preferred temperature, I placed snails in a glass trough measuring 91.4 cm by 20.3 cm by 15.2 cm and filled with 8.9 cm of water, mixed from each of the eight stock tanks (see Appendix A for information on the stock tanks), with an established temperature gradient created by heating one end of the trough and cooling the other

(similar to the methods noted in Diaz et al., 2006, 2011). To maintain the temperature gradient, I divided the trough into six sections using 2.5 cm thick by 7.6 cm tall, rigid polystyrene insulation (I cut the top edge into a trapezoid shape to reduce sharp edges) placed at 15.2 cm intervals such that 1.3 cm of foam extended on either side of the 15.2 cm mark (Figure 9). These foam sections helped to maintain different temperature zones by reducing the flow of hot water along the length of the trough. To generate the temperature gradients, I heated one end of the trough to approximately 45oC using an

26

Figure 9. Temperature Preference Trough. I placed five snails in the trough and left them for two hours before measuring the temperature of the water at each snail’s location. The trough measured 91.4 cm x 20.3 cm x 15.2 cm and was filled with 8.9 cm of water. I divided the trough into six sections using 2.5 cm, 7.6 cm tall, rigid polystyrene insulation (top edge cut into a trapezoid shape to reduce sharp edges) at 15.2 cm intervals such that 1.2 cm of foam extends on either side of the six-inch mark, to create a greater temperature gradient across the trough. I added air stones to each section to reduce the formation of thermoclines in the water. I heated one end to approximately 45oC using an immersion heater and reptile heating lamp with a

120W incandescent bulb, set on half power, and cooled the other end to approximately 5oC by placing two ice packs in section 6 and one ice pack in section 5. A) Polystyrene foam dividers;

B) Air Stones; C) Aquarium Air Tubing; D) Immersion Heater; E) Ice Packs; F) Air Flow Regulator; G) Plastic Air Hose Splitter; H) Water Level; I) Reptile Heat Lamp.

27 immersion heater and a reptile heating lamp with a 120W incandescent bulb, set to half power, and cooled the other end to approximately 5oC by placing two ice packs in the last section and one in the second last section (Figure 9). I chose this temperature range as this included the approximate temperatures in which P. wrighti can be found within in the

LHS (Te and Clarke, 1985; Lee and Ackerman, 1999; Salter, 2003; COSEWIC, 2008) plus offered the snails some temperatures they may not normally be exposed to. I replaced the ice packs when they were approximately 80% thawed and staggered replacement. As Diaz et al. (2006, 2011) initially reported isotherms in their temperature gradients, I added a small aquarium air stone (Grreat Choice®) to each section, plus one additional air stone to the heated section, and gently bubbled air through each section using a Top Fin® AIR-8000 air pump to help reduce the magnitude of the isotherms between the top and bottom layers of the water. Diaz et al. (2006, 2011) also used a similar method to mitigate the effect of the isotherms in their temperature gradient. I monitored the temperature at both ends of the trough using a Top Fin® Digital Aquarium

Thermometer.

I selected one snail from stock aquariums based on the ease with which I could reasonably remove the snail without disturbing the contents of the aquariums. I marked individual specimens on the back of the shell for identification using different colours of nail polish enamel (Fenwick and Amin, 1982; Clampitt, 1970, 1974), then placed the snails in a medium-size Marina® Hang On Holding and Breeding Box receiving bubbled water from one of the stock aquariums through the breeder box. I left the snails in the breeder box for approximately 22 hours to allow snails to recover after being marked. I added snails to the temperature gradient where the water temperature was closest to that 28 in the breeder box and the stock aquariums (i.e. approximately 30oC ± 2oC). Snails remained in the trough for two hours to allow them time to explore the range of water temperatures. At the end of this period, I recorded the water temperature at the position of each snail using a digital aquarium thermometer. Upon completion, I removed snails and returned them to their aquariums. I removed the water from the trough and placed the water back into each stock aquarium in equal amounts. I then cleaned the trough by rubbing the surfaces of the trough with a paper towel and rinsing the trough three times with warm tap water; I emptied the tap water into a sink after each rinse.

For each experimental replicate, I refreshed the water in the trough as noted above. I performed this procedure once per day, at the same time each day for six days, using a total of 30 snails (five snails per trial).

To determine the temperature preference for P. wrighti, I fit a Bayesian linear hierarchical model with aquarium and trial number as the predictors, a Gaussian distribution as the response, and assigned uninformative priors to the model parameters, using the package BRMS (Bayesian Regression Models using ‘Stan’) in R3.6.1. See

Chapter 3 for why Bayesian methods of analysis were chosen to analyze the data. The resulting output values allowed me to determine if there was a difference between tank and trial with regards to the preferred temperature. I used the following Markov chain

Monte Carlo (MCMC) diagnostic methods, which are used to verify the reliability of the results: R-hat values, effective sample sizes (ESS), and visual inspection of trace and posterior density kernel plots. MCMC diagnostics validated that the model had successfully converged; visual inspection of trace plots and posterior density kernel plots,

29 showed no trends, all R-hat values equalled one, and the ESS were sufficiently large (see

Appendix B for more information on diagnostic methods and an example of the trace and posterior density kernel plots produced during analysis).

I used the value for the preferred temperature to establish the range of test temperatures for the remaining experiments; 10oC above and below the preferred temperature of 23oC).

2.2.5 Observed Period of Greatest Activity – Varying Temperatures

To determine if different water temperatures could affect snail activity, I set up nine 2.5

US gallon aquariums, with the water temperature of three aquariums set to 33oC (hot water; 10oC above the preferred temperature), three set to the preferred temperature of

23oC (warm water), and three set to 13oC (cold water; 10oC below the snails preferred temperature). The water temperature for all aquariums varied by ±3oC. I selected 10 adult snails (for a total of 90 snails), of no specific size, to put in each of the aquariums,

(see Appendix A for detailed aquarium set up and maintenance).

I repeated the methods from the Observed Period of Greatest Activity – Constant

Temperature with a couple of alterations. First, I took qualitative observations of the snails in their aquariums with regards to their location within the aquariums, direction of movement, and lack of movement. Second, to ensure the scotophase was included in my observations, I generated 14 random observation times using Microsoft© Excel to schedule additional observations over one week. To record observations during the scotophase, I turned the lights on for < 5 minutes to see the snails and record observations.

30 2.2.6 Behavioural Observations of Physella wrighti

To determine if water temperature altered the behaviour of P. wrighti, I used the same snails and aquarium set up noted above for the Observed Period of Greatest Activity –

Varying Temperatures experiments. I observed snails for changes in behaviour including how often snails exited the water, the distance to which they moved out the of water, if snails congregated, irritability/twitching, avoidance of certain locations in aquariums, copulating or lack thereof, the occurrence of egg-laying, and the substrate on which egg masses were laid.

To measure the distance snails crawled out of the water, I used a 30 cm ruler placed against the outside of the aquarium and measured the distance from the water level to the snail. I observed snails daily during the observation period and also considered the same random times as noted for the Observed Period of Greatest Activity – Varying

Temperatures observations indicated above. No specific measurements were made for the duration that snails spent during any activity.

2.2.7 Determining Survivorship of Mature Physella wrighti

Using the same setup and snails noted in the Observed Period of Greatest Activity –

Varying Temperatures and Behavioural Observations of Physella wrighti experiments, I observed snails daily from the date they were introduced in the aquariums to the date I found snails deceased. I judged a snail as dead when it showed no response to external stimulus (i.e. gentle prodding with a blunt instrument). Only data for the snails that I could confirm as dead within the last 24 hours were considered in my

31 analysis. I removed dead snails from the aquarium and only replaced snails when the population of live adult snails in the aquarium declined to seven out of ten individuals.

The snails that I added were from the most recently collected specimens. I acclimated new snails as noted in Appendix A.

Survivorship of the snails in the hot (33oC) water treatments was low and required new snails be added to the experimental aquariums; however, I did not add new snails to the other temperature treatments as it was not necessary. Initially, I had not marked the original experimental snails in the aquariums, and thus was not able to track survivorship in the hot water aquariums accurately beyond the first replacement of snails; however, between the three tanks, I was able to get sufficient data for analysis. I prevented further confusion by marking snails in the warm and cold water aquariums for identification using nail enamel (Fenwick and Amin, 1982; Clampitt, 1970, 1974).

I analyzed the survivorship data to determine if there were clear differences in the number of days snails survived at the three different water temperatures. I fit a Bayesian generalized linear model with water temperature as the predictor variable, a gamma distribution for the response, and a log link using the package BRMS in R 3.6.1. Model parameters were assigned default, uninformative priors. A preliminary inspection supported the gamma distribution and log link, as the data were continuous and strictly positive. The MCMC diagnostics showed that the model had successfully converged

(based on visual inspection of trace plots and posterior density kernel plots (see Appendix

B for more information on diagnostic methods and example trace and posterior density kernel plots)), all R-hat equalled one, and effective sample sizes were sufficiently large.

32 2.3 Results

2.3.1 Observed Period of Greatest Activity – Constant Temperature

Physella wrighti did not show any obvious patterns of their location in the aquariums from visual observations for either the photophase or scotophase. Individuals remained relatively scattered throughout the aquariums and did not appear to congregate on any particular substrate. There were no visually discernible differences between the number of snails moving throughout the tank and those that remained stationary. Snails showed a range of movements from no movement, to moving slowly, to moving quickly. There did not appear to be any common direction of movement among snails. Overall, I did not observe there to be a photoperiod where snails were most active.

2.3.2 Determining the Temperature Preference for Physella wrighti

Physella wrighti prefer temperatures ranging from 15oC to 30oC (Figure 10), and P. wrighti showed the ability to tolerate a wide range of water temperatures. Snails in hot water crawled close to the heated end of the trough where the water temperature was

38.1oC to 39.1oC. On the other end of the temperature spectrum, I also observed snails crawling into colder water temperatures of 5.8oC and observed snails crawling directly on the ice packs in the trough. In one particular case, I observed one of the snails crawl into the hot water at 39.1oC and then into the cold water at 5.8oC within the two hour observation period, demonstrating P. wrighti’s ability to tolerate large, rapid changes in water temperature.

I found P wrighti preferred a temperature of 23.9oC (19.7-29oC, 95% credible interval); I 33

Figure 10. Frequency of water temperatures chosen by Physella wrighti. I placed one snail from five aquariums in a trough containing water with a temperature gradient of 5oC to 45oC at a position that corresponded to the temperature of the breeding box. I left snails to explore the trough for 2 hours, after which I recorded the temperature of the water at the position of each snail.

34 found no differences between trials and tanks. Knowing that temperatures vary a bit, the resulting value was rounded down and used as the preferred temperature for the remaining experiments.

2.3.3 Observed Period of Greatest Activity – Varying Temperatures

Physella wrighti remained active throughout each observation period. While some individuals showed no active movement during observation, I found no visually definitive pattern of inactivity, suggesting that individual snails rest only as required.

Additionally, I did not discern any patterns as to the direction of their movement within their aquariums; individuals appeared evenly distributed throughout the aquarium. Thus, there did not appear to be any time of day in which snails were most active, regardless of the water temperature.

2.3.4 Behavioural Observations of Physella wrighti

With the preferred temperature of P. wrighti established, I examined if there were any changes in behaviour within the different water temperatures of 13oC (cold water), 23oC

(warm water; snails’ preferred temperature), and 33oC (hot water). Generally, snails did not show any changes in behaviour in connection to the water temperature. Physella wrighti did not show any obvious preference for location in tank or surface material (e.g. plastic, sand, or glass) and showed a tendency to crawl anywhere they could get to, including on the heaters in the warm and hot water aquariums. I observed copulating in each of the temperature treatments (Figure 11) and snails laid egg masses throughout the aquariums, with the exception of the hot water aquariums (to be addressed in Chapter 3).

35 B

A

Figure 11. View of some of the male genitalia of Physella wrighti during copulation. The snail on the left is the acting female and the snail on the right is the acting male. A) Preputium; B) Penis sheath. Photo credit: Erika Helmond.

36 While snails in all aquariums laid egg masses on the glass aquariums walls, snails also laid egg masses on whatever they were able to crawl on, including, but not limited to the rubber air-line hose, plastic heater clips, silicone aquarium sealant, aquarium sand, sponge filter, and the heated portion of the aquarium heater (Figure 12). Snails in each tank congregated within the tanks when food was added, gathering around the food, and when copulating (during which I observed groups of 2-4 snails) (Figure13, 14). I also considered the time of day that I made observations, and found no obvious patterns there.

One aspect of the behaviour of P. wrighti that I did notice a difference in was their tendency to leave the water. I observed snails out of the water in 1.87% of my observations for all temperature treatments. Out of the total number of observations, snails in the hot water left the water more often than snails in either the cold or warm water. Out of the snails that left the water, snails in the hot water left the water 72.4% of the time compared to snails in cold water at 21.8%, and snails in warm water at 5.7%.

Additionally, snails in the hot water aquariums ventured farther out of the water than snails in both the cold and warm water aquariums at maximum distances of 7.5 cm, 5 cm, and 2.5 cm, respectively. I even found snails in hot water treatments crawling on aquarium lids (Figure 15).

2.3.5 Determining Survivorship of Mature Physella wrighti

Survival of field-collected snails varied among water temperatures. The first snail death occurred in the hot water aquariums, with death occurring within the first day of the experiment. I attempted to restock hot water aquariums with field snails from the new stock aquariums but was unsuccessful in keeping the hot water snails alive. As a result, 37

Figure 12. Physella wrighti egg mass laid on an aquarium heater in a warm water aquarium, indicated by the circle. Photo credit: Erika Helmond

38

Figure 14. Three copulating Physella wrighti, with penial complexes

visible. The uppermost snail is acting as a female, and the middle snail Figure 13. Three Physella wrighti copulating. Photo credit: Erika as both a male and female, copulating with the uppermost female snail Helmond but also being bred by the lowermost acting male snail. Photo credit:

Erika Helmond.

39

Figure 15. Physella wrighti on aquarium lid in a hot water aquarium (33oC), indicated by the circle. The hot water tanks were the only tanks in which I observed snails leave the water to this extent, suggesting they were experiencing thermal stress. Photo credit: Erika Helmond.

40 [A] [B] [C]

Figure 16. Survival of field-collected Physella wrighti snails in each of the tested water temperatures:

Cold (13oC), Warm (23oC), and Hot (33oC). The letters [A], [B], and [C] indicate that each of the treatments are clearly different from each other with > 95% certainty.

41 snails in the hot water aquariums did not survive past 60 days from the start of the experiments. The extended survival of snails in the cold water aquariums was a drastic contrast. While there were a few snails that died early, the majority survived much longer (Figure 16). By 276 days after the start of the experiment, 57% of the cold water snails were still alive, their life span extending beyond the 108 day observation period at an average of 240 days (174-342 days; 95% credible interval). Snails in the warm water aquariums survived better than those in the hot water aquariums (Figure 16), with an average survival length of 84 days and 6.9 days (38-189 days, 3-18 days; 95% credible interval), respectively. Several of the snails in the warm water lasted throughout the main observation period; however, the remaining snails in the warm water aquarium died shortly after the observation period. Snails in the cold water clearly survived longer than both the warm and hot water snails, with a life span 2.9 times longer than snails in warm water and 34.5 times than snails in hot water. Comparatively, the lifespan of snails in the warm water aquariums was 12.2 times longer than snails in the hot water aquariums.

2.4 Discussion

No two hot springs are the same, making each hot spring a niche ecosystem. The unique and extreme thermal environment characteristic of the Liard Hot Springs suggests that water temperature plays an important role in the survival of P. wrighti. Hot spring snails, such as P .wrighti, are particularly important to these environments as they provide services to the environment that cannot be easily replaced by other organisms, especially those unsuited to hot water conditions. Physella wrighti is Endangered and endemic to the hot springs (COSEWIC, 2008b). Thus, I wanted to develop a better understanding of

42 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.

Many, but not all, species of freshwater snails show a defined period of activity in response to photoperiod (Hutchison, 1947; Costil and Bailey, 1998; Lombardo et al.,

2010). In both the constant and altered temperature experiments, I found no evidence that P. wrighti has any point during a 24 hour period where they are more active. Snails remained relatively evenly distributed throughout their aquariums and they did not congregate anywhere in particular. There did not appear to be any migration of snails within the aquariums in response to day versus night stimuli, and snails did not show any visually discernible patterns of movement or lack thereof.

Bithynia leachii (Bithyniidae), Physella acuta (also referred to as Physa acuta),

Planorbis planorbis (Planorbidae), Galba truncatula (Lymnaeidae), Radix auricularia

(Lymnaeidae), and Valvata piscinalis (Valvatidae) (Lombardo et al., 2010) directly contrast with my observations of P. wrighti. Lombardo et al. (2010) found that 70-80% of snails the snails they observed were active during the day and that each species seemed to have a different time in the day during which they were most active. In contrast, the lack of change in activity level observed by Hutchison (1947) in the snail Viviparus malleatus (Viviparidae) (now known as Cipangopaludina malleata) and Costil and

Bailey (1998) observed in Planorbarius corneus (Planorbidae) is more similar to that of

P. wrighti. Viviparus malleatus and P. corneus are considered arrhythmic, exhibiting no patterns of activity with regards to photoperiod. As such, both are active and inactive throughout the day depending on the individual snail.

43 It may be possible to alter a snail’s period of activity by altering water temperature and creating a stressful environment. However, Hutchison (1947) noted that altering the water temperature did not affect the arrhythmic activity patterns for V. malleatus, lending support to my observations of P. wrighti in the varying temperatures; despite the temperature changes, I saw no difference in activity patterns.

A snail’s preferred water temperature plays a role in habitat selection (Vaidya and

Nagabhushanam, 1978; Reynolds, 1979). Thus, to aid in determining a snail’s population distribution, both within and outside of the surrounding geographical area, it is helpful to know its preferred temperature. I found P. wrighti’s preferred temperature to be approximately 23oC, which is near the lower end of the snail’s commonly observed temperature range of 23-40oC (Te and Clarke, 1985; Lee and Ackerman, 1999; Salter

2003; COSEWIC, 2008b). Thus, it is plausible that P. wrighti may only be tolerating the warmer water temperatures as opposed to requiring the warm water for survival.

However, my data suggests P. wrighti is capable of handling a wider range of water temperatures (between 5.8oC and 39.1oC). A team and I observed P. wrighti out of the water crawling on rocks and metal hand railings in Alpha Pool when the air temperature was below -22oC during field studies in the LHS in January 2018 and 2019. While P. wrighti appears to show a preferred water temperature, its apparent ability to tolerate such a wide range of water temperatures makes determining its population distribution more difficult. Clampitt (1970) found Physella gyrina (also referred to as Physa gyrina) and

Physella integra (also referred to as Physa integra) are both able to withstand a wide range of water temperatures, between 10oC and 40oC. Clampitt (1970) suggested that habitat selection and, thus, population distribution may not always be impacted by 44 preferred water temperature, rather selection is influenced by some other factors such as thermal maximum/minimum or requirement for surface air-breathing. Factors such as access to food, environmental stability, or chemical composition of the water (von

Oheimb et al., 2016) may influence habitat selection. Since P. wrighti seems to handle a wide range of temperatures, it is necessary to understand what other aspects may be influencing its distribution in the hot springs.

With the knowledge that P. wrighti can tolerate a wide range of temperatures, I wanted to determine how this species would respond to being kept in different water temperatures that were not of its choice. In response to temperature changes, organisms often exhibit behavioural responses, such as moving to find another temperature zone, entering an inactive state until conditions are more favourable, or attempting to tolerate the current temperature with possible displays of irritation (McClary, 1964; Garrity, 1984; Muñoz et al., 2005; Bae et al., 2015). The change in a snail’s behaviour with regards to water temperature is rather subtle. Throughout my observations, specimens of P. wrighti exhibited mostly the same behaviours between the different temperature treatments of

13oC, 23oC, and 33oC. Generally, P. wrighti reproduced in all temperatures, explored all areas of their aquarium, and remained mobile in all temperatures, although snails in the cold water moved more slowly than snails in the other temperatures. Additionally, P. wrighti did not demonstrate irritation in the form of shell twitching as noted by McClary

(1964) in Pomacea paludosa, nor did P. wrighti show any differences in floating in the water column, or orientating their shells, as has been observed in other species (Garrity,

1984; Muñoz et al., 2005; Bae et al., 2015). Physella wrighti did show some irritation in that snails left the hot water more frequently (72.4% of instances of snails leaving the 45 water) than in the warm and cold water, and moved farther away from the hot water

(mean 7.5 cm, compared to 2.5 cm and 5.0 cm in warm and cold water, respectively).

Taken together, this suggests that snails were experiencing some level of irritation or stress to the hot water temperature. In experiencing this stress, individuals may have left the water to cool down and prevent their internal body temperature from becoming too high. Desert invertebrates such as Ocymyrmex barbiger (Formicidae) and Eremogarypus perfectus (Garypidae) have been observed to climb up vegetation as air temperature increased during the day (Marsh, 1985; Herutault and Vannier, 1990; Clarke, 2014).

Clarke (2014) attributed this behaviour to the organism’s small size; thus, they have a greater surface area to volume ratio and low thermal mass, leading to quick changes in internal body temperatures. To minimize the effects of heat, the organisms climb up plants to reach cooler air. Perhaps, P. wrighti’s actions serve the same purpose in an aquatic environment as the actions of some invertebrates in a terrestrial environment.

Oxygen (DO) levels may be another factor contributing to P. wrighti leaving the water.

Lack of oxygen is a common characteristic of thermally heated waters in hot springs

(Brues, 1924). Snails are known to absorb DO through their skin and some species have additional respiratory structures that aid in facilitating the absorption of DO (Koopman et al., 2016). For example, some species of snail some snails have gills from which to acquire DO, such as Bithynia tentaculata (Koopman et al., 2016), others snails have a lung (a modified portion of the mantle cavity) (Brown, 2001), such as Planorbis planorbis (Koopman et al., 2016), and some snails have lung and accessory gill (i.e. pseudobranch) (Strong et al., 2008), such as Physa fontinalis (Physidae) (Koopman et al.,

2016). In water with low DO, snails that can breathe air may be able to maintain 46 physiological functions by leaving the water to access the atmospheric oxygen. Bae et al.

(2015) noted P. canaliculata to leave the water with increased water temperature. Bae et al. (2015) exposed specimens to temperatures ranging from 15oC to 30oC and noted that snails would breach the water to ventilate more often at 30oC than 15oC. Bae et al.

(2015) suggested that this was due to oxygen stress. Koopman et al. (2016) found pulmonates were sensitive to hypoxia (25% saturation) and that snails surfaced more frequently to breathe compared to normoxia (100% saturation) and hyperoxia (300% saturation). Koopman et al. (2016) also observed that, in water temperatures between

10oC and approximately 42oC at normoxia, pulmonate snails visited the surface more often at higher water temperatures. Van der Schalie et al. (1973) also observed snails leaving the water at higher temperatures, and noted this behaviour as a stress response to the increased temperature. Both Laurzier et al. (2011) and I have observed P. wrighti to leave the hot spring water. Of particular interest is that I have even observed snails leaving the water in January when the air temperature was well below freezing. While this could suggest temperature-related stress, it could also indicate the snails do not have access to sufficient dissolved oxygen. However, it is not known how reliant P. wrighti is on DO versus atmospheric oxygen (i.e. how might hypoxia affect P. wrighti) without the effects of temperature stress; P. wrighti is classified as a pulmonate snail (Turgeon et al.,

1998) but further research is required to determine if there are any potential interactions of temperature and DO levels and how P. wrighti responds to the varying conditions.

However, in my study of P. wrighti, I suggest this behaviour is more related to temperature-related stress because all aquariums were outfitted with aerators to ensure

47 oxygen was present in the water; thus, snails exiting the water is reflective of temperature stress at high temperature.

Generally, aquatic snails seem to survive best in cooler to mid-range temperatures than at warmer temperatures that are near or at their maximum survival temperature (Costil,

1994; El-Emam and Madsen, 1982; Vaidya and Nagabhushanam, 1978; van der Schalie et al., 1973). Since P. wrighti is typically found in water temperatures 23.5-36oC (Te and

Clarke, 1985; Lee and Ackerman, 1999; Salter 2003; COSEWIC, 2008b), it could be suggested that at temperatures near the mid-point and low end of this range will result in the greatest survival compared to if P. wrighti were raised in water temperatures below

23.5oC or closer to and above 36oC; I observed the opposite. Snails in the cold water aquariums survived the longest at an average of 240 days, while snails in the hot water survived the shortest amount of time at an average of 6.9 days. The snails in the warm water aquariums survived an average of 84 days. There is a clear relationship between the length of adult snail survival and the temperature of the water in which they are reared. My results suggest that P. wrighti survives the longest in water colder than what it is typically observed in and survival length decreases as water temperatures approach the range found in its natural habitat.

Ectotherms in cold conditions experience a reduced rate of biological functions because they are unable to maintain their internal body temperature. Hubendick (1958) notes that while cooler temperatures can have a significant effect on the life history of a snail, the effect is usually not fatal, and, instead, results in slowing down the snail’s life history.

The slower life history is due to the reduced molecular kinetic energy, which, in turn,

48 leads to slower physiological processes (Clarke, 2014). This is likely what happened for the specimens of P. wrighti in the cold water aquariums.

In general, hot temperatures stress organisms differently than cold temperatures; consequently, hot temperatures have a different effect on survival. As temperatures become too high for an organism, the organism starts to experience thermal denaturation of proteins in the body and may not be able to return the protein to the functional conformation (Clarke 2014). This eventually leads to systems shutting down and the death of the organism. This seems to be a plausible reason for the low survival of P. wrighti in the hot water aquariums. The hot water may have caused snails too much stress to survive for any length of time.

Warm water may have provided optimal conditions for the survival of the snails, such that they were not stressed by hot water, or slowed by cold water. The snails in the warm water lasted throughout the 108 days of the project, and only started to experience fatalities afterwards. It is possible that the parent P. wrighti were at the end of their life span and that this is the reason for the mortality in warm water snails. It is commonly observed that pulmonate snails experience high mortality rates following reproduction, i.e. they are semelparous (Calow, 1978; Dewitt, 1955; McMahon, 1975; Russell-Hunter,

1961a, 1961b); however, it is unknown if P. wrighti is capable of undergoing more than one period of reproduction. I suspect it is most likely that P. wrighti is semelparous.

The temperature of the water that a given species of snail is capable of tolerating usually relates to the habitat in which the species can survive. Snails with a narrow range of temperature tolerance are usually only found surviving well within specific habitats, and, 49 unless the habitat is abundant, the population remains relatively isolated. Conversely, species with a wide range of temperature tolerance are usually able to survive in a wide range of habitats and, thus, may be found in abundance. With Physella wrighti living in a very specialized environment, one assumes that the snail likely has a narrow tolerance range to water temperature and would not survive in other water temperatures; however, this does not seem to be the case. Thus far, P. wrighti’s ability to tolerate a range of water temperatures is perplexing, considering the specialized environment in which it is found. This seems to suggest that P. wrighti may only be tolerating the temperature of the hot springs and may actually be better suited to slightly cooler water.

50 CHAPTER 3: THE EFFECTS OF TEMPERATURE ON ASPECTS OF

REPRODUCTION OF PHYSELLA WRIGHTI

3.1 Introduction

Physella wrighti is a pulmonate snail belonging to the family Physidae (Turgeon et al.,

1998; Lee and Ackerman, 1999). Pulmonates are generally known to be simultaneous hermaphrodites, capable of self-fertilization (Dillon, 2004), oviparous (i.e. lay eggs), and semelparous (i.e. with a single reproductive period during lifespan) (Brown, 2001).

Additionally, pulmonates can be found in aquatic habitats that range from ponds and lakes to hot springs (Clench, 1926; DeWitt, 1955; van der Schalie et al., 1973; Te and

Clarke, 1985). With this diverse range of habitats comes potential exposure to a wide range of water temperatures. Some snails, such as Physella gyrina and Lymnaea stagnalis (Lymnaeidae), are commonly found in water that fluctuates in temperature due to the freeze/thaw cycles of the region (van der Schalie et al., 1973), whereas others, such as Physella johnsoni and P. wrighti, are found in water that remains warm and relatively stable all year, with the only source of fluctuation due to seasonal run-off (Grasby and

Lepitzki, 2002; Salter, 2003, British Columbia Ministry of Environment, 2014).

For poikilothermic animals, including aquatic snails, water temperature is known to be an important governing factor of the biological and chemical processes required for life

(Hubendick, 1958; Thomas and McClintock, 1990; Costil 1997; Britton and McMahon,

2004). Thus, when water temperatures become too cold or too hot there will tend to be adverse effects on survival, reproduction, and development (DeWitt, 1954b; van der

Schalie et al., 1973, Costil, 1997; Zukowski and Walker, 2009). 51 Physella wrighti is thought to be a hot water specialist snail (COSEWIC, 2008b), adapted to survive in hot water and unlikely to be capable of surviving in cooler water conditions

(Lee and Ackerman, 1999; DFO, 2010). The Canadian Department of Fisheries and

Oceans suggests that it is unlikely that P. wrighti is capable of surviving in water below

23oC indicating that P. wrighti is likely only capable of inhabiting a limited range of environments (DFO, 2010). Physella wrighti is thought to be endemic to a single habitat type in one hot spring system (COSEWIC, 2008b; DFO, 2010). To understand fully the capabilities of P. wrighti and its possible habitats, one must study how the snail responds to different water temperatures. However, there is a paucity of information on P. wrighti; only one study has been done on its life history (i.e. Lee and Ackerman, 1999).

There is much to learn concerning P. wrighti. This chapter explores aspects of P. wrighti’s reproductive success when reared under different water temperatures. The information here will help increase our knowledge of this species and help determine how sensitive the snail is to different water temperatures. To achieve this understanding,

I examined the relationship between water temperature and the number of egg masses produced, the number of eggs per mass, egg volume, viability of egg masses, and incubation period. I gathered data in these areas by exposing specimens of P. wrighti, to three different water temperatures for 108 days.

3.2 Experimental Methods

The experiments mentioned here used the same snails used in the Observed Period of

Greatest Activity - Varying Temperatures, Behavioural Observations of Physella

52 wrighti, and Determining Survivorship of Mature Physella wrighti experiments.

Aquarium conditions were the same as described in Chapter 2 (see Appendix A for a detailed aquarium set up). All observations were made daily for 108 days.

3.2.1 The Number of Egg Masses Produced by Physella wrighti

To determine the number of egg masses laid within each temperature treatment, I recorded egg masses that were immediately visible in the aquariums. I did not move any of the materials in the aquariums to find hidden egg masses to avoid disturbing the aquarium contents and snails. I recorded the location of egg masses on the exterior of the aquariums using window markers to prevent re-recording masses.

3.2.2 The Number of Eggs per Mass Produced by Physella wrighti

To determine the number of eggs per egg mass in each temperature treatment, I selected several masses from each of the experiment aquariums and counted the number of eggs per mass. I examined egg masses using three methods. For the first method, I removed several masses from each aquarium by firmly, but gently, scraping them off the deposited surface with my fingernail. I selected these masses based only on availability within each aquarium. I placed the masses, individually, in hinged plastic boxes measuring 2.2 cm by

2.9 cm by 2.9 cm, and floated the boxes at the top of aquariums. Each box had two holes covered with silkscreen to isolate the mass from the rest of the tank but allow for gas and water exchange into/out of the box. For the second method, I placed seven glass microscope slides in each of the aquariums, and removed the slides to examine masses deposited on the slides but refrained from removing the masses from the microscope

53 slides. The microscope slides allowed me to examine the masses on the surfaces they were laid, with minimal disruption to the eggs. These masses were also selected based on availability within each aquarium. For the last method, I counted eggs within undisturbed masses in the aquarium. I selected only those masses for which I was confident I could accurately count all the eggs. For the first two methods, I examined eggs using microscopes; I used a stereo microscope for egg masses that I removed from aquarium surfaces, and a compound light microscope for egg masses laid on microscope slides. After I finished examining the masses, I returned them to their respective aquariums. For the third method, I counted most eggs without magnification; however, some masses required magnification, which I achieved by taking a picture with a

Samsung Galaxy S7 phone and using the zoom feature to inspect the masses.

3.2.3 Determining Differences in the Size of Eggs Laid by Physella wrighti

To assess if there was a difference in egg size between snails reared in the different water temperatures, I examined several whole egg masses from each temperature based on the availability of masses. To avoid possible effects from the increased size a partly developed embryo might have on egg size, I measured eggs only if the embryo had not yet begun to revolve within the egg. I also included measurements of eggs that lacked an embryo. The egg masses in which I measured eggs consisted of a subset of the same masses collected in snap boxes and on microscope slides for the Number of Eggs per

Mass Produced by Physella wrighti experiment. Using a calibrated ocular micrometre,

I measured the length and width of eggs from the widest point of the inner membrane of each egg (Figure 17), as the outer membrane was not always visible, and calculated the

54

Figure 17. One of four eggs in an egg mass laid by Physella wrighti in a warm water aquarium (23oC).

This egg measures 0.69 mm long by 0.49 mm wide. The egg closely resembles a prolate ellipsoid. A.) the measured length of the egg; B.) the measured width of the egg; C.) the outer membrane of the egg; D.) the inner membrane of the egg; E.) an embryo of P. wrighti during early cell division. This picture was taken at 100x total magnification. Photo credit: Erika Helmond.

55 volume based on the equation of a prolate ellipsoid (1).

4 1 1 2 V= ( ) π* length* ( width) (1) 3 2 2

Although volume is commonly a measure of three dimensions, I could not measure the depth of the eggs. A volume based on a prolate ellipsoid allowed me to determine a relative measurement of egg size for all eggs, as I arbitrarily set egg depth equal to egg width. I used the same microscopes as described in the Number of Eggs per Mass

Produced by Physella wrighti experiment.

3.2.4 Viability of Eggs per Egg Mass Produced by Physella wrighti

I defined egg viability as the snails developing fully without any serious visible malformations (Figures18). I assessed embryos as being non-viable under two conditions: the point where the embryo started to degrade as evident by a fractured appearance of the embryo (which often occurred early in embryo development) (Figure

19 A, 19 B), or if the embryo developed a malformation followed by death within the egg

(which occurred later in development) (Figure 19 C to I). I examined viability using the same egg masses collected in snap boxes and on microscope slides, for the Number of

Eggs per Mass Produced by Physella wrighti experiment; the microscope equipment I used remained the same.

3.2.5 Physella wrighti Embryo Incubation Period

I defined the incubation time as the time it took from when an egg mass was laid to the point when the first snail hatched from an egg. The masses I examined were a subset of

56

o Figure 18. Examples of viable embryos of Physella wrighti in egg masses laid in warm (23 C) and cold water (13oC) aquariums. Photos are a general progression of viable embryo development following from left to right, top to bottom. Photo credits: Erika Helmond.

57

Figure 19. Various non-developed and non-viable embryos of Physella wrighti in egg masses laid in warm water (23oC) and cold water (13oC) aquariums. Photos ‘A’ and ‘B’ show embryos that died and degraded during development. Photo ‘C’ shows an embryo in early development that did not develop past the shown state. A normal embryo would be round to oval in shape. Photo ‘D’ shows an embryo in mid-development that has become non-viable. This embryo shows a general outline of a developing snail but has become transparent and lacks the cellular/tissue definition present in a normal embryo. Photo ‘E’ shows an embryo about three- quarters through development. Note the front edge of the shell has grown out and upward instead of growing down over the head of the embryo and the mantle of the embryo is beginning to balloon out from under its shell. Photo ‘F’ shows an embryo that has separated from its shell within the egg. This embryo is at approximately the same stage in development as in Photo ‘E’. Photo ‘G’ shows an almost fully developed but non-viable embryo that has developed a prolapsed radula (the protruding structure at the front of the specimen). Photo ‘H’ shows two non-viable embryos. In this case, the tissues of both embryos are joined. Photo ‘I’ show a non- viable embryo about three-quarters developed that has become non-viable. The shell remains definable, but the internal tissues of the specimen have ballooned out of the shell to fill the egg. Photo credits: Erika Helmond. 58 the masses collected in snap boxes and on microscope slides for the Number of Eggs per

Mass Produced by Physella wrighti experiment. I only included masses I was confident had been laid within 12-24 hours of collection. Masses that were laid later were not included, to keep incubation time within a maximum of one day.

3.2.6 Statistical Analysis

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). 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 us with the probability that our hypothesis is true only how likely we are to observe the data under certain conditions. Bayesian methods address the probability that our hypothesis is true based on the data (Ellison, 2004; Mason, 2011). The analysis produces a credibility interval that provides us with the probability that the parameter(s) of interest (e.g. the mean) is found within a range of values that could result in your data (Ellison, 2004). In my opinion, since the Bayesian methods are ultimately a more intuitive method of analysis, I chose to analyze the available data using this method. The following information includes the specific conditions and software that I used for my analysis.

I analyzed the effect of temperature on a) the number of egg masses, b) the number of eggs per mass, c) egg volume, d) incubation period, and e) egg viability using Bayesian generalized linear models with water temperature as the predictor variable. 59 To examine the effect of temperature on the number of egg masses and the number of eggs per mass I used a negative binomial distribution (as I had discrete data) and the standardly associated log link function. To examine the effect of temperature on egg volume and incubation period, a preliminary inspection supported the gamma distribution and log link, as the data were continuous and strictly positive. In the model for determining the effect of temperature on egg viability, I used a binomial distribution as this distribution is appropriate in analyzing data that occur under a “yes” or “no” state

(e.g. the egg is viable or the egg is not viable).

In all models, I assigned model parameters to default, uninformative priors. All statistics were performed using the package BRMS (Bayesian Regression Models using ‘Stan’) in

R 3.6.1. Markov chain Monte Carlo (MCMC) diagnostics showed that the model had successfully converged since all R-hat values were equal to one, effective sample sizes were sufficiently large, and visual inspection of trace plots and posterior density kernel plots showed no trends (see Appendix B for more information on diagnostic methods and example trace and posterior density kernel plots).

3.3 Results

3.3.1 The Number of Egg Masses Produced by Physella wrighti

Physella wrighti first began laying egg masses on different days for each water temperature. I first observed egg masses in the warm water aquariums two days after the introduction of snails into the aquarium, in the hot water eight days after introduction, and in the cold water 13 days after introduction. Physella wrighti laid 348 egg masses in

60 the warm water aquariums, whereas in the cold and hot water aquariums P. wrighti laid

39 and five masses, respectively (Figure 20). Only snails in the warm water produced egg masses in all three aquariums; those in the cold water only produced masses in two aquariums, whereas those in the hot water only produced masses in one aquarium. I found snails in the warm water aquariums laid more masses than either the cold and hot water aquariums, and the cold water aquariums laid more masses than the hot water aquarium with > 95% certainty. On average, snails in the warm water aquariums laid 117 masses per aquarium (42-340 masses; 95% credible interval), whereas snails in the cold water aquariums 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).

3.3.2 The Number of Eggs per Mass Produced by Physella wrighti

While the number of eggs per mass (EPM) varied both within and between temperature treatments (Figure 21), the minimum number of eggs I observed in a mass was one, laid in a warm water aquarium, and the maximum was 24, laid in a cold water aquarium

(Figures 22 and 23). I found that snails in the cold water aquariums produced as many as twice the number of EPM than snails in either the warm or hot water aquariums (> 95% certainty). Moreover, I found there to be no clear difference between the number of EPM between the warm and hot water aquariums (> 95% certainty). On average, snails kept in cold water laid 11 eggs per mass (9-13eggs/mass; 95% credible interval), snails in warm water laid 5 eggs per mass (3-8 eggs/mass; 95% credible interval), and snails in hot water laid 6 eggs per mass (3-12 eggs/mass; 95% credible interval).

61 [A] [B] [B]

[B]

[A] [C]

Figure 20. The total number of recorded egg masses laid by Physella Figure 21. The number of eggs per mass produced by specimens of wrighti in each water temperature. Only readily visible egg masses were Physella wrighti cultured at three different water temperatures. The water noted across all water temperatures to minimize disruptive activities in the temperatures are as follows: cold water = 13oC; warm water = 23oC; hot

o aquariums. The water temperatures are as follows: cold water = 13 C; water = 33oC. The letters [A], and [B] indicate that the cold water is clearly

o o warm water = 23 C; hot water = 33 C. The letters [A], [B], and [C] indicate different from the warm and hot water but the warm and hot water are not that each of the treatments are clearly different from each other with > 95% clearly different from each other with > 95% certainty. certainty. 62

Figure 23. An egg mass laid by Physella wrighti containing 24 eggs, the

maximum number contained within one egg mass. This mass was found in

a cold water aquarium. Photo credit: Erika Helmond.

Figure 22. An egg mass laid by Physella wrighti containing one egg, the minimum number contained within an egg mass. This egg mass was laid in a warm water aquarium. Photo credit: Erika Helmond.

63 3.3.3 Determining Differences in the Size of Eggs Laid by Physella wrighti

For the most part, the eggs P. wrighti laid approximated the shape of a prolate ellipsoid

(Figure 17). However, snails also laid some slightly irregularly shaped eggs, which I still included in the volume calculations The minimum and maximum egg volumes of 0.055 mm3 and 0.521 mm3, respectively, both came from eggs laid in the warm water aquariums; however, egg volume varied between and within water temperatures (Figure

24). The length and width measurements of the eggs that corresponded to the minimum volume was 0.48 mm by 0.46 mm, and the maximum volume was 1.08 mm by 0.96 mm.

The average egg volumes for cold, warm, and hot water were 0.16 mm3 (0.15-0.18 mm3;

95% credible interval), 0.17 mm3 (0.14-0.20 mm3; 95% credible interval), and 0.19 mm3

(0.14-0.27 mm3; 95% credible interval), respectively. While I found that the average volume of eggs produced by P. wrighti in hot water was marginally larger than the cold and warm water, overall I determined that there was no clear difference in the average egg volume between temperatures (> 95% certainty).

3.3.4 Viability of Eggs per Egg Mass Produced by Physella wrighti

The viability of egg masses ranged from 0-100% across all temperature treatments

(Figure 25), and showed no obvious trend for the time it took an embryo to become non- viable. The average percent viability of egg masses laid in cold water was less than half the viability of warm water at 31.2% (23-40.1%; 95% credible interval) and 73.1% (52.7-

86.5%; 95% credible interval), respectively. Egg masses laid in hot water clearly had greater viability than those in cold water but viability was still clearly lower than in warm water with an average viability of 56.7% (27.7-81.1%; 95% credible interval) in hot 64 [A] [B] [C] [A] [A] [A]

Figure 24. A comparison of egg volume (mm3) for eggs produced by Figure 25. Comparison of the percent viability of egg masses laid by specimens of Physella wrighti cultured at three different water specimens of Physella wrighti in three different water temperatures. The

o o temperatures: cold water = 13oC, warm water = 23oC, and hot water = 33oC. water temperatures are as follows: cold water = 13 C, warm water = 23 C,

o Egg volume was calculated using the equation for a prolate ellipsoid. The and hot water = 33 C. The letters [A], [B], and [C] indicate that each of the letter [A] indicates that each of the treatments are not clearly different from treatments are clearly different from each other with > 95% certainty. each other with > 95% certainty. 65 [A] [B] [C]

Figure 26. Comparison of the incubation period for embryos of Physella wrighti cultured in three different water temperatures. The water temperatures are as follows: cold water = 13oC, warm water = 23oC, and hot water = 33oC. The letters [A], [B], and [C] indicate that each of the treatments are clearly different from each other with > 95% certainty.

66 water. I found that egg masses laid in warm water clearly had the greatest viability and the egg masses laid in the cold water had the lowest viability (> 95% certainty).

3.3.5 Physella wrighti Embryo Incubation Period

I defined incubation period as the time from when an egg mass was first laid to the time when the first snail hatched from an egg. I observed that the incubation period differed greatly between the water temperatures (Figure 26). The minimum incubation period I observed occurred in hot water and took four days, whereas the maximum incubation period occurred in cold water and took over 45 days. While the incubation period between temperatures varied, the incubation period within each temperature remained relatively clustered. I found that as temperature increased, the developmental period decreased across the three temperature treatments (> 95% certainty). I found that cold water, on average, prolonged the developmental period of P. wrighti approximately 6.8 times compared to hot water. Moreover, incubation time in hot water was still approximately1.6 times shorter than in warm water. While incubation in warm water was not as fast as hot water, incubation time in warm water was still approximately 4.1 times faster than in cold water. The average incubation time of embryos in cold, warm, and hot water was 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.

3.4 Discussion

The ability of P. wrighti to tolerate a range of water temperatures is surprising considering the specialized hot-water environment in which it is found. By exploring

67 how water temperature could impact this species’ life history, I found that the incubation period for eggs was the shortest in hot (33oC) water and P. wrighti reared in hot water produced fewer egg masses, relative to cold (13oC) and warm water (23oC).

Additionally, I found P. wrighti produced fewer eggs per mass in warm and hot water relative to cold water; however, P. wrighti in cold water demonstrated reduced egg viability. In contrast, I found no difference in egg size among temperature treatments.

These results suggest that P. wrighti may only be tolerating the high temperatures of the hot springs, and may instead have better reproductive output in slightly cooler water temperatures than the conditions in its current habitat.

Water temperature indirectly influences egg-laying because if the water temperature is not in the right range, the rate of development for the new generation will not occur in sync with the seasons (Duncan, 1959). Water temperature directly affects egg-laying due to the effects on the biological function of the parent generation. Reproduction by snails in cold temperatures can be slowed or halted entirely. By exposing wild-caught specimens of Physa fontinalis to three temperatures in the lab, Duncan (1959) demonstrated that when exposed to 18oC water, P. fontinalis produced egg masses regularly; but, egg mass production was reduced at 8oC, and slowed to near zero when the same snails were exposed to 4oC. I found similar patterns in P. wrighti, as snails in the warm water laid egg masses consistently, whereas those in the cold tanks laid far fewer egg masses. At low enough temperatures, snails have been shown to put more energy into growth than reproduction (e.g. Physella virgata (also referred to as Physa virgata), Britton and McMahon, 2004) as the rate of metabolic function is reduced to the point where organisms are simply surviving (Hubendick, 1958). Once the threshold is 68 met so that snails do not expend energy solely on growth and survival, snails will begin producing offspring with reproduction increasing as the biological function increases. In my experiments, I found egg masses in only two of the three cold water aquariums.

Notably, these two aquariums with egg masses were the ones located on the top shelf in the temperature-controlled refrigerator which measured 12oC-14oC, while the third tank, with no egg masses laid, was on the bottom shelf and measured 2oC-4oC cooler (8oC-

11oC). It is, therefore, feasible that the lower temperature may have been too cold for snails to produce egg masses, but was not sufficiently cold to cause death as these snails seemed to otherwise behave similarly to all the other snails. These results indicate a possible reproductive threshold between the 13oC water and the 8oC-11oC water for P. wrighti, but further study is needed at a lower water temperature range to verify.

When water temperature becomes too high, the reproductive output may be reduced due to thermal stress. Van der Schalie et al. (1973) found egg mass production in L. emarginata increased as water temperature increased to the optimal survival temperature for the snail. Once the water temperature reached or exceeded the snail’s tolerance range, egg mass production ceased. Physella wrighti seems to have produced egg masses similarly such that the number of egg masses laid increased with water temperature to a point; then, in the hot water, egg-laying almost completely stopped. Britton and

McMahon (2004) found that when water temperature became too high for P. virgata, to deal with the increased requirement for biological maintenance, the energy was redirected from reproduction and growth to survival. Hence, the lack of egg masses in the hot water aquariums is suggestive of thermal stress. Another example suggesting thermal stress comes from a study on P. wrighti (Lee and Ackerman, 1999), which found that 69 specimens kept in 28oC water more regularly laid eggs above the water level, where the temperature was 25oC. The warmer water may have been too hot for snails to reproduce, but because snails were able to find a suitable temperature within their environment, reproduction still occurred. The lack of egg mass production at 28oC indicates that even a water temperature five degrees lower than the hot water (33oC) I tested may be too hot for P. wrighti to produce egg masses. This supports the idea that the lack of egg masses produced by P. wrighti in the hot water temperature is most likely a result of thermal stress. It seems that the water temperature of the Liard Hot Springs may be at the maximum limit for the reproductive capabilities of P. wrighti.

The number of eggs laid per egg mass contributes to the reproductive capacity of snails in general. While I did not expect to find a difference in the number of EPM as a response to temperature treatment, I found that snails kept in the cold water laid as many as two times more eggs per mass than snails kept in the warm and hot water treatments, but no difference between the warm and hot water treatments. The relationship between the number of EPM may be species dependent. McMahon (1975) studied two natural populations of Physella virgata; one living in artificially warmed water from heated discharge and the other living in unaltered water, and found those in the warmer water produced fewer EPM than those in cooler water. By contrast, van der Schalie et al.

(1973) found that the number of EPM increased as water temperature increased for populations of L. stagnalis studied in different water temperatures in the lab. Physella wrighti somewhat shows an inverse relationship between EPM and water temperature as the EPM decreased as temperature increased from cold to warm. However, the lack of difference between warm and hot water does not aid in supporting this trend. It is 70 possible that when snails are approaching death they may be producing as many eggs as physiologically possible (Emlen and Zimmer, 2020). I tested snails at three different temperatures spanning 20 degrees. I may not have tested enough different temperatures to show fully the inverse relationship between temperature and EPM, but it was enough to indicate the relationship existed. The lack of difference between warm and hot water treatments may be due to the snails being thermally stressed, resulting in snails producing as many eggs as possible. Thus, the snails produced the same average number of EPM in both warm and hot water. Further testing is required to understand the relationship or lack thereof between EPM and water temperature.

The decreased number of EPM in warmer water could indicate that P. wrighti is better suited to a cooler environment than found at LHS; more EPM means more offspring, assuming the egg mass are all viable. Despite the evidence that P. wrighti produces more eggs per mass in cold water, the reproductive capabilities of this snail are still lower in cold water when taking into consideration the overall number of eggs laid in each water temperature. Physella wrighti produced the lowest number of eggs in the hot water and the highest in the warm water. While the number of eggs per mass was greater in the cold water environment, the snails were still capable of laying more egg masses in the warm water environment. Additionally, even though there was no difference between the number of eggs per mass between the warm and hot water, snails in the warm water produced more eggs than in the hot water. It is becoming more evident that P. wrighti may be better suited to surviving in water temperatures that are warmer than in most environments in Canada, but not as hot as the water in which it currently resides.

71 Incidentally, I found that the number of eggs laid per egg mass varied in general, regardless of the water temperature. I reviewed the previous study performed on P. wrighti by Lee and Ackerman (1999) to determine how similar or different my results compared to one of the few previously observed aspects of P. wrighti’s life history. Lee and Ackerman (1999) found that a small population of P. wrighti in 28oC water laid egg masses containing 6-18 eggs. Notably, the eggs were laid above the waterline where the air temperature was 25oC. The range of EPM observed by Lee and Ackerman (1999) is narrower than the number that I observed across all temperatures, ranging from 1-24 eggs per mass. To provide a more direct temperature comparison between the numbers of eggs per mass, snails in the warm water treatment (which corresponds closest to the temperature provided by Lee and Ackerman (1999)) laid 1-14 eggs per egg mass. I believe this to be reasonably similar to Lee and Ackerman’s (1999) findings and suggest that the difference is a reflection of the difference between the cultured populations.

Even so, it is evident that, overall, P. wrighti is capable of laying a greater range of eggs than indicated previously, and it is necessary to examine the snail’s life history in a range of water temperatures.

I found there to be no difference between the egg volumes for any of the temperatures; hot water eggs were slightly larger but were not clearly different. The slight increase in size may have been from the lower number of eggs sampled, as egg size did range from large to small in both the cold and warm water tanks. Lam and Calow (1989) studied egg volume in Lymnaea peregra and found the eggs produced in warmer temperatures were significantly larger than those from one of the two natural sites in one year but found no difference between all three sites in the following year. Overall, they concluded that egg 72 size was unlikely affected by temperature and that any differences may have been due to other conditions or chance. These results agree with my observations, as they are effectively mirrored by the lack of difference in the size of eggs produced by P. wrighti across the temperature treatments.

In contrast to egg size, egg viability is impacted by water temperature, with the highest viability in warm conditions. Researchers such as Lam and Calow (1989), Costil (1997), and Vaidya and Nagabhushanam (1978) have examined the percent of snails hatched from egg masses with regards to temperature, which closely relates to how I assessed viability for P. wrighti. In considering the relationship between percent hatching rate and water temperature, it is apparent that the hatching rate may or may not be related to water temperature depending on the species of snail. For instance, L. peregra, Planorbarius corneus, and Planorbis planorbis did not exhibit a relationship between water temperature and hatching rate (Lam and Calow, 1989; Costil, 1997). Alternatively,

Indoplanorbis exustus did show a relationship between water temperature and hatching rate, such that the hatching rate increased with water temperature, and when the water temperature becomes too hot for I. exustus, the hatching rate decreases (Vaidya and

Nagabhushanam, 1978). This is similar to what I observed in P. wrighti. Moreover, it would make sense to observe a difference in mass viability with regards to temperature, for the same reasons discussed in the number of egg masses produced by P. wrighti. If water temperature is too high or too low, then the biological processes required for development become disrupted or altered (Hubendick, 1958; Thomas and McClintock,

1990; Britton and McMahon, 2004).

73 In further consideration of percent viability, it is possible that the values I obtained for average percent viability are an underestimate. While studying L. stagnalis, Vaughn

(1953) found that when egg masses were placed in cylindrical tubes (covered on either end with fine mesh cloth and floated in aquariums), the percent hatch of egg masses was not optimized, but when masses were instead placed in cellophane dialyzing tubing the percent hatch was nearly 100%. Vaughn (1953) attributed this to insufficient aeration in the original tubes, whereas the dialyzing tubing allowed for better air penetration. I attempted to ensure that oxygen was available in all temperature treatments by aerating all the aquariums; however, the containers I used to keep egg masses may not have allowed for sufficient aeration of the egg masses. However, I ensured all aquariums were aerated and used the same types of egg mass holding containers in all aquariums.

Vaughn (1953) notes dissolved oxygen (DO) as being a factor in lowering hatch rate, thus even if my viabilities were low, the viability would still be relative across all temperature treatments and does not explain the differences in viabilities that I observed.

Thus, it is possible that my estimates of viability are low, but the relationship I observed of the greatest viability in warm water remains relative across temperature treatments.

Further examination of the impact of DO on egg mass viability should be considered to better understand the sensitivity of developing snails to different levels of DO.

If reproductive output is measured as the total number of eggs multiplied by egg viability, snails had the highest fitness in the warm temperatures. In cold water, snails laid two times more eggs than warm and hot water snails, but the overall number of egg masses laid was clearly less than that of warm water but greater than hot water. Earlier, I suggested that warm water appears more optimal based on the total number of eggs laid; 74 this combination far outnumbered that of the cold and hot water. Based on the current observed trend in percent viability, even if the snails in all tanks had laid a similar number of egg masses, resulting in cold water laying two times more overall eggs, the viability of those eggs is more than two times lower in the cold water than in the warm water, and two times lower than the hot water, resulting in fewer viable offspring than the snails in the warm water. Continuing this comparison with the hot water, one sees that snails in hot water would still reproduce less when compared to warm water.

Interestingly, snails in the hot water may produce an equal number of viable offspring compared to the snails in the cold. The overall number of eggs laid was greater in the warm water; therefore, P. wrighti living in warm water will produce more offspring than

P. wrighti living in cold or hot water and will produce more offspring in cold water than in hot water. This suggests that hot water is potentially the least suitable habitat for snail reproduction.

In studies of aquatic snails, water temperature inversely affects the incubation time of gastropods, within the survival limits for the species (DeWitt, 1954b; Duncan, 1959;

Thomas and McClintock, 1990; Costil, 1997; Zukowski and Walker, 2009). Based on my observations, and those of Lee and Ackerman (1999), we can conclude that the incubation period of P. wrighti in water 23-28oC is approximately eight days, with the length of time increasing as temperature decreases. Cold water prolongs the development period of snail embryos, whereas warm and hot water are very effective at reducing the incubation period. Similar to P. wrighti, the incubation period for P. gyrina in room temperature water (20oC-30oC) is approximately seven to eight days, but drops to approximately 5.5 days when the water temperature is 30ºC (DeWitt, 1954b). Thomas 75 and McClintock (1990) demonstrated the inverse relationship of water temperature and incubation period with Physella cubensis by showing the longest incubation period took

31 days at 10oC, the shortest took nine days at 30oC, with an incubation period at 20ºC of

13 days. Duncan (1959) declared that, for P. fontinalis, the water temperature was the most important aspect affecting development speed. Physella acuta, Glyptophysa gibbosa (Planorbidae), P. corneus, and P. planorbis also show the same relationship between incubation period; the former studied by Zukowski and Walker (2009), and the latter by Costil (1997). Increased temperature causes an increased rate of biochemical processes during embryo development (Costil, 1997), whereas decreased temperature reduces this rate (Hubendick, 1958), thus altering incubation periods. Consequently, it is more advantageous for most snails, P. wrighti included, to produce eggs in warmer water than cooler water (excluding CTmin and CTmax) as decreased incubation time increases population numbers more quickly.

Taken together, P. wrighti has greater reproductive capacity in warm water than in either cold or hot water; it lays more egg masses, has greater percent viability for masses, and a generally short incubation period. Because P. wrighti’s reproduction is reduced in hot water, water that is slightly cooler than that in which it currently lives may be more advantageous for the survival of the species. The currently reported water temperatures in which P. wrighti exist and copulate do not appear to be specific to the exact spots in which snails are found, rather the temperatures appear to be applied to the overall water body in general (e.g. Alpha Pool, and Alpha Stream) Liard Hot Springs (Te and Clarke,

1985; Lee and Ackerman, 1999; British Columbia Ministry of Environment, 2014; personal observation). This is relevant because water bodies are known to form 76 thermoclines (Birge, 1897 as cited in Sundram and Rehm, 1971), and I have noted there to be thermoclines in Alpha pool, particularly during January (personal observation);

With the temperature differential between the incoming water temperature of ~50oC and an air temperature of -30oC, there are going to be different thermal habitats throughout the pool (Wetzel, 2001). Hence, there may be microhabitats forming in the hot spring pools and outlet that may actually be slightly cooler in temperature. If this is the case, then P. wrighti may appear to reproduce in warm water when, in reality, the water temperature is cooler. Already, the trends I presented in this chapter have implications for the designation of P. wrighti as a hot water specialist snail (COSEWIC, 2008b); they may not require hot water to survive. Determining the presence of microhabitats may further alter our understanding of P. wrighti, and would be useful in understanding the distribution of the snail within its current environment.

77 CHAPTER 4: GENERAL DISCUSSION AND CONCLUSION

4.1 Discussion

Physella wrighti is a challenging but rewarding species to be able to study. The following discussion dialogues some of the challenges of performing life history studies, including the challenges I faced in rearing P. wrighti and briefly notes some of the current research occurring on this snail. Also addressed are ecological considerations for this snail; how it may contribute to the hot springs, and the potential threat of invasive species on the existing population and the threat this species may pose as an invader. I wrap up the discussion with a summary of the overall results, a consideration of how the results may impact the population dynamics of this snail, and my thoughts on future research to be accomplished.

4.1.2 Challenges of Studying Aspects of Life History in Gastropods

When attempting to establish a knowledge base of ecological information for species, there will be challenges in gathering desired and comparable data simply because nature is unpredictable and subject to many influences. Gathering information on gastropods is no exception. For example, van der Schalie et al. (1973) had some difficulty culturing

Helisoma anceps (Planorbidae), Helisoma campanulatum (now known as Planorbella campanulatum), and Amnicola limosa (Amnicolidae) (now known as Amnicola limosus) in laboratory conditions. While van der Schalie et al. (1973) did not explicitly state a particular cause for the culturing difficulties, they suggested it could have been from the presence of toxic algae or other organisms in the aquariums; Dillon (2004) suggested the

78 difficulties experienced by van der Schalie et al. (1973) may have been due to unsuitable diet or water conditions. Lee and Ackerman (1999) maintained Physella wrighti in a lab setting for over a year. However, they had difficulty maintaining tank conditions because the substrate from the Liard Hot Springs (LHS) that they used was almost impossible to clean, resulting in an almost complete loss of snails. Unfortunately, Lee and Ackerman

(1999) did not describe the population metrics that they measured in their studies, so I was unable to compare the success of my experiments to theirs.

Throughout my study I found it difficult to rear P. wrighti in the lab. I attempted to replicate water conditions from the LHS by including populations of the algae Chara vulgaris¸ which was present in the snail’s habitat, and a small amount of substrate from the hot springs (to mitigate the difficulties noted by Lee and Ackerman (1999)) and tufa

(a type of porous rock formed by the precipitation of calcium carbonate (Ford, 1989)) in the aquariums, but population size remained low. Generally, I maintained five to ten adult snails per stock aquarium; attempts to stock more than 10 snails resulted in the death of individuals, and a reduction to 10 surviving individuals suggesting a carrying capacity of approximately 10 adult snails per aquarium, although this was a much lower density than observed in the field. However, I did observe juveniles in the aquariums, suggesting snails were reproducing. It seems possible that something was missing from my set up that is required by P. wrighti for a larger population to be established; however, what this is remains unknown. Despite this, I was successful at rearing P. wrighti for periods of sufficient length to collect data to address aspects of this snail’s life history.

Another challenge in understanding gastropod life history is that laboratory-based studies

79 may not be reflective of a snail’s true biology; they may not be repeatable with any degree of accuracy, showing only how the snail responds to its particular environment at that moment in time (Dillon, 2004). This was demonstrated in a study by van der Schalie et al. (1973) that examined how temperature alters the growth and reproduction of several different species of snails. Despite running a series of lab experiments on effects of temperature on life history, data on the number of egg masses and eggs produced at temperatures that differed by less than 1ºC, were vastly different (van der Schalie et al.,

1973). DeWitt (1954a) encountered similar difficulties in replicating experiments;

Physella gyrina reared in a lab had different egg mass numbers, eggs per snail, and viabilities than snails in the field. Van der Schalie et al. (1973) also examined P. gyrina within the lab and their lab results differed from the lab results of DeWitt (1954a). More pertinent to this study is the difference in the number of eggs per mass that I observed P. wrighti produce (1-24 eggs per mass), compared to Lee and Ackerman’s (1999) observed

6-18 eggs per mass. It is apparent that one lab-based life history study alone is not definitive regarding the true biology for any snail species; however, I suggest that if we run many of these life history studies, each study contributes, and brings us closer, to the true biology of a species. Lymnaea stagnalis, Lymnaea palustris (now known as

Stagnicola palustris), P. gyrina, and Physella integra are all reported to have a yearly juvenile population dynamic that is characterized as increasing with juvenile recruitment then decreasing sharply with juvenile mortality (Brown, 1979). Thais lamellosa

(Muricidae) (now known as Nucella lamellosa) was reported to have a <1% hatchling survival rate even after accounting for predation and environmental stress in a natural setting (Spight, 1975). Costil (1994) examined juvenile survival in different temperatures

80 for Planorbarius corneus and Planorbis planorbis and found mortality to be high across all temperatures. Thus, it is plausible that the high juvenile mortality rate for P. wrighti is natural, and that a greater number of juvenile snails would result in enough surviving juveniles to assess the aforementioned elements of P. wrighti’s life history.

4.1.3 Current Opinions on Taxonomy and Physella wrighti as a Distinct Species

Te (1978) believed that there were five major groups within the snail family Physidae: the “Aplexa”, “fontinalis”, “gyrina”, “acuta”, and “cubensis” groups. He distinguished these groups by penial morphology and shell characteristics; and, based on such characteristics, he classified P. wrighti as being most closely related to species in the cubensis group. Te (1978) characterized the cubensis group as having a shell with a total length typically no longer than 10 mm, appearing polished or shiny, and a shape that is elongate-ovate, with an expanded penultimate whorl, ear-shaped aperture, and acutiform spire. The penial morphology was classified as physa type ‘bc,’ which was considered to exhibit characteristics of both the gyrina group penial morphology physa type ‘b,’ and the acuta group penial morphology physa type ‘c.’ Te (1978) described the penial morphology as having a preputial gland noted to have a two-part (i.e. glandular and non- glandular) penial sheath. The glandular portion is very short and the non-glandular potion is a thin, transparent wall, is turgid in appearance, ends in a solid bulb, and has a sheath up to 1.5 times the length of the preputium. Since Te’s (1978) classification, there have been multiple studies performed on the genetic relationship between P. wrighti and other snail species, including Physella johnsoni and P. gyrina (Remigio et al., 2001;

Wethington and Guralnick, 2004; Wethington and Lydeard, 2007).

81 In examining the available research on the genetic relationship between P. wrighti and other snails, one can see there is debate on the relationship of P. wrighti to other snail species, specifically whether P. wrighti is a separate species, or a subpopulation of

Physella gyrina that has adapted to living in a hot spring environment (Remigio et al.,

2001; Wethington and Guralnick, 2004; Wethington and Lydeard, 2007). Te and Clarke

(1985) believed that P. wrighti was not closely related to any other species of physid, and has existed as a species from before the last glacial period, making the snails important for studying evolutionary history. Remigio et al. (2001), using the mitochondrial DNA gene cytochrome c oxidase I (COI) and the 16S ribosomal RNA gene, suggest that P. wrighti is likely a more recently derived species that evolved from a different species of snail that survived in the glacial refugium surrounding what is now the LHS.

Additionally, Remigio et al. (2001) concluded that, while P. wrighti is a more recently evolved species, P. wrighti may be the ancestral species for P. gyrina and P. johnsoni, which have also evolved recently. Remigio et al. (2001) suggested this relationship based on the levels of genetic divergence between P. wrighti and P. gyrina (1.2-1.7% for

COI, 0.6-1.4% for 16S), and P. wrighti and P. johnsoni (1.4-1.9% for COI, 0.6-1.6% for

16S) being greater than the divergence between P. gyrina and P. johnsoni (0.5-1.2% for

COI, 0.04-0.4% for 16S). Wethington and Guralnick (2004), also using COI and 16S genes, suggest that each of P. wrighti (LHS), P. johnsoni [Banff Hot Springs (BHS)],

Physella aurea (Hot Springs, Bath County, Virginia, USA), Physella wolfiana (Hot

Springs, Colorado, USA), and P. gyrina (non-hot springs species) are all P. gyrina because their genetic divergence is not enough (approximately 6%) to justify each of the noted species as separate. Thus, Wethington and Guralnick (2004) concluded that P.

82 wrighti is not ancestral to P. gyrina, and is, therefore, not a relict species. More recently,

Wethington and Lydeard’s (2007) phylogenetic analysis suggests that P. gyrina is the parent species to both P. wrighti and P. johnsoni, supporting that P. wrighti is not an old species. Remigio et al. (2001) stated that there is no set measurement in genetics for how much difference between genes is required to have separate species. An interpretation presented in the COSEWIC (2008b) report for P. wrighti suggested that the genetic sequences are so similar because P. wrighti, P. gyrina, and P. johnsoni are all relatively recently evolved. Placing P. wrighti as the ancestral species to P. gyrina and P. johnsoni explains both the genetic similarities and differences observed between the three species, as species that evolved earlier exhibit greater genetic distance than related species that are more recently diverged (COSEWIC, 2008b).

To understand the relationship of P. wrighti with other snail species better, there is genetic sampling currently occurring on P. wrighti and other snail specimens collected from areas surrounding LHS. These findings are being compared with genetic samples from throughout western North America (Sheffield, 2020, personal communication).

4.1.3 Ecological Considerations for the snail Physella wrighti

4.1.3.1The Potential Ecological Roles of Physella wrighti in the Liard Hot Springs

Ecosystem

Currently, we know very little about the ecological role that P. wrighti plays in the LHS

(Lee and Ackerman, 1999; Salter 2001; COSEWIC, 2008b; DFO, 2010). Due to the close relationship between the Banff Springs Snail (P. johnsoni) and P. wrighti (Remigio

83 et al., 2001; Wethington and Guralnick, 2004; Wethington and Lydeard, 2007), and the similarity of the habitat they exist within, considerations for P. johnsoni are often used as substitutions for missing information on P. wrighti (Heron, 2007; Laurzier et al., 2011;

British Columbia Ministry of Environment, 2014). It has been suggested that P. johnsoni could be providing significant services to the BHS through grazing that impacts the microbial populations of the hot springs (Hebert, 1997, as cited in Lepitzki et al., 2002;

Lepitzki, 2002; Lepitzki and Pacas, 2010). Lepitzki et al. (2002) also suggest that the microorganisms in the hot springs could be dependent on the waste and shell materials produced by P. johnsoni. They suggest that losing these snails would cause significant damage to the hot spring ecosystem. In this way, P. johnsoni could be considered a keystone species (Lepitzki, 2002), a species whose loss would result in a significant alteration to the ecology of an environment (Meffe and Carroll, 1997). Clearly, P. johnsoni is considered an important organism within the BHS, and as such, P. wrighti is likely just as important in the LHS.

Since a major ecological role of P. johnsoni may come from the snail’s diet, I posit that this would also be the case for P. wrighti within the LHS. While it is currently unknown what exactly P. wrighti eats, it is accepted that it consumes a variety of microorganisms

(Lee and Ackerman, 1999, Laurzier et al., 2011). Hence, P. wrighti could be significantly altering the microbial populations present in the LHS. Through its excretions, P. wrighti may also be providing nutrients to both plants and microorganisms present. The dead shells of P. wrighti might be important to this ecosystem, through the materials that dissolve out of the shell, or from the shells providing habitats for other organisms. 84 Additionally, P. wrighti may act as competitors for nutrients and habitat with other snails present in the LHS, namely Gyraulus circumstriatus (Planorbidae), Stagnicola elodes

(Lymnaeidae) (now known as Ladislavella elodes), and Promenetus umbilicatellus

(Planorbidae) (Salter, 2001; Salter, 2003). These snails have been documented at various locations throughout the warm water swamp, making P. wrighti a potential competitor.

However, it is currently believed that the habitat of the other snails does not overlap with that of P. wrighti and thus do not present competition (Heron, 2007). This supposition is likely to change as more surveys of the hot springs are made. For example, colleagues and I recently observed the presence of a snail within the same habitat as P. wrighti (see

Appendix C for photos). We inadvertently brought back specimens of this snail to the lab in Chara samples, and unintentionally reared them in some of the stock aquariums with

P. wrighti. Interestingly, in aquariums where I saw hair algae (species not identified) develop, these snails appeared to survive better than P. wrighti, as the hair algae seemed to entangle P. wrighti, but not the other snails, which had a more narrow profile.

Specimens of this other snail have been collected, but, to my knowledge, have not yet been identified. With this in mind, P. wrighti could be a keystone species within the LHS by providing microbial population control, nutrients, habitats, and competition for other organisms.

While our knowledge to date is largely speculation, and we truly know very little about the role of P. wrighti within the LHS, progress is being made. In 2016, the first discovery of the presence of the hot spring mite Thermacarus nevadensis in Canada was published (Heron and Sheffield, 2016). This mite is an enigma, as it has no confirmed host (Heron and Sheffield, 2016; Heather Proctor, 2019, personal communication). 85 Several specimens of P wrighti collected in the field carried the larval stage of T. nevadensis which I observed upon placing snails in aquariums. Further examination indicated the larva were attached to the snails, which was tentatively confirmed to be the case and that the larva appeared to engorged on the snail (Heather Proctor, 2019, personal communication). Photos of specimens attached to P. wrighti can be found in Appendix

D. It has been tentatively confirmed that T. nevadensis is using P. wrighti as a host during larval development (Heather Proctor, 2019, personal communication). More research, such as histological analysis and aquarium studies, needs to be done to confirm this, but the early observations are promising (Heather Proctor, 2019, personal communication).

4.1.3.2 Invasive Species – Potential Threats to the Population of Physella wrighti with a note on the Potential of Physella wrighti as the invader

Introduced species can have negative impacts on native species through competition for habitat and nutrition, predation, diseases, and parasites may be a primary area of concern for the longevity of P. wrighti in LHS. Invasive species commonly enter an ecosystem through hitchhiking on equipment, an unsuspecting individual, and/or a transport/recreation vessel, as was the case with the introduction of Zebra Mussels

(Dreissena polymorpha) in the Great Lakes (Herbert et al., 1988). Other ways that species get introduced into an ecosystem are through attempts to manage other organisms, and through uninformed, but well-intentioned, pet owners. According to

Pullin (2002), native species that are isolated from predation and competition are the species most likely to be negatively impacted by the introduction of a non-native species

86 into an ecosystem. Physella wrighti seems to fall into this classification because it is seemingly endemic to a unique environment (COSEWIC, 2008b), has no documented direct predators (Lee and Ackerman, 1999; Salter, 2001; COSEWIC, 2008b; Fisheries and Oceans Canada, 2017), and the effects of competition remain unknown. Thus, the introduction of non-native species into the LHS could potentially have a direct and/or indirect impact (Fisheries and Oceans Canada, 2017). For example, a new predator could directly impact the population of P. wrighti by reducing or causing the extinction of the population. Snails, in general, have a long list of predators (Brown, 2001; Salter, 2001), so introducing new and potentially threatening organisms to this environment could greatly impact populations of P. wrighti; this snail may not be sufficiently adapted to survive active predation. Indirect negative impacts from a non-native species could come in the form of competition (Fisheries and Oceans Canada, 2017), resulting in less habitat and food than what is currently available for P. wrighti. To date, there have been two reported introductions of turtles into the LHS (Heron, 2007). Even though the species of turtles were not indicated, it is conceivable that the introductions were the result of pet owners trying to re-home their animals. This demonstrates that the introduction of other organisms to the hot springs is a real concern and that more introductions could, unfortunately, be a reality. Presumably, P. wrighti is a species that would be very sensitive to the introduction of non-native species into the LHS ecosystem. Thus, it is extremely important to prevent such incidences as the impact may be large and occur extremely quickly, leaving little to no opportunity for intervention.

There is also the potential that P. wrighti could be introduced into another habitat and become an invasive species. Based on my findings that P. wrighti survives and 87 reproduces in a range of water, I believe that this snail has the capability of surviving to reproduce in a range of habitats; however, it is not yet known if it would be able to survive in water of a different mineral content and chemical profile, or if predation will occur. There is potential for this snail to end up in personal aquariums due to its ability to survive at warmer water temperatures. This would only become detrimental if those people decided to shut down their aquarium and dump the contents into a water body other than the LHS; such incidents are not uncommon. I suggest that consideration should be given to the possibility of the public removing snails from the hot springs and the potential that this species could end up invading other ecosystems.

4.1.4 Summary of Findings

I began my study on this snail by looking for differences in diurnal and nocturnal behaviours. I concluded that there were no differences; P. wrighti seems to show arrhythmic activity by remaining equally active during dark and light periods. I next examined if P. wrighti displayed a preference for a particular water temperature, and found it is capable of withstanding a variety of temperatures, but generally seems to prefer water temperatures ~23oC. I established a series of aquariums set up at three different water temperatures, 13oC (cold), 23oC (warm), and 33oC (hot), and reared specimens of P. wrighti, again looking for any apparent diurnal/nocturnal differences in behaviours but found none. I found that snails in the hot water seemed to exhibit behaviours related to heat stress. P. wrighti more frequently left the water and crawled farther from the water than snails in the cold and warm temperatures. Snails in the cold water aquariums may also have been stressed, as they also left the water more frequently

88 than snails in the warm water. This behaviour may represent an attempt to move to a warmer location. Snails in the warm water did not appear to exhibit any different types of behaviour compared to snails in the other temperatures. Additionally, these snails rarely left the water, suggesting that those few instances when they did were simply exploratory activities. The overall behaviours appear to indicate a preference for warm water over hot or cold water.

I also examined how water temperature affected snail survival and aspects of its reproductive capabilities including the number of egg masses produced, number of eggs per mass, egg volume, egg viability, and embryo incubation period. Physella wrighti survived and reproduced in each of 13oC, 23oC, and 33oC water temperatures; however, snails cultured in the 33oC did not perform as well, overall, when compared to snails reared in 13oC or 23oC water temperatures. Snails reared in the hot water had the shortest survival (dying before the experiment finished), produced the lowest number of egg masses, and had the second-highest viability when compared to snails in the cold and warm water. I also found no difference in the number of eggs laid per mass between the snails in the hot and warm water. Notably, snails in the hot water had the shortest incubation period; but, from the lack of offspring produced, it is clear that hot water had a negative impact on P. wrighti’s fecundity. Comparatively, snails in the warm water survived the full length of the experiment (with the second-longest length of survival), had the second-fastest incubation time, produced the most egg masses, and had the greatest viability. Snails in the cold water survived the longest, produced the most eggs per mass, the second greatest number of egg masses, but had the lowest viability and the longest incubation time. There was no apparent difference in the egg volume between 89 treatments, suggesting that there is no effect of water temperature on the size of the eggs that P. wrighti produces.

Snails reared in the hot water experienced stress from the greater temperature. This is evident in their exiting the tank, lack of egg laying, and shorter survival time. It is common that as temperature increases, the rate of development and viability increases, but only to a point. Once this point is reached, both decrease (Vaidya and

Nagabhushanam, 1978; Thomas and McClintock, 1990; Costil, 1997; Zukowski and

Walker, 2009). There are contrasting trends among different species of snails when it comes to the number of eggs laid per mass. Some snails show an inverse relationship between the number of eggs per mass (EPM) and temperature, while others show an increase in EMP with an increase in temperature (van der Schalie et al., 1973; McMahon,

1975). Physella wrighti kept in the cold water clearly laid more eggs per mass compared to warm water. On its own, this could indicate that P. wrighti has an inverse relationship with the number of EPM and temperature. If this was the case, the hot water snails should lay fewer EPM compared to the warm water snails, assuming snails are not at the maximum of their temperature tolerance range. I did not observe any difference in the number of EPM laid by snails in the warm and hot water. It may be that, since the hot water appeared to have multiple negative effects on P. wrighti, stress on the snails caused the maximum physiologically output possible at high temperatures before death. Thus, snails produced more EPM resulting in the same EPM as warm water. Alternatively, the difference in EPM between warm and cold water may represent the maximum difference in the number of EPM that could be exhibited by P. wrighti. Hence, an inverse relationship may be observed at a range of temperatures between 13oC and 23oC. 90 The results from my research suggest that 33oC may be the threshold for the reproduction of P. wrighti. Although, at this temperature, the incubation time was reduced, egg viability was low, fewer egg masses were produced, adult snail survival was low, and stress behaviours were evident. This evidence suggests that the naturally hot water of the

LHS is not beneficial for the survival and reproduction of P. wrighti. This could be indicative that snails are migrating to different temperatures and that this snail does not require the hot water to carry out its life history.

Physella wrighti reared in cold water appeared more capable of carrying out their life history than I expected. The life span of these snails increased 34.5 times compared to those in hot water and 2.9 times compared to those in warm water. The cold water appeared to impact the snail’s reproductive capabilities negatively compared to the warm water snails but did not create the level of stress seen in snails in the hot water. The cold water snails laid fewer egg masses than the warm water snails, and showed, overall, the lowest viability, and longest incubation period. This pattern follows a common trend among snails, as decreases in water temperature slows physiological processes or completely prevent said processes from occurring (DeWitt, 1954b; Hubendick, 1958; van der Schalie et al., 1973; Clarke, 2014). Overall, P. wrighti raised in the cold water still produced more offspring than snails in the hot water, again supporting the idea that it might not require the hot water of the LHS.

Physella wrighti reared in the warm water had the greatest overall reproductive capacity of the tested temperatures. These snails had the greatest viability and produced the most egg masses, resulting in the overall greatest production of offspring compared to snails

91 reared in both cold and hot water. Although the incubation period of snails in the warm water was slower than the snails in the hot water, it was still considerably faster than snails in cold water. The number of eggs per mass was significantly less than in cold water, although no different than hot water; however, this had little impact on the reproductive capacity of P. wrighti in warm water as their viability was almost three times greater than that of snails in cold water. The snails in the warm water survived longer than snails in the hot water; however, the snails in the warm water did not survive for as long as those in the cold water. The warm water did not result in the longest survival; however, it did give snails an advantage in reproductive capacity.

In considering the results for survival, behaviour, number of egg masses, number of eggs per mass, egg volume, egg mass viability, and incubation period, hot water temperatures appear to cause the fastest development but otherwise appear to negatively impact P. wrighti’s life history. There seems to be a trade-off regarding cold and warm water; snails live much longer in cold water but have the greatest reproductive capacity in warm water. This, along with the behaviours of P. wrighti in the different temperatures, indicates that this species can handle a wide range of temperatures, and is better suited to surviving at water temperatures lower than its current habitat. Therefore, it is possible that P. wrighti merely tolerates the hot water temperatures, and there is another reason for its presence in the LHS.

4.1.5 Potential Impact of Temperature on Physella wrighti Reproductive Potential

Why P. wrighti tolerates the hot water temperatures of the LHS has many potential answers, including minimal predation, exclusive access to food and habitat, and/or 92 reproductive benefits over snails in non-hot spring environments. While reproductive benefits may seem contradictory to the evidence I have provided, snails within the hot spring may be reproducing in spots where there are unrecognized microclimates.

Although my research did not examine how P. wright would directly benefit from reduced predation and exclusive access to food/habitat, I can provide a rough estimate of the total recruitment over a year for P. wrighti compared to the closely related snail P. gyrina, using the general equations (2) through (4).

average % Recruitment average number average number mass viability = ( ) ( ) ( ) (2) per Generation of eggs per mass of masses 100

Generations 365 Days = ( ) (3) per Year Total Development Time

Generations Total Recruitment Recruitment per Year = (4) per Year per Generation

In equation (2), Recruitment is to the potential number of offspring produced by one snail in one generation (not considering factors of mortality), the average number of eggs per mass is the average number of eggs per mass that are produced by one snail, the average number of masses is the average number of masses that are produced by one snail, and

푎푣푒푟푎푔푒 % (푚푎푠푠 푣푖푎푏푖푙푖푡푦) is the average percent viability of a mass expressed as a decimal. In 100 equation (3), Generations per year is the total number of generations that could be produced over one year assuming there are no effects of seasonality on snail reproduction and Total development time is to the length of time it takes for offspring to develop from

93 when the parent snail laid the egg to when the offspring reach reproductive maturity. In equation (4), Total Recruitment per Year is the potential total number of offspring that a single snail could produce in one year, not considering factors of mortality.

To calculate the total number of offspring that one snail could produce in one year (4), I multiplied the potential recruitment produced by one snail in one generation by the total number of generations (2) that could occur per year (3). If there is a seasonal effect on the number of generations per year a snail could produce and the total generations per year is known, then Equation 3 is substituted for the already specified number of generations (e.g. in a snail’s natural habitat it normally only produces one generation per year, thus Total Recruitment per Year is equal to Recruitment per Generation * 1)

Most pulmonate snails have a life span of approximately one year, during which they reach maturity, reproduce, and die (Brown, 2001; Dillon, 2004). Additionally, many species experience seasonal pressures that result in the production of only one generation of offspring per year (Dillon, 2004), which effectively reduces annual offspring production. While several aspects of P. wrighti’s life history remain unknown, because of its close relationship to P. gyrina and P. johnsoni, I will use information from studies on P. gyrina reared in a laboratory setting (DeWitt, 1954a, 1954b, 1955) to extrapolate to

P. wrighti. With this information, I compare the reproductive capacity of the closely related P. gyrina with that of P. wrighti and hypothesize about the reproductive benefit that P. wrighti may experience by tolerating the warmer hot spring environment. I acknowledge that this is an extremely rough estimate as it does not account for aspects

94 such as naturally occurring mortality, predation, effects of parasitism, etc., and assumes that P. wrighti is reproducing in locations in the hot springs are approximately 23oC.

Physella gyrina generally inhabit water bodies that are exposed to seasonal temperatures, which limits this species to one generation per year. Physella gyrina is also semelparous, so once the mature snails reproduce they die. On average, P. gyrina produces approximately 18 egg masses (14 eggs per mass or 253 eggs total) per snail, with 73% viability. Thus, in one year, one snail is capable of producing approximately 184 offspring. Development to maturity typically takes 127 days (from when eggs are laid), with reproduction occurring in the spring, and snails reaching maturity by fall. This is significant because water temperature begins declining in the fall, which prevents the new generation from reproducing until water temperatures increase again in spring.

The number of generations of P. wrighti per year is unknown (Lee and Ackerman, 1999).

However, since there are no seasonal temperature limitations, and eggs and juveniles of the closely related P. johnsoni have been found year-round (Lepitzki, 2002), it is likely that P. wrighti is not limited to one generation per year, and snails of each generation will begin reproducing as soon as maturity is reached. P. wrighti is also, presumably, semelparous (Lee and Ackerman, 1999), thus limited to one reproductive period in its life span (Te, 1978; Te and Clarke, 1985); most freshwater pulmonate snails are semelparous

(Brown, 2001). There is also no documented information on the length of time it takes P. wrighti to reach maturity (Lee and Ackerman, 1999). For P. johnsoni, it is estimated that maturity is reached in approximately 69 days (63 days from hatching plus six days for incubation) (COSEWIC, 2008a), so it is reasonable to suggest that time to maturity is

95 similar for P. wrighti. Hence, at 23oC, a single specimen of P. wrighti is capable of producing 12 egg masses (60 total eggs), with a viability of 73%, resulting in 44 total offspring. Given that the total time to maturity is 71 days (63 days plus six days for incubation) and there are 365 days in a year, one parental snail can produce at least

164,916,224 new snails from one parent snail. Not all these snails would be present at the same time, as it is likely that the life span of P. wrighti is shorter compared to that of

P. gyrina due to the faster time to maturity, and semelparous nature of P. wrighti.

Physella wrighti is likely hindered by the warmer water temperature in terms of its life span, and, most importantly, total fecundity compared to P. gyrina, 58 eggs and 253 eggs respectively. Viability is approximately the same for both and, thus, the difference in the offspring produced in one generation remains comparable. Physella wrighti likely produce fewer offspring in one generation than P. gyrina; however, P. wrighti is not inhibited by seasonality and, thus, can reproduce year-round. Consequently, P. wrighti could naturally out-produce P. gyrina by 710,278:1. Such a fast generation time in P. wrighti would allow for greater genetic diversity within the population, and a faster rate of evolution, both of which are necessary for species survival in niche environments. It has been recorded for P. gyrina that if the seasonal temperature constraints are removed, the new generation of P. gyrina will reproduce as soon as maturity is reached (DeWitt,

1954a; Sankurathri and Holmes, 1976). Taken together, these observations indicate that, while there are some negative impacts on the individual, P. wrighti, as a species, benefits from the warmer water temperatures; thus, its presence in the hot spring environment is a function of the snail’s reproductive strategy.

96 4.1.6 Considerations for Future Research on Physella wrighti

There is still much to learn about P. wrighti. Additional studies on the thermal thresholds of P. wrighti may be useful in identifying potential habitats for this snail. For instance,

Clampitt (1970) found that, even though there was no particular difference in temperature preference between P. gyrina and P. integra, gyrina was better able to tolerate higher temperatures longer than P. integra, which perhaps excludes P. integra from some of the habitats in which P. gyrina is found. This concept may apply to wrighti and allow us to determine where it will definitely not survive.

In addition to the reproductive benefits that P. wrighti may be experiencing in the LHS, research needs to be done on the snail’s food source. In most cases, physids consume detritus and algal materials (Dillon, 2004). Studying the gut content of P. wrighti would offer some knowledge of what it is consuming and if they are direct competitors with other snail species. This information may also provide us with knowledge of specific areas or aspects of the LHS that need more rigorous monitoring to help protect this snail.

Throughout my visits to the LHS, I regularly observed P. wrighti leaving the water.

Since this is an aquatic snail species (Te and Clarke, 1985), leaving the water for prolonged periods potentially puts it at risk of desiccation. Behavioural studies may help us understand why P. wrighti behaves in this manner and the risks and benefits it may experience. Additional risks to desiccation come from dam and weir maintenance

(Heron, 2007), and complete structural failure (COSEWIC, 2008b). Understanding how

P. wrighti responds to drying could, for instance, influence how maintenance is

97 performed on the structures present in the Alpha Pool. Moreover, we may be able to develop a population recovery strategy for P. wrighti in the event of structural failure.

Not only would dam/weir failure expose snails to desiccation, but it would also result in sudden changes in temperature. Snails in the Alpha Pool would suddenly be cooled and snails downstream could suddenly be inundated with warmer water from Alpha pool.

Performing plop tests (i.e. exposing snails to sudden changes in water temperature (van der Schalie et al., 1973)), such as performed by van der Schalie et al. (1973), would help to assess any risk of temperature shock to P. wrighti by showing what temperatures shock is induced at and showing P. wrighti’s capacity to recover from such shock.

Further work is also needed on how the Thermacarus mite could be affecting P. wrighti.

Increasing our knowledge in this area has the potential to expand knowledge regarding parasitism of snails. Although we also need a better understanding of other ecological interactions that P. wrighti contributes to in this hot spring, studying how these two species interact will give us an indication of their significance to each other.

Through my research, I demonstrated that P. wrighti appears better suited to survive in water that is at the low end of the range and potentially cooler than the water it is currently found in. In addition to my methods, the optimum temperature can also be examined via gonad development (van der Schalie et al., 1973). It is possible to examine the point at which temperature has deleterious effects on gonad development (van der

Schalie et al., 1973). For example, L. stagnalis has no to very little gonad development at 6oC and 12oC and very high temperatures. Thus, even if egg masses and snails can

98 survive these temperatures, their ability to reproduce is hindered (van der Schalie et al.,

1973). Hence, this information would provide insight into the reproductive development of P. wrighti in different water temperatures.

In my experiments, I also attempted to explore how water temperature altered life span, growth rates, and time to sexual maturity in a subset of juvenile snails. Unfortunately, due to high juvenile mortality, I was unable to collect any data in these areas. Further studies on these aspects of P. wrighti’s life history would help increase the overall understanding of this snail. It would also help us determine how P. wrighti operates within the hot spring environment. High juvenile mortality appears to be a common trait in snails (Costil, 1994), so taking the time to study this aspect would allow us to understand better how the population of P. wrighti fluctuates over the course of a year.

The population of P. wrighti fluctuates greatly (Salter, 2001; Laurzier et al., 2011; British

Columbia Ministry of Environment, 2014; personal observation). Many factors, such as generational turnover, parasitism, and predation, could be affecting this. Population assessments following the outline of the British Columbia Ministry of Environment

(2014) study should continue so we can increase our understanding of how the population fluctuates throughout the year and the magnitude of these fluctuations. This will provide us with information on the stability of P. wrighti; however, with frequent and regular assessments, we may also be able to observe ecological interactions, such as predation, as well as other interactions that cannot be readily observed in a lab.

While I have attempted to fill in the knowledge gaps surrounding P. wrighti, many still remain, and we still have a poor understanding of this snail. Based on lab and field 99 studies of other snails, my results and how P. wrighti operates within its natural environment will likely differ. To understand this snail fully, it may be necessary to study it in situ, because a simulated environment is never the same as the natural environment. Clearly, there is still much work yet to be done, but my studies provide insight and offer further direction into research and management of this snail.

4.2 Final Conclusion

The Endangered hot spring snail Physella wrighti is a unique species of snail endemic to the Liard Hot Springs, Liard River Hot Springs Provincial Park, British Columbia,

Canada. The goals of my study were to help increase our knowledge of this mysterious snail, evaluate whether it is truly a hot water specialist snail, and provide information that can aid researchers and conservation specialists working with this amazing species.

Generally speaking, P. wrighti was believed to be a hot water specialist snail

(COSEWIC, 2008b), unable to tolerate or survive in water temperatures colder than 23oC

(DFO, 2010). I found that P. wrighti is not nearly as sensitive to cooler water temperatures as previously thought, and may actually prefer slightly cooler water temperatures than occurs in the LHS. Consequently, P. wrighti’s ability to exist in the

LHS may be a result of it tolerating the hot water at the small cost of one aspect of its life history; living in the hot water may decrease the reproductive capacity of one generation of snails, but the snails can produce many generations per year.

P. wrighti is potentially ecologically and evolutionarily significant. Due to P. wrighti being listed as Endangered, and endemic to the LHS, it is a fragile species and at risk of

100 extinction (Fisheries and Oceans Canada, 2017). Thus, it is extremely important to study and understand how this snail operates within its environment. Not only will this work help increase our scientific understanding of this snail, but it may also help increase our understanding of how other organisms operate within hot springs environments and set the framework for further studies of hot spring snails and snails in general.

101 REFERENCES

Arnold, W. 1965 [Accessed: Mar. 2020]. A Glossary of a Thousand-and-One Terms

Used in Conchology. The Veliger, 7(suppl.): 1-50. Available from: https://ia800708

.us.archive.org/6/items/veliger71965berk/veliger71965berk.pdf.

B.C. Conservation Data Centre. 2008 [Accessed: Jun. 2020]. Conservation Status

Report: Physella wrighti. B.C. Minist. of Environment. Available from: http://a100.gov

.bc.ca/pub/eswp/speciesSummary.do?id=19941.

B.C. Parks. 2010 [Accessed: 2017]. Liard River Hot Springs Provincial Park.

Available from: http://www.env.gov.bc.ca/bcparks/explore/parkpgs/liard_rv_hs

/liardriver.pdf.

B.C. Parks. 2020a [Accessed: Jun. 2020]. Summary of the Parks and Protected Areas

System. Available from: http://bcparks.ca/about/park-designations.html#:~:text=The

%20majority%20of%20the%20provincial,and%20enjoyment%20of%20the%20public.&t ext=Class%20A%20parks%20can%20be%20established%20by%20two%20means.

B.C. Parks. 2020b [Accessed: Jun. 2020]. Liard River Hot Springs Provincial Park.

Available from: http://bcparks.ca/explore/parkpgs/liard_rv_hs/nat_cul.html#History.

Bae, M., Chon, T., and Park, Y. 2015. Modeling Behavior Control of Golden Apple

Snails at Different Temperatures. Ecological Modelling, 306: 86-94.

102 Bal, N., Kumar, A., Du, J., and Nugegoda, D. 2016. Prednisolone impairs embryonic and posthatching development and shell formation of the freshwater snail, Physa acuta.

Environmental Toxicology and Chemistry, 35(9): 2339-2348.

British Columbia Ministry of Environment. 2009. Peace Region Management Plan for

Liard River Corridor Provincial Park and Protected Area and Scatter River Old Growth

Provincial Park - Draft.

British Columbia Ministry of Environment. 2014. 2012 and 2013 Hotwater Physa

(Physella wrighti) field studies at Liard River Hotsprings Provincial Park. British

Columbia Ministry of Environment, Victoria, British Columbia. viii + 57pp.

Britton, D. and McMahon, R. 2004. Seasonal and Artificially Elevated Temperatures

Influence Bioenergetic Allocation Patterns in the Common Pond Snail Physella virgata.

Physiological and Biochemical Zoology, 77(2): 187-196.

Brown, K. 1979. The Adaptive Demography of Four Freshwater Pulmonate Snails.

Evolution, 33(1): 417-432.

Brown, K. 2001. Mollusca: Gastropoda. In J. Thorp and A. Covich (Ed.), Ecology and

Classification of North American Freshwater Invertebrates (2nd ed.). Academic Press,

California: pp. 297-329.

Brues, C. 1924. Observations on Animal Life in the Thermal Waters of Yellowstone park, with a Consideration of the Thermal Environment. Proceedings of the American

Academy of Arts and Sciences, 59(15): 371-438.

103 Calow, P. 1978. The Evolution of Life-Cycle Strategies in Fresh-Water Gastropods.

Malacologia, 17(2): 351-364.

Campos, D., Gravato, C., Fedorova, G., Burkina, V., Soares, A., and Pestana, J. 2017.

Ecotoxicity of two organic UV-filters to the freshwater caddisfly Sericostoma vittatum.

Environmental Pollution, 228: 370-377.

Camsell, C. 1956. Son of the North. (3rd printing). The Ryerson Press, Toronto: pp. 71.

Clampitt, P. 1970. Comparative Ecology of the Snails Physa Gyrina and Physa Integra

(Basommatophora: Physidae). Malacologia, 10(1): 113-151.

Clampitt, P. 1974. Seasonal Migratory Cycle and Related Movements of the Fresh-

Water Pulmonate Snail, Physa integra. American Midland Naturalist, 275–300.

Clarke, A. 1981. The Freshwater Molluscs of Canada. National Museum of Natural

Sciences, National Museums of Canada, Ottawa.

Clarke, A. 2014. The Thermal Limits to Life on Earth. International Journal of

Astrobiology, 13(2): 141-154.

Clarke, A. 1974 [Accessed: Nov. 2018]. A Survey of British Columbian Freshwater

Mollusks: Preliminary Results. The American Malacological Union, Inc. Page 42.

Available from: https://www.biodiversitylibrary.org/item/217491#page/204/ mode/1up.

Clench, W. 1926. Three new species of Physa. Occasional Papers of the Museum of

Zoology, University of Michigan, 168: 1-8.

104 COSEWIC. 2008a. COSEWIC assessment and update status report on the Banff Springs

Snail Physella johnsoni in Canada. Committee on the Status of Endangered Wildlife in

Canada. Ottawa. vii + 53 pp.

COSEWIC. 2008b. COSEWIC assessment and update status report on the Hotwater

Physa Physella wrighti in Canada. Committee on the Status of Endangered Wildlife in

Canada. Ottawa. vii + 34 pp.

Costil, K. 1994. Influence of temperature on survival and growth of two freshwater planorbid species, Planorbis corneus (L.) and Planorbis planorbis (L.). Journal of

Molluscan Studies, 60: 223-235.

Costil, K. 1997. Effect of temperature on embryonic development of two freshwater pulmonates, Planorbarius corneus (L.) and Planorbis planorbis (L.). Journal of

Molluscan Studies, 63: 293-296.

Costil, K. and Bailey, S. 1998. Influence of Water Temperature on the Activity of

Planorbarius corneus (L.) (Pulmonata, Planorbidae). Malacologia, 39(1-2): 141-150.

DeWitt, R. 1954a. Reproductive Capacity in a Pulmonate Snail (Physa gyrina Say).

The American Naturalist, 88(840): 159-164.

DeWitt, R. 1954b. Reproduction, Embryonic Development, and Growth in the Pond

Snail, Physa gyrina Say. Transactions of the American Microscopical Society, 73(2):

124-137.

105 DeWitt, R. 1955. The Ecology and Life History of the Pond Snail Physa gyrina.

Ecology, 36(1): 40-44.

DFO. 2010. Recovery Potential Assessment for Hotwater Physa (Physella wrighti).

DFO Can. Sci. Advis. Sec. Sci. Advis. Rep. 2009/072

Diaz, F., Re, A., Medina, Z., Re, G., Valdez, G., and Valenzuela, F. 2006. Thermal preference and tolerance of green abalone Haliotis fulgens (Philippi, 1845) and pink abalone Haliotis corrugata (Gray, 1828). Aquaculture Research, 37(9): 877-884.

Diaz, F., Salas, A., Re, A., Gonzalez, M., and Reyes, I. 2011. Thermal preference and tolerance of Megastrea (Lithopoma) undosa (Wood, 1828; Gastropoda: Turbinidae).

Journal of Thermal Biology, 36(1): 34-37.

Dillon, R. 2004. The Ecology of Freshwater Molluscs. Cambridge University Press,

Cambridge.

Duncan, C. 1959. The Life Cycle and Ecology of the Freshwater Snail Physa fontinalis

(L.). Journal of Animal Ecology, 28(1): 97-117.

El-Emam, M. and Madsen, H. 1982. The Effect of Temperature, Darkness, Starvation and Various Food Types on Growth, Survival and Reproduction of Helisoma duryi,

Biomphlaria alexandrina and Bulinus truncates (Gastropoda: Planorbidae).

Hydrobiologia, 88(3): 265-275.

Elevate Environmental Inc. 2012. Environmental Menagement Recommendations –

Liard River Hot Springs Pool Deck and Change Room Construction Project. Prepared

106 for Bear Mountain Construction Ltd and BC Parks Ministry of Enviornment. BC Parks

File: 2945537. 6 pp.

Ellison, A. 2004. Bayesian inference in ecology. Ecology Letters, 7: 509-520.

Emlen, D. and Zimmer, C. 2020. Evolution: Making Sense of Life (3rd ed). Macmillan

Learning, Austin, Texas.

Fenwick, A. and Amin, M. 1982. Marking snails with nail varnish as a field experimental technique. Annals of Tropical Medicine and Parasitology, 77(4): 387-390.

Fisheries and Oceans Canada. 2017. Action Plan for the Hotwater Physa (Physella wrighti) in Canada [Proposed]. Species at Risk Act Action Plan Series. Fisheries and

Oceans Canada, Ottawa. iv + 22 pp.

Ford, T. 1989. Tufa: a freshwater limestone. Geology Today, 5(2): 60-63.

Formline Architecture. 2012 [Accessed: Nov. 2018]. Liard River Hot Springs

Replacement Facility. Available from: https://formline.ca/LIARD-RIVER

-HOTSPRINGS.

Garrity, S. 1984. Some Adaptions of Gastropods to Physical Stress on a Rocky Shore.

Ecology, 65(2): 559-574.

Getz, L., Chichester, L., and Burch, J. 2017. Land Mollusks of Northeastern United

States and Southeastern Canada. Malacological Review, 45/46: 227-285.

107 Gibson, K., Miller, J., Johnson, P., and Stewart, P. 2016. Toxicity of Sodium Dodecyl

Sulfate to Federally Threatened and Petitioned Freshwater Mollusk Species. Freshwater

Mollusk Conservation Society, 19(1): 29-35.

Grasby, S. and Lepitzki, D. 2002. Physical and Chemical properties of the Sulphur

Mountain thermal springs, Banff National Park, and implication for endangered snails.

Canadian Journal of Earth Sciences, 39: 1349-1361.

Grasby, S., Hutcheon, I., and Krouse, H. 2000. The influence of water-rock interactions on the chemistry of the thermal springs in western Canada. Applied Geochemistry, 15:

439-454.

GW Solutions Inc. 2010. Liard Hotsprings Groundwater Regime. A report prepared for

Fisheries and Oceans Canada, Vancouver British Columbia by GW Wolutions Inc.

Nanaimo, British Columbia. September. 17pp.

Heiler, K., von Oheimb, P.V., Ekschmitt, K., and Albrecht, C. 2008. Studies on the

Temperature Dependence of Activity and on the Diurnal Activity Rhythm of the Invasive

Pomacea canaliculata (Gastropoda: Ampullariidae). Mollusca, 26(1): 73-81.

Herbert, P., Muncaster, B., and Mackie, G. 1988. Ecological and Genetic Studies on

Dreissena polymorpha (Pallas): A New Mollusc in the Great Lakes. Canadian Journal of Fisheries and Aquatic Sciences, 46: 1587-1591.

108 Heron, J. 2007. Recovery Strategy for the Hotwater Physa (Physella wrighti) in Canada.

Species at Risk Act Recovery Strategy Series. Fisheries and Oceans Canada, Vancouver, vii + 27 pp.

Heron, J. and Sheffield, C. 2016. First Canadian record of the water mite Thermacarus nevadensis Marshall, 1928 (Arachnida: Acariformes: Hydrachnidiae: Thermacaridae) from hot springs in British Columbia. Biodiversity Data Journal, 4: e9550. DOI:

10.3897/BDJ.4.e9550.

Heurtault, J. and Vannier, G. 1990. Thermorésistance chez deus pseudoscorpions

(Garypidae), l’un du desert de Namibie, l’autre del la region de Gênes (Italie). Acta

Zoologica Fennica, 190: 165-172.

Hubendick, B. 1958. Factors Conditioning the Habitat of Freshwater Snails. Bulletin of the World Health Organization, 18(5-6): 1072-1080.

Hutchison, L. 1947. Analysis of the Activity Fresh-Water Snail, Viviparus Malleatus

(Reeve). Ecology, 28(4): 335-345.

Kavaliers, M. 1980. A Circadian Rhythm of Behavioural Thermoregulation in a

Freshwater Gastropod, Helisoma trivolis. Canadian Journal of Zoology, 58(11): 2152-

2155.

Koopman, K.R., Collas, F., van der Velde, G., and Verberk, W. 2016. Oxygen can limit heat tolerance in freshwater gastropods: differences between gill and lung breathers.

Hydrobiologia, 763: 301-312.

109 Kruschke, J. 2015. Doing Bayesian Data Analysis (2nd ed.). Elsevier – Avademic Press,

Amsterdam.

Lam, P. and Calow, P. 1989. Intraspecific Life-History Variation in Lymnaea peregra

(Gastropoda: Pulmonata). I. Field Study. Journal of Animal Ecology, 58(2): 571-588.

Laurzier, R., Davis, S., and Heron, J. 2011. Recovery Potential Assessment for Hotwater

Physa (Physella wrighti). Department of Fisheries and Oceans Canadian Science

Advisory Secretariat Research Document 2011/027, vi + 28 p.

Lee, J. and Ackerman, J. 1999. Status of the Hotwater Physa, Physella wrighti Te and

Clarke 1985. Committee on the Status of Endangered Wildlife in Canada. Ottawa. iii +

22 pp.

Lepitzki, D. 2002. Status of the Banff Springs Snail (Physella johnsoni) in Alberta.

Alberta Sustainable Resource Development, Fish and Wildlife Division, and Alberta

Conservation Association, Wildlife Status Report No. 40, Edmonton, AB. 29 pp.

Lepitzki, D. and Pacas, C. 2010. Recovery Strategy and Action Plan for the Banff

Springs Snail (Physella johnsoni), in Canada. Species at Risk Act Recovery Strategy

Series. Parks Canada Agency, Ottawa. vii + 63 pp.

Lepitzki, D., Pacas, C., and Dalman, M. 2002. Resource Management Plat for the

Recovery of the Banff Springs Snail (Physella johnsoni) in Banff National Park, Alberta.

Plan prepared for and Approved by Parks Canada, Banff National Park. 22 March.

48pp. + Appendix I (26pp), Appendix II (19pp), and Appendix III (13pp).

110 Lombardo, P., Miccoli, F., Giustini, M., and Cicolani, B. 2010. Diel Activity Cycles of

Freshwater Gastropods Under Natural Light: Patterns and Ecological Implications.

International Journal of Limnology, 46: 29-40.

Marsh, A. 1985. Thermal Responses and Temperature Tolerance in a Diurnal Desert

Ant, Ocymyrmex barbiger. Physiological Zoology, 58(6): 629-639.

Masson, M. 2011. A tutorial on a practical Bayesian alternative to null-hypothesis significance testing. Behavior Research Methods, 43(3): 679-690.

McClary, A. 1964. Surface Inspiration and Ciliary Feeding in Pomacea paludosa

(Prosobranchia: Mesogastropoda: Ampullariidae). Malacologia, 2(1): 87-104.

McMahon, R. 1975. Effects of Artificially Elevated Water Temperatures on the Growth,

Reproduction and Life Cycle of a Natural Population of Physa Virgata Gould. Ecology,

56(5): 1167-1175.

Meffe, G. and Carroll, C. 1997. Principals of Conservation Biology (2nd ed.). Sinauer

Associates, Sunderland, Massachusetts.

Muñoz, J., Finke, R., Camus, P., and Bozinovic, F. 2005. Thermoregulatory Behaviour,

Heat Gain and Thermal Tolerance in the Periwinkle Echinolittorina peruviana in Central

Chile. Comparative Biochemistry and Physiology, 142: 92-28.

NatureServe Explorer. 2020 [Accessed: Jun. 2020]. Physella wrighti Hotwater Physa.

NatureServe, Arlington, Virginia. Available from: https://explorer.natureserve.org

/Taxon/ELEMENT_GLOBAL.2.116672/Physella_wrighti.

111 Neuparth, T., Martins, C., de los Santos, C., Costa, M., Martins, I., Costa, P, and Santos,

M. 2014. Hypocholesterolaemic pharmaceutical simvastatin disrupts reproduction and population growth of the amphipod Gammarus locusta at the ng/L range. Aquatic

Toxicology, 155: 337-347.

Parks Canada. 2018 [Accessed: Nov. 2018]. Banff Upper Hot Springs – Water Flow.

Available from: https://www.pc.gc.ca/en/voyage-travel/promotion/sources-springs/ banff/debit-flow.

Peepre, J., Jordan, P., and Nathan, J. 1990. Liard River Hotsprings Provincial Park

Master Plan. Ministry of Parks, British Columbia.

Perez, K. and Cordeiro, J. 2008. A Guide for Terrestrial Gastropod Identification.

American Malacological Society-Terrestrial Gastropod Identification Workshop,

Southern Illinois University, Carbondale, IL.

Pullin, A. 2002. Conservation Biology. Cambridge University Press, Cambridge.

Reiser, S., Herrmann J.P., and Temming, A. 2014. Thermal Preference of the Common

Brown Shrimp (Crangon Crangon, L.) Determined by the Acute and Gravitational

Method. Journal of Experimental Marine Biology and Ecology, 461: 250-256.

Remigio, E., Lepitzki, D., Lee, J., and Hebert, P. 2001. Molecular systematic relationships and Evidence for a recent origin of the thermal spring endemic snails

Physella johnsoni and Physella wrighti (Pulmonata: Physidae). Canadian Journal of

Zoology, 79: 1941-1950.

112 Reynolds, W. 1979. Perspective and Introduction to the Symposium: Thermoregulation in Ectotherms. American Zoologist, 19(1): 193-194.

Reynolds, W. and Casterlin, M. 1979. Behavioural Thermoregulation and the Final

Preferendum Paradigm. American Zoologist, 19: 211-224.

Roskov, Y., Ower, G., Orrell, T., Nicolson, D., Bailly, N., Kirk, P., Bourgoin, T.,

DeWalt, R., Decock, W., Nieukerken, E. van, Zarucchi, J., and Penev, L. (eds.). 2019.

Species 2000 & ITIS Catalogue of Life, 2019 Annual Checklist – About the Catalogue of

Life. Species 2000: Naturalis, Leiden, the Netherlands. ISSN 2450-884X. Available from: http://www.catalogueoflife.org/annual-checklist/2019/info/about.

Roth, A. 1960. Aspects of the Function of the Bursa Copulatrix and Seminal Receptacle in the Prosobranch Snail Oncomelania formosana Pilsbry and Hirase. Transactions of the American Microscopical Society, 74(4): 412-419.

Russell-Hunter, W. 1961a. Annual Variations in Growth and Density in Natural

Populations of Freshwater Snails in the West of Scotland. Proceedings of the Zoological

Society of London, 136: 219-253.

Russell-Hunter, W. 1961b. Life Cycles of Four Freshwater Snails in Limited

Populations in Loch Lomond with a Discussion of Intraspecific Variation. Proceedings of the Zoological Society of London, 137: 135-171.

113 Salter, S. 2001. Management of Hot Water Physa (Physella wrighti) in Liard River Hot

Springs. Report submitted to British Columbia Parks Peace-Liard District, Ministry of

Environment, Lands and Parks. 23pp.

Salter, S. 2003. Invertebrates of Selected Thermal Springs of British Columbia. Final

Report, Habitat Conservation Trust Fund Project File #8-72, Contract #CTF02081.

Victoria, British Columbia. 90 pp.

Sankurathri C. and Holmes, J. 1976. Effects of thermal effluents on the population dynamics of Physa gyrina Say (Mollusca: Gastropoda) at Lake Wabamun, Alberta.

Canadian Journal of Zoology, 54: 582-590

Schmitt, C., Oetken, M., Dittberner, O., Wagner, M, and Oehlmann, J. 2008. Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus. Environmental

Pollution, 152: 322-329.

Seuffert, M., Burela, S., and Martín, P. 2010. Influence of water temperature on the activity of the freshwater snail Pomacea canaliculata (Caenogastropoda: Ampullariidae) at its southernmost limit (Southern Pampas, Argentina). Journal of Thermal Biology, 35:

77-84.

Species at Risk Act, S.C. 2002, c.29. Available from: https://laws-lois.justice.gc.ca/eng

/acts/s-15.3/page-1.html

Spight, T. 1975. On a Snail’s Chances of Becoming a Year Old. Oikos, 26(1): 9-14.

114 Stephens, P., Buskirk, S., Hayward, G., and Martìnez Del Rio, C. 2005. Journal of

Applied Ecology, 42: 4-12.

Strong, E., Gargominy, O., Ponder, W., and Bouchet, P. 2008. Global diversity of gastropods (Gastropoda; Mollusca) in freshwater. Hydrobiologia, 595: 149-166.

Sundram, T. and Rehm, R. 1971. Formation and Maintenance of Thermoclines in

Temperate Lakes. American Institute of Aeronautics and Astronautics Journal, 9(7):

1322-1329.

Te, G. 1978. A Systematic Study of the Family Physidae (Basommatorphora:

Pulmonata). Ph.D. diss., University of Michigan.

Te, G. and Clarke, A. 1985. Physella (Physella) wrighti (Gastropoda: Physidae), A

New Species of Tadpole Snail from Liard Hot Springs, British Columbia. The Canadian

Field-Naturalist, 99(3): 295-299.

Thomas, D. and McClintock, J. 1990. Embryogenesis and the Effects of Temperature on

Embryonic Development, Juvenile Growth Rates, and the Onset of Oviposition in the

Fresh-Water Pulmonate Gastropod Physella cubensis. Invertebrate Reproduction and

Development, 17(1): 65-71.

Trumpener, B. 2019 [Accessed: Jun. 2020]. “‘Frustrated’ wheelchair user calls for ramp at hot springs after he can no longer access pools”. CBC News. Available from: https://www.cbc.ca/news/canada/british-columbia/liard-river-hot-springs-no-wheelchair

-access-for-disabled-1.5200664.

115 Turgeon, D., Quinn J. Jr., Bogan, A., Coan, E., Hochberg, F., Lyons, W., Mikkelsen, P.

Neves, R., Roper, C., Rosenberg, G., Roth, G., Scheltema, A., Thompson, F., Vecchione,

M., and Williams, J. 1998. Common and Scientific Names of Aquatic Invertebrates from the United States and Canada: Mollusks (2nd e.d). American Fisheries Society Special

Publication, 26. Bethesda, Maryland.

Vaidya, D. and Nagabushanam, R. 1978. Laboratory Studies on the Direct Effect of

Temperature on the Freshwater Vector Snail, Indoplanorbis exustus (Deshyes).

Hydrobiologia, 61(3): 267-271. van der Schalie, H., Berry, E., and Mount, D. 1973. The effects of temperature on growth and reproduction of aquatic snails. Ecological Research Series. No. PB-221549;

EPA-R-3-73-021. Michigan Univ., Ann Arbor (USA).

Vaughn, C. 1953. Effects of Temperature on Hatching and Growth of Lymnaea stagnails appressa Say. The American Midland Naturalist, 49(1): 214-228. von Oheimb, V.P., Landler, L, and von Oheimb, K. 2016. Cold Snails in Hot Springs:

Observations from Patagonia and the Tibetan Plateau. Malacologia, 59(2): 313-320.

Wethington, A. and Guralnick, R. 2004. Are Populations of Physids for Different Hot

Springs Distinctive Lineages. American Malacological Bulletin, 19 (1/2): 135-144.

Wethington, R. and Lydeard, C. 2007. A Molecular Phylogeny of Physidae

(Gastropoda: Basommatophora) Based on Mitochondrial DNA Sequences. Journal of

Molluscan Studies, 73 (3): 241-257.

116 Wetzel, R. 2001. Limnology: Lake and River Ecosystems (3rd ed). Elsevier - Academic

Press, California.

White, D. 1957. Thermal Waters of Volcanic Origin. Bulletin of the Geological Society of America, 68: 1637-1658.

Zukowski, S. and Walker, K. 2009. Freshwater snails in competition: alien Physa acuta

(Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South

Australia. Marine and Freshwater Research, 60: 999-1005.

117 APPENDIX A –AQUARIUM SET-UP AND MAINTENANCE

A.1.1 Stock Aquarium Set-Up

I cultured specimens of P. wrighti in fully filled 10 gallon aquariums (Figure A.1). I maintained the water temperature for snails used in the Observed Period of Greatest

Activity - Constant Temperature and Determining the Temperature Preference for

Physella wrighti experiments at 30oC ± 2oC, as this was the middle of the field temperatures noted by Lee and Ackerman (1999). I kept the water temperature in stock tanks for the remaining experiments at 23 ± 2oC, because I determined this was the snails’ preferred temperature. I maintained the temperature using Fluval® M100 fully submersible aquarium heaters.

I lit the stock aquariums with one Sun BlasterTM T5HO bulb per aquarium to maintain a natural light spectrum. I kept all aquariums on a 12h light/dark cycle.

To prevent snails from crawling outside the aquariums, I added a liberal layer (2.5 cm wide) of Vaseline® 100% Petroleum Jelly to the top edge of each tank. This was unsuccessful; snails still crawled over the jelly layer and the jelly left a film on the surface of the water when splashed. As a result, I removed the jelly layer from all the aquariums. To prevent water from evaporating from the aquariums too quickly, I initially covered the aquariums with 6.35 mm thick clear acrylic plastic lids. This reduced water evaporation, but also reduced the amount of light entering the aquariums. To mitigate this, I cut the lids to allow for a 20 cm wide gap at the center of each lid. This allowed better lighting in the aquariums and reduced the rate of water evaporation. 118 Figure A.1 Various setups of the stock

aquariums used to keep Physella wrighti.

Photo A shows the stock tanks that

contained snails used for the Observed

Period of Greatest Activity - Constant

Temperature and Determining the

Temperature Preference for Physella

wrighti experiments. Photo B shows one

of the two stock tanks established for the

remaining experiments just before the

addition of the polishing pad (noted on

page 118). Photo credits: Erika

Helmond.

119 I used clean filter sand, purchased from a local aquarium supply store, as the substrate in the aquariums for the Observed Period of Greatest Activity - Constant Temperature and Determining the Temperature Preference for Physella wrighti experiments. I used silt from the LHS and clean filter sand in two of the aquariums but found both substrates exceedingly difficult to keep clean, as I could not easily separate either substrate from the snail poop. I placed a piece of Malaysian driftwood, purchased from aquarium stores, and Chara in some of the aquariums. Neither the presence nor absence of the driftwood and Chara, alone, or in combination, appeared to have any effects on the snails. Consequently, I added nothing to the stock aquariums containing snails for use in the remaining experiments; this was the most efficient way to maintain the stock aquariums, especially with the added maintenance of the experimental aquariums.

To maintain water quality and dissolved oxygen levels in the stock aquariums for the

Observed Period of Greatest Activity - Constant Temperature and Determining the

Temperature Preference for Physella wrighti experiments, for each stock aquarium, I initially used one Fluval®C2 filter, which contained a Fluval® C2 Poly/Foam Pad,

Fluval® C-Nodes (aka bio media), 45g of Fluval® Activated Carbon, and a Fluval® Bio-

Screen Pad. I set the filters to low power to reduce water currents and the number of snails sucked into the filters. Snails sucked into the filters survived; I regularly removed them during tank maintenance. I initially maintained filters by replacing the Poly/Foam

Pad and Carbon every five weeks. In aquariums containing Chara, I found it best to remove the Poly/Foam Pad, because it quickly became clogged with Chara particulates and required frequent changes. After discussions with an aquarium specialist in Regina,

S.K., and the Greater Vancouver Zoo, B.C., I removed the carbon from the filters; I 120 deemed it unnecessary in maintaining the aquariums. I observed no change in snail health after I removed the foam pad or carbon. Instead, I used a Fluval® Water Polishing

Pad wrapped in silk screening to provide a surface for beneficial bacteria growth

(typically a function of the substrate aquarium filter) and an airline and air stone to maintain dissolved oxygen and gentle water flow within the aquariums.

I performed weekly water changes with either reverse osmosis (RO), or a combination of

RO water and tap water treated with Seachem® Prime®, on all the stock aquariums. The water changes consisted of a repeating schedule of a 10% water change on weeks one and two and a 20% water change on week three, with larger water changes done as I deemed necessary. Initially, I only used a combination of RO and treated tap water during water changes; however, in the interest of creating water chemistry that better reflected the LHS water chemistry (as noted in Section 1.1 of Chapter 1), I supplemented the aquariums

® during water changes with CaSO4 and NYOS Alkalinity+. I judged these to be the most important factors of the water chemistry. I found these additions provided no benefit to the snails; they did not seem to respond to increases or decreases the measured level of said supplements. As a result, I ceased supplementing the aquariums and, instead, only used treated tap water to keep the measured water chemistry at levels I deemed reasonable.

I monitored water chemistry for ammonia (NH3), pH, general hardness (GH), nitrite

- ® 2+ (NO2 ), and phosphate (PO4), with test kits from Nutrafin , and for calcium (Ca ),

- - carbonate hardness (alkalinity (KH) or HCO3 ), and nitrate (NO3 ) with test kits from

® 3- - - Nyos . I tested aquariums for NH3, pH, PO4 , NO2 , NO3 , and GH every two weeks,

121 2+ 3- and Ca and KH once a week. I tested for PO4 regularly, but as levels remained

3- consistent during the first 8 months, I tested for PO4 only as I felt necessary.

Additionally, I tested for Ca2+ and KH after water changes to allow for accurate supplementation, but I also tested before water changes as I deemed necessary.

I removed solid waste materials from the aquarium using a fine-mesh fish net and a turkey baster. I rinsed all waste over silk screening to prevent the loss of live snails. I removed dead shells from the aquariums and preserved them in 60% ethanol.

A.1.2 Experimental Aquarium Set-up

To assess the period of greatest activity at varying temperatures, behaviour, survivorship, number of egg masses, number of eggs per mass, egg size, egg viability, and incubation period, I kept the snails in nine 2.5 US gallon aquariums. I fit six aquariums with a 50

Watt fully submersible aquarium heater and heated three to 23oC (warm) and three to

33oC (hot) (Figure A.2). I cooled the remaining aquariums to 13oC by keeping them in a

Marathon Deluxe 8.5cu.ft. All Refrigerator, modified with a Penn A421ABC-02 temperature controller to maintain the water temperature (Figure A.3). To each of the aquariums, I added approximately 2.5 cm of filter sand and a sponge filter. The sponge filter was composed of a Top Fin® Filter Cartridge (for Top Fin® Multi-stage Internal

Filter 10) with the activated carbon removed and replaced with an air stone (Figure A.4).

This provided gentle water currents and maintained oxygen in all the aquariums. To aid in maintaining water temperatures and preventing snails from escaping into other aquariums I separated aquariums with 2.5 cm thick pieces of expanded polystyrene foam.

122

Figure A.2. The heated aquariums used in the hot and warm water experiments. The three aquariums on the right were heated to 33oC

(hot water) and the three on the left were heated to 23oC (warm water). Each aquarium was heated with a 50 Watt fully submersible heater and contained a sponge filter. The aquariums were separated with 2.5 cm thick pieces of expanded polystyrene foam and illuminated with a 121.92 cm long T5HO light suspended 12.5 cm above the aquariums. Photo credit: Erika Helmond.

Figure A.3. The aquariums used for the 13oC (cold water) experiments. The aquariums are separated with 2.5 cm thick pieces of expanded polystyrene foam and are lit with a 45.72 cm long T5HO light suspended 12.5 cm above the aquariums. Due to the limited size of the refrigerator, one aquarium was placed on a lower shelf from the other two aquariums and the lights were set at an angle.

Photo credit: Erika Helmond

123

Figure A.4. A sponge filter used for aeration in one of the experimental aquariums. I fit an air stone and airline, indicated by the arrow, where the activated carbon had been prior to removal. The sponge often floated to the water surface because of trapped air bubbles. Thus, I weighed it down with small, aquarium- safe stones. Photo credit: Erika Helmond.

124 I initially filled each aquarium with 500 mL of water from the stock aquariums, 500 mL tap water (treated with Seachem® Prime®), and the remainder with RO water supplemented with CaSO4; the water was 10.2 cm deep. No further supplementation was added to any aquarium because there was an adequate level of Ca in the aquariums. The stock aquariums water used here came only from the aquariums containing snails for the experiments carried out in the 2.5 US gallon aquariums. These stock aquariums were initially set up nine days prior to addition of the new snails from the LHS, 12 days prior to set up of experimental aquariums, and consisted of 90% RO water supplemented with

CaSO4, 10% treated tap water, plus the added transport water from the transport containers). I allowed the experimental aquariums to run for six days before adding snails. I added 10 snails by placing them, along with water from the stock tank, in a container, which I floated in the desired aquarium for at least one hour to allow snails to acclimate to the aquarium temperature. I then poured the snails and water from the container into the aquarium. The next day, I added seven microscope slides on which snails could lay their eggs to each aquarium. (Figure A.5).

I kept each experimental aquarium on a 12 hour light/dark cycle with the 23oC and 33oC aquariums lit via a single 121.9 cm Sun BlasterTM T5HO light (Figure A.2) and the 13oC aquariums with a 45.7 cm Sun BlasterTM T5HO light (Figure A.3). All lights were suspended from 12.5 cm above the aquariums. I covered the top, front, and back of the aquariums with lids, but left the middle of the aquarium open to the light (Figure A.6) to allow sufficient light to enter the aquarium. This also ensured the snails were not stressed by the light levels by providing locations of reduced light. I added lids to reduce the rate

125

Figure A.6. The Plastic aquarium covers used on the experimental

Figure A.5. One of the microscope slides present within an experimental aquariums. Photo credit: Erika Helmond. tank. I suspended the slide with thread to prevent it from falling during observation and maintenance activities. Photo credit: Erika Helmond.

126 of water evaporation from the aquariums. Water chemistry was monitored via weekly

- - 2+ testing for NH3, pH, NO2 , NO3 , GH, KH, and Ca . I performed water changes on all the aquariums as I deemed necessary. I typically replaced water using a mixture of half

RO, half treated tap water, but used only RO or only treated tap water as I deemed necessary to maintain aquarium conditions. To mitigate evaporation, I frequently topped up the aquariums using RO water.

127 APPENDIX B: CONVERGENCE PLOTS

The following describes the Markov chain Monte Carlo (MCMC) diagnostics for the

Bayesian analysis of the data in the Determining Temperature Preference for Physella wrighti, Determining Survivorship of Mature Physella wrighti, The Number of Egg

Masses Produced by Physella wrighti, The Number of Eggs Per Mass Produced by

Physella wrighti, Determining Differences in the Size of Eggs Laid by Physella wrighti, Viability of Eggs per Egg Mass Produced by Physella wrighti, and Physella wrighti Embryo Incubation Period experiments. The MCMC diagnostics used consisted of R-hat values, effective sample sizes, 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 (Figure B.1) 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 Figure B.1). 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 ) (Figure B.2) 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.

128

Figure B.1. Markov chain Monte Carlo diagnostic trace plot for the effect of hot water produced during the assessment of the difference in the length of survival for wild snails with regards to temperature. I fit a

Bayesian generalized linear model with water temperature as the predictor variable, a gamma distribution for the response, and a log link using the package BRMS in R 3.6.1. Model parameters were assigned default, uninformative priors. Each colour in the plots represents a different chain and shows no trends in the posterior distribution for the parameters, demonstrating that chains have converged.

Figure B.2. MCMC diagnostic posterior density kernel plot for the effect of hot water produced during the assessment of the difference in the length of survival for wild snails with regards to temperature. I fit a

Bayesian generalized linear model with water temperature as the predictor variable, a gamma distribution for the response, and a log link using the package BRMS in R 3.6.1. Model parameters were assigned default, uninformative priors. Each colour in the plots represents a different chain and shows the frequency of parameter values for each chain. All chains show similar frequency indicating that chains have converged.

129 Each chain is indicated by a different colour and should appear similar to the other runs

(e.g. see Figure B.2). 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 my statistics were equal to one.

Using the three different methods of diagnostics on chain convergence verified that I had not made an error in assessing chain convergence for each of my statistical analysis.

The effective sample size (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). If we were 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, we can be confident that we have enough independent predictions to get information from the posterior distribution (Kruschke, 2015). A desirable ESS is at least 1000 (Vanderwel, 2018, personal communication); however, there are differing opinions on the minimum ESS value. All of the ESS in my analysis were greater than

1000.

130 Figures B.1 and B.2 are an example of a diagnostic trace and posterior density kernel plot produced during my statistical analysis. These are two of the plots produced for the analysis of Determining Survivorship of Mature Physella wrighti; the remaining plots produced for Determining Survivorship of Mature Physella wrighti, as well as the plots produced during the statistical analysis for the Determining Temperature

Preference for Physella wrighti, The Number of Egg Masses Produced by Physella wrighti, The Number of Eggs Per Mass Produced by Physella wrighti, Determining

Differences in the Size of Eggs Laid by Physella wrighti, Viability of Eggs per Egg

Mass Produced by Physella wrighti, and Physella wrighti Embryo Incubation Period experiments were similar in appearance, so have not been presented here.

131 APPENDIX C: UNIDENTIFIED SNAIL SPECIMENS FROM LIARD HOT

SPRINGS ALPHA STREAM

Figure C.1. Several Specimens of the unidentified

Snail in Alpha Stream, Liard Hot Springs, Liard River

Hot Spring Provincial Park, indicated by the circles. I

observed these snails within the first 50m of the

Alpha Stream. The image was taken with a Samsung

Galaxy S7 and resulted in a pink/red reflection on the

surface of the water due to the phone case. Photo

Credit: Erika Helmond.

Figure C.2. One of the unidentified snails

from Liard Hot Springs, Liard River Hot

Spring Provincial Park, indicated by the

arrow. Several of the unidentified snails

were unintentionally collected with samples

of Chara from the hot springs resulting in

several individuals present in stock

aquariums during my studies of Physella.

wrighti. Several specimens of P. wrighti are

also present in this photo. Photo Credit:

Erika Helmond.

132

Figure C.3. Various angles of the unidentified snail from Liard Hot Springs, Liard River Hot Spring

Provincial Park. The same specimen was used in each of the 5 photos present here. All photos were taken at 8X total magnification using an OMAXTM A355U microscope camera. Photos 1 and 2 are dorsal and ventral views (respectively) of the shell. Photos 3-5 were taken at different angles to provide a perspective of the contours of the shell. The diameter of the shell measures approximately 4.47 mm in Photo 1. Photo

Credits: Erika Helmond.

133 APPENDIX D: LARVAL STAGE FOR THE HOT SPRING MITE

THERMACARUS NEVADENSIS

Figure D.1. One specimen of

presumed Thermacarus

nevadensis during its larval

stage present under the shell of

its presumed host Physella

wrighti, indicated by the arrow.

Photo taken April 2018 by

Erika Helmond.

Figure D.2. The inferred larval

stage for Thermacarus

nevadensis, indicated by the

arrows, present under the shell

of its presumed host Physella

wrighti. This is the same snail

as shown in Figure D.1 and,

from this viewpoint, appears to

be host to two mites. Photo

taken in April 2018 by Erika

Helmond.

134 Figure D.3. The inferred

larval stage for Thermacarus

nevadensis present under the

shell of its presumed host

Physella wrighti, indicated

by the arrow. This is the

same snail as shown in

Figure D.1. Photo taken

April 2018 by Erika

Helmond.

A. B.

Figure D.4. The larval stage for Thermacarus nevadensis present on presumed host Physella wrighti. This snail had two larval mites present on it, but only one is clearly visible in these images. Photo A: The visible mite appears to be engorged on the snail (Proctor, 2019, personal communication). Photo B: The mite from Photo ‘A’ is clearly visible, as well as the second mite is visible as a red tint on the right side at the front edge of the snail shell, indicated by the arrow. This mite measured 0.58 mm in length. Photos were taken at 8x total magnification in January 2019 by Erika Helmond.

135

Figure D.5. The larval stage for Thermacarus nevadensis present on the presumed host Physella wrighti.

This snail specimen had one larval mite present on it that measured 0.59 mm in length. The mite appeared engorged on the snail (Proctor, 2019, personal communication). Photo taken at 56x total magnification,

January 2019 by Erika Helmond.

Figure D.6. The larval stage

for Thermacarus nevadensis

present on its presumed host

Physella wrighti. This snail

and mite pictured here are

the same specimens shown

in Figure F.5. Photo taken

January 2019 by Erika

Helmond.

136