Forestry, Faculty of
DSpace
UBCV
Fisher, Jennifer
Master of Science - MSc
University of British Columbia
White Sturgeon (Acipenser transmontanus) are from an ancient lineage and many of their populations are listed as endangered. Recruitment failure, a general term for a low or a lack of survival to sexual maturity, is the primary contributor to this endangerment. In the Nechako River of British Columbia, White Sturgeon embryos and yolk sac larvae are not surviving past their first month of life, despite successful spawning events taking place every year. Changes in benthic sediment composition because of flow regulation appear the likely cause of this lack of early life history success. However, it remains uncertain precisely in what way which these events have impacted these early life stages, and what the most important factors are for survival in the first month of life of White Sturgeon. Few studies have examined yolk sac larvae (YSL), and even fewer have studied embryos. No studies have thus far examined the interaction between predaceous stonefly nymphs and early life stage White Sturgeon, despite them being found in the same environments. I investigated how different substrate sizes act in conjunction with invertebrate predators to influence the early life history success of White Sturgeon embryos and larvae. I also tested how sand-coating impacts embryos. I discuss three experiments, each addressing different aspects of these interactions. I found no evidence that predatory stonefly nymphs consume White Sturgeon embryos, nor that their presence and sand coating are good predictors of embryo survival to hatch. However, I did find that there was a significant effect of invertebrate presence on hatch timing, shifting hatch timing slightly later. I found that the condition factor of YSL reared over sand was significantly lower than that of YSL reared over coarse gravel. However, substrate size did not influence the vulnerability of YSL to predation by predatory stonefly nymphs. I also found that substrate size had a significant impact on time to emergence for larvae, but that invertebrate presence did not. Overall, I found that predatory stonefly nymphs did not appear to prey White Sturgeon embryos, that they lowered survivorship of larvae, and that coarser substrate led to increased condition factor.
Early life history interactions of White Sturgeon (Acipenser transmontanus) with substrates and predaceous invertebrates by Jennifer Fisher B.Sc. (Hons), University of British Columbia, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2023 © Jennifer Fisher, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Early life history interactions of White Sturgeon (Acipenser transmontanus) with substrates and predaceous invertebrates submitted by Jennifer Fisher in partial fulfilment of the requirements for the degree of Master of Science in Forestry Examining Committee: John Richardson, Professor Emeritus, Forestry, UBC Co-supervisor Steve McAdam, Adjunct Professor, Institute for Oceans and Fisheries, UBC Co-supervisor Brett Eaton, Professor, Geography, UBC Supervisory Committee Member Patricia Schulte, Professor, Zoology, UBC Additional Examiner iii Abstract White Sturgeon (Acipenser transmontanus) are from an ancient lineage and many of their populations are listed as endangered. Recruitment failure, a general term for a low or a lack of survival to sexual maturity, is the primary contributor to this endangerment. In the Nechako River of British Columbia, White Sturgeon embryos and yolk sac larvae are not surviving past their first month of life, despite successful spawning events taking place every year. Changes in benthic sediment composition because of flow regulation appear the likely cause of this lack of early life history success. However, it remains uncertain precisely in what way which these events have impacted these early life stages, and what the most important factors are for survival in the first month of life of White Sturgeon. Few studies have examined yolk sac larvae (YSL), and even fewer have studied embryos. No studies have thus far examined the interaction between predaceous stonefly nymphs and early life stage White Sturgeon, despite them being found in the same environments. I investigated how different substrate sizes act in conjunction with invertebrate predators to influence the early life history success of White Sturgeon embryos and larvae. I also tested how sand-coating impacts embryos. I discuss three experiments, each addressing different aspects of these interactions. I found no evidence that predatory stonefly nymphs consume White Sturgeon embryos, nor that their presence and sand coating are good predictors of embryo survival to hatch. However, I did find that there was a significant effect of invertebrate presence on hatch timing, shifting hatch timing slightly later. I found that the condition factor of YSL reared over sand was significantly lower than that of YSL reared over coarse gravel. However, substrate size did not influence the vulnerability of YSL to predation by predatory stonefly nymphs. I also found that substrate size had a significant impact iv on time to emergence for larvae, but that invertebrate presence did not. Overall, I found that predatory stonefly nymphs did not appear to prey White Sturgeon embryos, that they lowered survivorship of larvae, and that coarser substrate led to increased condition factor. v Lay Summary White Sturgeon are part of an ancient lineage and some populations are endangered due to a lack of survival of their embryos and larvae. The exact causes of this are unknown, but there is a link between changes in substrate composition because of flow modification and the onset of recruitment failure. I tested whether different substrate compositions would impact the behaviours of these life stages, and if predaceous invertebrates would consume White Sturgeon embryos and larvae. I found that predatory stonefly nymphs are not predators of White Sturgeon embryos, but that they had a significant effect on larval survival. I also found that larvae reared over sand had poorer health than larvae reared in coarser gravel. vi Preface Jennifer Fisher is the primary author of this thesis, with editing by Steve McAdam and John Richardson. Both Steve McAdam and John Richardson contributed to conceptualization. Assistance with fish husbandry and experimental monitoring was provided by Paulina Chalmers, Aline Isabelle, Gretchen MacNaughton, Mathew Appelbaum, and Cole Munro. A version of this thesis will be submitted for manuscript publication with Steve McAdam and John Richardson as co-authors. All work done for this thesis was done in compliance with UBC Animal Care Protocol A22 - 0043 and Species at Risk (SARA) permitting. vii Table of Contents Abstract ........................................................................................................................................................ iii Lay Summary ................................................................................................................................................. v Preface ......................................................................................................................................................... vi Table of Contents ......................................................................................................................................... vii List of Tables ................................................................................................................................................ ix List of Figures ................................................................................................................................................ x List of Abbreviations ...................................................................................................................................xiii Terminology ................................................................................................................................................ xiv Acknowledgements ..................................................................................................................................... xv Dedication ................................................................................................................................................... xvi I Introduction ................................................................................................................................................ 1 I.I Freshwater Biodiversity Under Peril ......................................................................................................... 1 I.2 Acipenseriformes ..................................................................................................................................... 2 I.3 Population Dynamics and Recruitment Failure ....................................................................................... 3 I.4 White Sturgeon Range and Status ........................................................................................................... 4 I.5 General Life History of White Sturgeon ................................................................................................... 7 I.6 Substrate Composition and Early Life History Success ............................................................................ 9 I.7 Predation on Early Life Stages of White Sturgeon ................................................................................. 10 I.8 Thesis Objectives and Research Questions ............................................................................................ 13 I.8.I Hatch Plasticity Experiment ......................................................................................................... 13 I.8.2 Predation and Substrate Size Experiment .................................................................................. 14 I.8.3 Larval Emergence Timing Experiment......................................................................................... 14 2 Methods ................................................................................................................................................... 16 2.I Embryo Sourcing and Animal Husbandry .............................................................................................. 16 2.2 Hatch Plasticity Experiment .................................................................................................................. 17 2.2.I Experimental Design ................................................................................................................... 17 2.3 Predation and Substrate Size Experiment ............................................................................................ 19 2.3.I Experimental Design ................................................................................................................... 19 2.3.2 Condition Factor and Yolk Sac Area Measurements .................................................................. 21 2.4 Emergence Timing Experiment ............................................................................................................. 21 2.4.I Experimental Design: .................................................................................................................. 21 2.5 Data Analysis ......................................................................................................................................... 23 viii 3 Results ...................................................................................................................................................... 25 3.I Hatch Plasticity Experiment ................................................................................................................... 25 3.I.I Invertebrate Identification ........................................................................................................... 25 3.I.2 Survivorship................................................................................................................................. 25 3.I.3 Hatch Timing ............................................................................................................................... 27 3.2 Predation and Substrate Size Experiment ............................................................................................ 31 3.2.I Invertebrate Identification .......................................................................................................... 31 3.2.2 Predation Rate ........................................................................................................................... 31 3.2.3 Larval Size and Condition ........................................................................................................... 32 3.3 Emergence Timing Experiment ............................................................................................................. 35 3.3.I Invertebrate Identification .......................................................................................................... 35 3.3.2 Survivorship................................................................................................................................ 35 3.3.3 Proportion of Larvae That Emerged ........................................................................................... 37 3.3.4 Time to Emergence .................................................................................................................... 39 4 Discussion ................................................................................................................................................. 42 4.I Hatch Plasticity Experiment ................................................................................................................... 43 4.I.I Limitations .................................................................................................................................... 47 4.2 Predation and Substrate Size Experiment ............................................................................................ 48 4.2.I Limitations ................................................................................................................................... 51 4.3 Emergence Timing Experiment ............................................................................................................. 52 4.3.I Limitations ................................................................................................................................... 54 4.4 Synthesis and Future Directions ........................................................................................................... 55 5 Conclusion ................................................................................................................................................ 59 References .................................................................................................................................................. 60 Appendix ..................................................................................................................................................... 72 ix List of Tables Table 1 Results of post-hoc Tukey test comparing the proportion of embryos hatching overnight or during the day between treatment groups. Comparisons are denoted by dashes ................ 29 x List of Figures Figure 1 Schematic of egg rearing and transportation device. Eggs are on the side of the mesh facing into the cover. Mesh sheets each had four tabs that folded over corners of the protective covers (repurposed take-out containers) and were secured with zip ties for transport .............. 18 Figure 2 Percent survivorship to hatch for each treatment group. Maternal family of each replicate is indicated by a shape. Unknown maternal family is either maternal family 1, 2, or 3, but is uncertain. N = 9 for all treatments. Combined refers to embryos coated in sand and exposed to a predaceous invertebrate, while in the control embryos were not coated in sand and no invertebrates were present............................................................................................... 26 Figure 3 Mean proportion of hatch occurring on each day post fertilization. N = 9 for all treatments, and aquaria with unknown maternal family are included. Dashed red lines show the weighted mean date of hatch for each treatment group, measured in days post fertilization.... 27 Figure 4 Proportion of hatch occurring at each time of day across all treatment groups. N = 9 for all groups. Letters show results of a post-hoc Tukey test ............................................................. 30 Figure 5 Number of larvae missing at the end of the experiment (7 days post hatch). The number of larvae missing is presumed to be due to predation and all aquaria started with the same number of larvae (50). N = 9 for sand and mixed gravel, while N = 10 for coarse gravel and small gravel. Black points represent group means and error bars are the standard error .......... 32 Figure 6 A: Total larval length (cm) B: Larval wet mass (mg). C: Fulton’s Condition Factor (K). D: Yolk sac area (mm2). N = 9 for sand and mixed gravel, while N = 10 for coarse gravel and small xi gravel. The measurements for each replicate are an average for a sample of larvae from that aquarium. Substrate sizes are color-coded; sand is in purple, small gravel in blue, mixed gravel in green, and coarse gravel in yellow. Letters, where present, denote significant differences in group means that were determined by post-hoc Tukey tests ...................................................... 33 Figure 7 A) Estimated survivorship (%) by treatment group. N = 8 for large gravel with invertebrate present, N = 9 for small gravel with invertebrate, and N = 10 for large gravel and small gravel. B) Number of larvae missing by treatment group. N = 9 for large gravel with invertebrate and small gravel with invertebrate, N = 10 for large gravel and small gravel. Arrows and corresponding values indicate the value of outliers above the chart area ........................... 36 Figure 8 A) the Proportion of larvae that emerged by 21 days post hatch for all experimental treatments. Letters show the results of Tukey tests on square root-transformed data. N = 10 for large Gravel and small gravel, N = 9 for large gravel with an invertebrate and small gravel with invertebrate. B) the proportion of surviving larvae that emerged for all treatment groups. N = 9 for large gravel, small gravel, and small gravel with invertebrate, N = 8 for large gravel with invertebrate. The y-axis can also be interpreted as the proportion of surviving larvae that emerged ........................................................................................................................................ 37 Figure 9 Proportion of emergence occurring on each day post hatch. Dashed red bars show the mean date of emergence within that treatment that emerged on that day. N = 10 large gravel and small gravel, N = 9 for large gravel and invertebrate and for small gravel and invertebrate.41 xii Figure A1 Respiration rate per embryo from each treatment group. N = 3 for control and sand coated, N = 4 for invertebrate and combined. Combined refers to embryos being reared both coated in sand and in the presence of a predatory invertebrate ................................................. 75 xiii List of Abbreviations DPH days post hatch DPF days post fertilization YSL yolk sac larvae xiv Terminology The terms ‘free embryo’ and ‘yolk sac larvae’ and occasionally “eleutheroembryo” are used to refer to post-hatch larvae which have not begun exogenous feeding. While the predaceous invertebrates used for my experiments and referred to throughout this thesis are in fact larvae themselves, I never refer to them as such without specifying with the word 'invertebrate’. The word ‘larvae’ within this thesis is meant to refer to white sturgeon larvae. I did not feed any larvae used in these experiments, so all larvae used are assumed to not yet be considered feeding larvae but rather YSL. xv Acknowledgements There are many people who contributed to actualizing what is presented in this thesis. Thank you to my supervisors John Richardson and Steve McAdam for their expertise and guidance throughout this process. I learned a lot from working with you two. Thank you to Paulina Chalmers, Gretchen Macnaughton, Aline Isabelle, Mathew Appelbaum, and Cole Munro for their help in the field and in the lab. Thank you also to the team at the Nechako White Sturgeon Conservation Centre Hatchery; namely Mike Manky, Fraser Linza, Jake Zimich and Kara Geary. Funding for this research was provided by the BC Ministry of Water, Land and Resource Stewardship, NSERC and the Department of Fisheries and Oceans. xvi Dedication I am very lucky to be surrounded by supportive friends and family who have been crucial to this process. Completing a master’s thesis is quite challenging and I would not have completed this without the encouragement of my family and friends. Thank you to my people. I Introduction I.I Freshwater Biodiversity Under Peril Freshwaters contain an astounding proportion of global biodiversity and a high proportion of all described fish species. When compared by global land cover, freshwaters cover about 1% of Earth’s surface, in stark comparison to marine waters which cover closer to 71% of the earth (Barbarossa et al. 2020, Costello et al. 2010). Despite this, conservative estimations of freshwater biodiversity approximate that about 9.5% of global animal biodiversity resides in freshwater (Balian et al. 2008). Moreover, it is estimated that 45% of all described fish species live in freshwaters (Lévêque et al. 2008). Freshwater conservation is therefore of the utmost importance given that freshwaters support such a high proportion of global biodiversity. Unfortunately, freshwater biodiversity is declining at a rapid rate and is currently under threat due to anthropogenic factors. There are greater predicted extinction rates for freshwater species than marine and terrestrial species globally (Ricciardi and Rasmussen 1999). For example, the freshwater fishes of North America are facing extinction rates that are 877 times higher than historical background extinction rates (Burkhead 2012). Another report indicates that there has been a population decline of 83% in monitored freshwater species across many taxa since 1970 (World Wildlife Fund 2022). Dudgeon et al. (2006) categorizes threats to freshwater systems into five main categories: pollution, over-exploitation, flow modification, habitat degradation, and invasive species. Among the many species threatened by human activities are the sturgeons and paddlefishes of the order Acipenseriformes. 2 I.2 Acipenseriformes The exploitation of freshwater systems has been particularly challenging for members of the order Acipenseriformes. Acipenseriformes typically live in large river systems, many of which have been subject to flow modification from dam construction (Billard and Lecointre 2001). Moreover, many Acipenseriformes are or have been heavily exploited by commercial fishing, usually for their caviar (Raymakers 2006). These factors have resulted in the members of Acipenseriformes being among the million species of plants and animals facing endangerment in the present day (World Wildlife Fund 2022). This is exemplified by the appearance of all 27 members of the order Acipenseriformes on the IUCN Red List. Therein, sixteen species of Acipenseriformes are listed as Critically Endangered, while all others have some recognized status as either Vulnerable or Endangered (IUCN 2023, Haxton et al. 2016). Furthermore, two species are recently categorized as Extinct: first, the Yangtze River Sturgeon, which is extinct in the wild, and the Chinese Paddlefish. Therefore, the generalization can be made that all Acipenseriformes species face some level of endangerment. Acipenseriformes is an ancient and diverse order of fishes. In fact, Acipenseriformes diverged from other fishes approximately 200 million years ago (Peng et al. 2007). Acipenseriformes exists naturally only in the Northern Hemisphere and is made up of 25 extant sturgeon species from the family Acipenseridae and 2 paddlefish species of the family Polyodontidae, of which nine live in North America (Birstein 1993, Billard and Lecointre 2001). Common to all sturgeon species are their late sexual maturity (between 5-30 years), long life spans, and spawning in freshwaters (Billard and Lecointre 2001). 3 I.3 Population Dynamics and Recruitment Failure Population growth is often limited by survival of young life stages, and recruitment failure is a term which broadly describes a low, or lack of, survival of young life stages to sexual maturity. Recruitment failure is documented and of interest in many taxa; for example, freshwater mussels, tropical tree species, tropical corals, and black abalone (Strayer & Malcom 2012, Vlam et al. 2014, Hughes & Tanner 2000, Miner et al. 2006). In the example of freshwater mussels, recruitment failure is evident in a population of Elliptio complanata in Webatuck Creek where the majority of individuals are over 50 years, and there are no individuals under 20 years old (Strayer and Malcom 2012). Based on data collected for North Sea demersal fishes it is hypothesized that larval mortality is particularly high at two critical life transitions: the transition from the YSL stage to exogenous feeding and the metamorphosis of feeding larvae into juveniles (Beverton and Holt 1993). Fishes tend to have type-III survivorship curves, which means that mortality rates are very high in young life stages, then decrease and remain stable later in life (Rauschert 2010). This means that survivorship is always low for early life stages, but species with this type-III survivorship curve tend to produce many offspring to account for this. While there is naturally a great variation in larval and egg survival of fishes (McGurk 1984), this can be of concern if recruitment, the number of individuals reaching sexual maturity, is consistently lower than the mortality rate of breeding adults. Predation is the most important factor driving mortality rates in non-feeding early life stages of fishes. Most mortality in early life stages of fishes is believed to be caused by starvation and predation (Paradis et al. 1996). Perhaps rather obviously, McGurk (1984) points out that fish eggs are not able to defend themselves from predators. Overall, predation has been considered 4 the most important cause of larval mortality since it impacts all life stages, whereas starvation is limited to a short period of time (Paradis et al. 1996). Size is an important trait for predicting early life survival and recruitment in fishes. Larger body size is correlated to high survival in the first year of life, and subsequently high recruitment for some populations of cod and haddock (Stige et al. 2019). It is suggested that larvae that grow and develop faster will be better at foraging (Pepin 1991). Moreover, increased size of feeding larvae is associated with decreased daily mortality rates in marine fishes (Pepin 1991). It has also been experimentally observed that larger capelin (Mallotus villosus) larvae are less vulnerable to predation by stickleback (Gasterosteus aculeatus) than smaller conspecifics (Pepin et al. 1992). Altogether, size can be a useful metric for predicting vulnerability of fish larvae to mortality by starvation and predation, with larger size widely believed to confer a survival benefit. There can be a trade-off between growing in size and avoiding predation (Werner and Gilliam 1984, Werner and Anholt 1993). I.4 White Sturgeon Range and Status The White Sturgeon (Acipenser transmontanus, Richardson 1863) is the largest freshwater fish in North America (Hildebrand et al. 2016, Peng et al. 2007). As a species, they are thought to have diverged from other Acipenseriformes about 46 million years ago (Peng et al. 2007). White Sturgeon are found only in western North America from as far south as Baja California to as far north as the Gulf of Alaska (Hildebrand et al. 2016, Haxton et al. 2016). White Sturgeon are not strictly anadromous because they do not require visits to marine water to complete their life cycle such as in the Nechako River; however, certain populations such as the Lower 5 Fraser River have individuals that occasionally undergo marine movements, the exact purpose(s) of which are unknown (Hildebrand et al. 2016). Similar to other sturgeon species, White Sturgeon within Canada have been greatly impacted by human activities. There are six populations of White Sturgeon in Canada, all within British Columbia. Of these six populations, four are listed as Endangered (Nechako River population, Kootenay River population, Upper Columbia River population, and Upper Fraser River population) while the Lower Fraser River population is assessed as Threatened. The Middle Fraser River population is not listed under SARA but is assessed as endangered by the Committee on the Status of Endangered Wildlife in Canada (Fisheries and Oceans Canada 2023, COSEWIC 2012). The Kootenai River population of White Sturgeon is also listed as Endangered in the United States (the White Sturgeon in the Kootenay River south of the United States- Canada border) (U.S. Fish and Wildlife Service 2019). The status of White Sturgeon is attributed to overfishing, habitat modifications, especially dam constructions, and pollution (Hildebrand et al. 2016). Presently, the most pressing threat to White Sturgeon is recruitment failure (Coutant 2004, Hildebrand et al. 2016, Kock et al. 2006). For White Sturgeon specifically, the largest bottleneck for survival appears to be happening very early on, sometime during the embryo and larval stages (Coutant 2004, Hildebrand et al. 2016, Kock et al. 2006). The embryo stage appears to be the most vulnerable, with low catch rates of wild White Sturgeon beyond this life stage. This is demonstrated by embryos being commonly captured in the wild on the Kootenai and Nechako rivers, while juveniles and larvae are rarely caught (Hardy et al. 2020, McAdam personal communication, Paragamian et al. 1998). Specifically, on the Kootenai River between 1994 and 6 2002, embryos were captured in the range of 75 to 483 individuals every year while only one larva was captured in this 8-year time frame (Paragamian 2012). Even so, very few studies have examined the ecological factors leading to embryo success. While the exact mechanism(s) behind recruitment failure is unknown, there is a link between recruitment failure and flow regulation due to dam construction on most rivers. In situations where recruitment is altered by flow modification, the cause appears to be the altered riverbed substrate (McAdam et al. 2005, McAdam 2011). The Nechako River has been greatly impacted by the construction of the Kenney Dam in 1952, which diverts more than half of the historical flow out of the watershed. Recruitment failure on the Nechako River occurred shortly after in 1967 (McAdam et al. 2005, McAdam 2012). There is still uncertainty surrounding why this 15-year gap between dam construction and the onset of recruitment failure occurred (Northwest Hydraulics Consultants 2003). Current discharges in the Nechako River post dam construction are not sufficient to move the larger sediments trapping fines so there is no way to clear out the fine sediments from the riverbed (Gauthier-Fauteux 2017). This leads to an accumulation of fine sediments and a sandy riverbed. Conversely, the lower Columbia River below the Bonneville dam hosts a stable population of White Sturgeon experiencing successful recruitment, indicating that the relationship between flow modification and White Sturgeon recruitment is context dependent (Hildebrand et al. 2016). Further research is needed to fully understand the interactions between early life stages of White Sturgeon and flow modification/substrate to enact effective conservation management. 7 I.5 General Life History of White Sturgeon Some of the life history traits of White Sturgeon make them especially vulnerable to endangerment. Their general life history traits include late maturation, yet high fecundity with an absence of paternal/maternal care, and a long-life span. In fact, White Sturgeon are thought to be able to live for over a hundred years (Scott and Crossman 1973, McPhail 2007). The age of sexual maturity varies by population, but can be between 15-32 years for females and as early as 12 years for males (Hildebrand et al. 2016). White Sturgeon spawning events happen every year, although each individual female may not spawn every year (Scott and Crossman 1973, Hildebrand et al. 2016, Parsley et al. 2002, McPhail 2007). Spawning usually occurs when rivers are between 8 ˚C and 20 ˚C, and adult White Sturgeon appear to choose deep areas of high flow velocity for releasing gametes in broadcast (Hildebrand et al. 2016, Paragamian et al. 2009). Parents produce many gametes, and one female typically produces in the order of hundreds of thousands of eggs, and in the Columbia River specifically, estimates are between 39 400 and 713 000 eggs produced per female (Hildebrand et al. 2016, Wydoski and Whitney 2003). Some of these life history traits, such as late sexual maturity and no parental care make the species vulnerable to endangerment through recruitment failure. There are five recognized life stages for White Sturgeon. In order of occurrence, they are egg/embryo, yolk sac larva (YSL, also known as free embryo), feeding larva, juvenile, and adult (Hildebrand et al. 2016). A YSL is differentiated from a feeding larva due to the presence of yolk-sac and lack of exogenous feeding, and once a fish has successfully begun exogenously feeding it moves from being considered a YSL to feeding larva. Feeding larva still appear quite different from adults and have fewer distinct feeding and swimming structures compared to a juvenile 8 which has better developed fins and appears more similar to an adult (Hildebrand et al. 2016). The outermost layer of White Sturgeon eggs becomes adhesive shortly after contact with freshwater and due to their negatively buoyant nature, fertilized embryos will adhere to the bottom of the river (Cherr and Clark 1985, Hildebrand et al. 2016, Scott and Crossman 1973, McPhail 2007). Once these embryos hatch, YSL will initiate a hiding period where they hide in the benthic substrate until they are ready to begin searching for food, which happens around the time that the yolk-sac has been absorbed (McAdam 2011, Utter et al. 1983). The effects of an altered flow regime, and in turn benthic substrate, on the use of benthos by embryos and YSL is the subject of investigation and is critical to understanding recruitment failure (Baker et al. 2014, Boucher et al. 2018, Kynard et al. 2013, Parsley and Kofoot 2013). The timing of drift and emergence (when YSL emerge from hiding as feeding larvae) for White Sturgeon has been subject to investigation, often without consideration of how other species may influence this key behaviour (Coulter et al. 2023, Brannon et al. 1984, McAdam 2012), but the precise mechanisms affecting emergence or drift timing remain uncertain. Our current understanding from laboratory studies is that YSL hide in interstitial spaces upon hatch when porous substrate is available (McAdam 2011). However, drift timing of White Sturgeon has not been investigated in the context of the presence of predaceous invertebrates, which live within interstitial spaces and could potentially therefore alter drift timing by motivating YSL towards the water column prematurely. There is evidence that stonefly nymphs make other invertebrates more vulnerable to fish predators, likely by motivating these invertebrates to drift (Soluk and Richardson 1997). 9 I.6 Substrate Composition and Early Life History Success Substrate composition is an important variable for the early life stages of many fishes and understanding the substrate needs of each life stage is crucial for effective conservation. Conservation managers can add substrate to rivers to target a particular life stage or process, for example gravel addition to facilitate salmon redd building (Merz et al. 2019). In field studies with Chinook salmon, smaller gravel is associated with increased abundance of leeches (predators), and this increased abundance of leeches appears to be responsible a 17% lower survival of Chinook salmon embryos reared in smaller gravel relative to larger gravel (Merz et al. 2019). Moreover, fine sediments can alter oxygen consumption of early life stages. Atlantic salmon (Salmo salar) embryos have 41% lower oxygen uptake across the chorion when coated in clay (see appendix) (Greig et al. 2005a, Greig et al. 2005b). Altogether, understanding how early life stages interact with different substrates is important for many species, and effective substrate restoration efforts should balance the substrate needs of the many organisms in the ecosystem. Substrate composition is confirmed to be an important factor in success of both the embryo and larval stages of White Sturgeon, with current knowledge indicating that interstitial spaces are the key component to success. Even in the absence of predators, a survival advantage appears to exist for larvae reared in gravel, with larvae reared in gravel having close to 20% higher survival to those reared without (Boucher et al. 2014). Additionally, it has been well-established that larvae reared in gravel or artificial Bioballs™ attain larger body size and have increased growth when compared to larvae that are reared in tanks with no substrate (Boucher et al. 2014, Baker et al. 2014, Boucher et al. 2018, Bates et al. 2014). In one study, the wet mass 10 of larvae reared in gravel was 12% greater than for larvae reared without (Boucher et al. 2018). Sand as a rearing substrate appears to compare to bare tanks; larvae reared over sand versus gravel are smaller in size, have lower condition factor and decreased rate of gut development (Baker et al. 2014). Furthermore, sand has been indicated as an unsuitable benthic substrate for embryos to adhere to (Parsley and Kofoot 2013). Limited studies on sand coating and infilling for White Sturgeon embryos suggest that sand coating does not appear to have an impact on survival or size; however, being buried beneath 5 mm-20 mm of sediment does impact size and survival (Jason McLellan, fish biologist for the Colville Tribe, personal communication, Kock et al. 2006). Similarly, survival of Pallid Sturgeon (Scaphirhynchus albus) and Shovelnose Sturgeon (Scaphirhynchus platorynchus) embryos is reduced by both partial (hatch reduced by 23% - 63%) and complete burial (hatch reduced by 65% - 80%) in sand or fine sediment (Chojnacki et al. 2023). However, in the both the Kootenai (Paragamian et al. 2001) and Nechako rivers, embryos are found coated in sand and not necessarily buried, the effects and nuance of which is unknown. I.7 Predation on Early Life Stages of White Sturgeon Porous substrates are not only beneficial for larval growth, but also for evading predators. Laboratory findings have shown porous substrates can provide cover for White Sturgeon larvae from fish predators (McAdam 2011). Larvae exposed to sculpins (predators) experienced between zero and less than 20% survival (survival varied by days post hatch) when over sand, whereas survival was much higher (between ~17% and ~77% survival) for larvae that were able to hide (McAdam 2011). Access to interstitial spaces would therefore seem essential to avoiding predation. 11 Larvae can also respond to predator presence through behavioural changes. In studies with Lake Sturgeon, larvae seemingly prefer being on sandy substrate to being in the water column, but altered their behaviour in response to the presence of a crayfish predator. When crayfish were not present, approximately 55 - 83% of larvae were observed in the sand, but in the presence of a crayfish only 10 - 30% of larvae were observed in the sand and were instead in the water column (Crossman et al. 2018). However, larvae had the opposite reaction to fish predators in the same study, preferring the substrate to the water column in response to fish predator presence (Crossman et al. 2018). This study provides good evidence that sturgeon larvae can respond to perceived predation risk by avoiding predators and changing their behaviour. Recently, the interaction between benthic invertebrates such as predatory stonefly larvae, and Lake Sturgeon larvae has been investigated. These limited studies (Walquist et al. 2022, Wassink et al. 2021) identify stonefly larvae from the family Perlidae to be predators of Lake Sturgeon embryos and larvae. Lake Sturgeon embryos show lower egg diameter in the presence of perlids, 2 times lower survival to hatch, and earlier timing of 100% hatch relative to other invertebrate (Isonychiidae, Heptageniidae and Helicopsychidae) and control treatments (Walquist et al. 2022). The reduced egg diameter observed for embryos in the presence of perlids was predicted to be due to perlids consuming outer portions of the chorion (Walquist et al. 2022). Similarly, Lake Sturgeon YSL experience between 11% - 13% higher mortality in the presence of perlids compared to isonychiid (mayfly) and control treatments. When later exposed to stressors, the larvae exposed to perlids had cortisol levels nearly 2x that of control (Wassink et al. 2021). These studies have not addressed substrate as a factor in the interaction 12 between perlids and Lake Sturgeon larvae, and similar studies with White Sturgeon embryos or larvae have never been done, despite similar predaceous invertebrates living throughout the White Sturgeon species range. Hatch timing can be altered by the presence of predators. Differences in hatch timing for frogs in response to predators have been documented numerous times (Ireland et al. 2007, Schalk et al. 2002, Chivers et al. 2001). Hatch timing of green frog (Rana clamitans) embryos has been observed to be altered by the presence of different predators. The presence of freshwater leeches (Nephelopsis obscura) caused hatch to occur earlier, meaning that tadpoles were smaller and earlier in development. The opposite was seen in the presence of dragonfly nymphs (Aeshna canadensis), where hatch occurred later than in controls and therefore tadpoles were larger and later in development (Ireland et al. 2007). Changing hatch timing in response to different predator cues can be seen as a way of maximizing the chances of survival in early life stages. Adhesive eggs appear in many taxa, and there are competing ideas as to the advantage that this trait provides for survival, although very few studies have actually investigated this. The most referenced of these ideas is that adhesive coatings evolved to give embryos the ability to adhere to benthic substrates and remain stationary. Alternatively, adhesive coatings could serve as a deterrent to predation (Mike Parsley, US Geological Survey, personal communication). This is exemplified in yellow perch, whose egg jelly deters predators despite it not being toxic. In the lab, fish predators such as Brook Trout and stickleback, will reject and learn to ignore Yellow Perch eggs that have a jelly coat and occur as egg masses (Newsome and Tompkins 1985). White Sturgeon have adhesive embryos that can adhere to a variety of substrates (Parsley and 13 Kofoot 2013). However, it has never been experimentally tested whether this adhesive property could serve another purpose such as in deterring predation as seen in Yellow Perch. Common practice in fish culture is to coat embryos with silt for de-adhesion; therefore, potentially biasing our understanding of early life history development. Overall, it is unknown exactly what benefits there are to having adhesive eggs beyond the ability to adhere to benthic substrates. I.8 Thesis Objectives and Research Questions There three experiments discussed in this thesis address a thus-far ignored area, the interaction between predaceous stonefly nymphs and White Sturgeon embryos and larvae. Previous studies on the interaction between Lake Sturgeon embryos and larvae and predaceous invertebrates have failed to include natural substrate. Therefore, I put these interactions into the context of substrate composition and predation risk, to understand how larvae may change their behaviour in response to interstitial predators. I.8.I Hatch Plasticity Experiment This experiment tested whether Perlidae nymphs prey on White Sturgeon embryos, whether predaceous invertebrate presence could alter hatch timing of White Sturgeon embryos, and whether sand coating has any impact on the level of predation. I reared White Sturgeon embryos to hatch either in the presence or absence of a perlid with the added variable of sand coating the embryos. I hypothesized that sand coating could increase the vulnerability of White Sturgeon embryos to predation by perlids by making them less adhesive. This hypothesis is informed by my previous observations that predaceous invertebrates can become adhered to White Sturgeon embryos. An alternative hypothesis would be that sand coating makes White Sturgeon embryos less vulnerable to predation by 14 predatory stonefly nymphs, perhaps through camouflage. I also hypothesized that hatch timing would be earlier for embryos in contact with predators because of mechanical disturbance to the egg sheath resulting in YSL becoming dislodged earlier. This was based on my previous observations that slight disturbances from water changes caused YSL to come free of their egg sheath. This is supported by findings from Walquist et al. (2022) who found that hatch of Lake Sturgeon embryos in the presence of perlids occurred on average 1.5 days earlier than for those without. I did not anticipate a difference in hatch timing in response to sand coating, although I predicted lower survival of eggs coated in sand could be possible, despite unpublished results (Jason McLellan, fish biologist for the Colville Tribe) indicating no difference in survival for embryos coated in sand. I.8.2 Predation and Substrate Size Experiment I aimed to determine whether larval stoneflies were predators of White Sturgeon YSL and whether this level of predation would be influenced by substrate size. I reared YSL in four substrate size treatments: sand, small gravel, large gravel, and mixed gravel. Each was in the presence of predatory stonefly nymph. I hypothesized that there could be a negative impact of finer substrates, such as sand, on the ability of White Sturgeon larvae to hide from predators I also aimed to further the understanding of the effects of substrate size on larval growth by providing a wider variety of gravel treatments when compared to previous studies by Boucher et al. (2014) and Bates et al. (2014). I.8.3 Larval Emergence Timing Experiment The final experiment investigated whether White Sturgeon YSL altered their hiding behaviour in response to perlids, and whether substrate size had an impact on this interaction. I 15 hypothesized that larger interstitial spaces could make White Sturgeon YSL more vulnerable to predation or disturbance, due to assumed increased permeability or maneuverability of stonefly predators in a larger substrate. Under the assumption that this would initiate drift in the wild, I recorded emergence timing of yolk sac larvae in the presence or absence of a perlid in two substrate size treatments: small gravel 8 mm- 16 mm, and larger gravel: 16 mm – 31 mm. I predicted that in the presence of predaceous invertebrates White Sturgeon YSL would emerge from the substrate earlier and that more YSL overall would emerge from the larger gravel treatment than the smaller gravel treatment. 16 2 Methods 2.I Embryo Sourcing and Animal Husbandry Details of embryo sourcing and husbandry were similar among experiments and those common elements are summarized in this section. Fish were sourced from the Nechako White Sturgeon Conservation Centre hatchery in Vanderhoof, BC. For both the predation and substrate size experiment and the emergence timing experiment embryos were fertilized and de-adhered (coated with fine clay) in accordance with hatchery protocol and reared in Macdonald jars (standard hatchery devices for rearing White Sturgeon embryos). Both the hatch plasticity and larval emergence studies were carried out in environmental chambers at the InSEAS facility at the University of British Columbia, and for these experiments, embryos were transported from Vanderhoof to Vancouver in coolers with sufficient air (approximately 2:1 ratio of air:water) and water. The environmental chambers at InSEAS were kept at 14 ˚C with a photoperiod of 12 hours light: 12 hours dark, and water used was dechlorinated municipal water. The predation and substrate size experiment was done at the hatchery in Vanderhoof. For all experiments, aquaria had Marina® air stones with constant bubbling of room air into the water. Predaceous invertebrates for all experiments were captured by kicknetting. Invertebrates were selected for large body size (relative to the other invertebrates captured) and were not identified to the genus level until the end of the experiments using the taxonomic key from Merritt et al. (2008) (see results). For both the hatch plasticity and larval emergence experiments, invertebrates were captured from the Malcom Knapp Research Forest in Maple Ridge, B.C between the 17th and 19th May, 2023. All genera used are either present in the Nechako River or are analogous to other Perlidae species found in the Nechako. For the 17 predation and substrate size experiment, invertebrates were captured from the Nechako River near Vanderhoof. Checks for invertebrate condition and water changes were done twice a day throughout all experiments. At each check, half of the water in each aquarium was refreshed and embryos/YSL and invertebrate condition were monitored for general wellbeing. Embryos or YSL that died or had grown fungus were removed. If a predaceous invertebrate died during the experiment, they were replaced with an invertebrate from a holding tank maintained under analogous conditions. At the end of the experiments, or when an invertebrate died during the experiment, they were preserved in 70% Ethanol for identification. 2.2 Hatch Plasticity Experiment 2.2.I Experimental Design This experiment was conducted in static water aquaria, 37.85 L in volume. Fourteen litres of dechlorinated city water were added to each aquarium before the start of the experiment. A two-by-two factorial design was used to test both the individual and interactive effects of predaceous invertebrates and sand coating. Each of these two factors had two levels: absent or present. There were nine replicates per treatment for a total of 36 aquaria. On May 19, 2023, predators were added randomly to aquaria being treated with invertebrates. Invertebrate head width and length were measured using standard calipers as they were added to aquaria. The largest individuals present in the holding tank were used. The predators were allowed to acclimate to the aquaria for six days and were left until the start of the experiment. Only one predator was added to each invertebrate positive aquarium. 18 Eggs from three females were crossed with the pooled milt of 6 males. Maternal families were kept separate. Females were ovulated at staggered times between 2:40 pm and 7:20pm on May 24, 2023, at the Nechako White Sturgeon Conservation Centre Hatchery. Eggs were divided into small groups prior to fertilization and mixed with milt prior to the addition of water. Then, eggs were spread over egg mats and allowed to sit in water and become adhesive prior to coating with sand (grain size between 500 µm - 2 mm). The term ‘egg mat’ refers to mesh sheets (2.38 mm PECAP, Dynamic Aqua Supplies) used for embryo incubation (Figure 1). Pore size of this mesh was ideal so that embryos were ‘cradled’ within a pore and adhered to the mesh. Twelve egg mats were seeded with embryos for each maternal family. Six of the twelve egg mats seeded with each female’s eggs were subjected to the sand coating treatment. As sand was added to the eggs in small handfuls, excess sand was shaken off. Embryos from the Figure 1: Schematic of egg rearing and transportation device. Eggs are on the side of the mesh facing into the cover. Mesh sheets each had four tabs that folded over corners of the protective covers (repurposed take-out containers) and were secured with zip ties for transport. 19 non-coated treatment were also shaken to control for any effect of the sand coating process. Egg mats were secured with a protective cover to ensure eggs remained on egg mats and that they did not become crushed during transport. Eggs were then transported to UBC InSEAS and added to experimental aquaria at 1 day post fertilization (DPF). Once the embryos arrived at the environmental chambers at InSEAS, the number of eggs on each egg mat was counted visually and photos were taken to verify counts (using the multi-point tool in FIJI (ImageJ)). The number of eggs on each egg mat at the start of the experiment ranged from 45 - 85. Embryos adhered to the mats in clumps were removed gently by hand. At the morning checks, egg mats were photographed as an additional measure to monitor the number of eggs present. Hatch was counted as the time when the YSL were partially outside of the egg sheath, since at this point slight disturbances, such as water refills, are enough to cause the YSL to come free from the egg sheath. It is assumed at this point that in a river the embryo would be completely free-swimming and ‘hatched’ because of the flowing water. Hatch detected at the morning checks were considered to have occurred overnight, while hatch noted at the evening checks were considered to have occurred during the day. The experiment ended once 100% hatch was reached. YSL were then euthanized according to an approved animal care protocol (see acknowledgements for protocol number). 2.3 Predation and Substrate Size Experiment 2.3.I Experimental Design This experiment was conducted in 11.4 L rectangular glass aquaria at the Nechako White Sturgeon Conservation Centre. Aquaria were placed in plastic tubs with water up to a depth of ~10 cm that served as a static water bath to maintain a more stable temperature in 20 experimental aquaria. Water in the water baths was exchanged when necessary for regulating temperature, with a target water temperature of between 13 – 15 ˚C, which was monitored at least twice daily. The location within the hatchery where this experiment was conducted was not photoperiod or temperature regulated, however the water temperature in the aquaria remained within the range of 10.6 – 17.3 ˚C. The water used was from the hatchery system which is water taken from the Nechako River and filtered. The four treatments for this experiment evaluated predation by invertebrates in the presence of the various sizes of benthic substrate used. The treatments were: Sand (250 µm - 2 mm), small gravel (4 mm - 12.7 mm), coarse gravel (12.7 mm - 4.5 cm), and a mixture of small and coarse gravel (50:50 volume ratio). Each aquarium was filled with 1.5 L of substrate followed by 5 L of water. One predaceous invertebrate was present in each aquarium, and ten replicates were conducted per treatment, for a total of 40 aquaria. YSL ranged from zero to one day post hatch (DPH) at the start of experiment. YSL were from crosses between four females and three males. Each aquarium received 50 YSL. In contrast to the other experiments, predaceous invertebrates for this experiment were captured from the Nechako River. Invertebrates were added to the aquaria on May 24, 2022, and allowed four days to acclimate to the aquaria prior to the start of experiment. The experiment was initiated with the addition of the YSL to each aquarium. After five days, the experiment was ended by the removal of all the YSL and predaceous invertebrates from each aquarium. At this time, YSL were counted and euthanized according to animal care protocol prior to being preserved in 10% neutral buffered formalin (epredia, Netherlands) for further measurements. 21 2.3.2 Condition Factor and Yolk Sac Area Measurements Five YSL from each aquarium were sampled for the following measurements. YSL were removed from formalin five at a time and allowed to dry on a paper towel for 5 minutes before they were blotted with paper towel, measured, and weighed. YSL were weighed individually using a microbalance (Sartorius M2P, accuracy ±0.001 mg; Goettingen, Germany). Evaporation made a stable reading of the balance impossible, so all wet masses were taken sixty seconds after placing larva on the scale. Total length was measured from the tip of the snout to the end of the tail using a ruler. To calculate condition factor, Fulton’s condition factor equation was used as follows, where K is condition factor, L is total body length (cm), and W is body mass (g) (Froese 2006): 𝐾 = 100𝑊𝐿3 To measure yolk sac area, YSL were photographed using an AmScope MU500 microscope camera in frame with an objective micrometer (Nikon). Photos were analyzed using FIJI (ImageJ) (Schindelin et al. 2012). by visually tracing the margins of the yolk sac and using the built-in area measurement function. 2.4 Emergence Timing Experiment 2.4.I Experimental Design: Glass aquaria with a total volume of 11.4 L with static water were used for this experiment. Each was filled with substrate to a depth of 4 cm and 5 L of water. Spawning occurred on May 17, 2023. Eggs from three females were crossed with the pooled milt of seven males. Embryos were reared in the hatchery until six DPF, at which point they were transported by air freight to UBC InSEAS. Upon arrival, embryos were transferred into aquaria separated by 22 maternal family. Aquaria contained 8 L of dechlorinated municipal water. Half of the water in each aquarium was exchanged twice a day for clean water at which point embryos were checked and embryos showing signs of fungus were removed. This was a two-way factorial experiment with gravel size (small or large) and predaceous invertebrates (one present or absent) as the two factors, resulting in four treatment levels. Substrate was measured with metal sieves into the two size classes: 8mm - 16 mm (small gravel), and 16 mm - 31 mm (large gravel). The experiment was started at zero DPH, at which point the maternal families were pooled, and fifty YSL were counted and added into each aquarium. Experimental monitoring began the next morning at 1 day post hatch (DPH) to allow YSL time to hide within the substrate. Throughout the experiment, checks were done twice a day. Once in the morning, and once in the evening. At each check, it was first noted how many YSL were visible and swimming in the water column (defined as above the 4cm gravel mark) before removing these YSL that were deemed as ‘emerged’. Removal of emerged YSL was done to ensure that the same YSL was not counted as ‘emerged’ more than once. Occasionally, a YSL would swim back into the substrate before it could be removed. It is assumed that in a natural environment, once the larva has come above the gravel line, natural flow would be sufficient to carry this larva downstream and we would consider it to have drifted. If visible, invertebrate condition was noted and if an invertebrate died throughout the experiment it was preserved in 70% ethanol for identification, then replaced. After monitoring, half of the water in each aquarium was refreshed to maintain water quality. When invertebrates were identified, there were some invertebrates that were not identifiable due to being mid-emergence or being badly coated in 23 fungus. However, all invertebrates selected for this experiment were similar in appearance to the naked eye. Survivorship throughout the experiment was approximated at the end of the experiment based on presence or absence of fungus and obvious deformities. YSL that were coated in fungus were assumed to be dead and YSL without fungus were assumed to be alive. 2.5 Data Analysis Data were analyzed in R (R Core Team 2022, version 4.2.2). Results are reported as mean ± standard deviation, except in the case of dates of hatch and emergence, which are expressed as weighted means. Weighting was done using the weighted.mean function in R. Box-plots show the median (horizontal line), the interquartile range (box), and the bars extend to the lowest or highest points that do not exceed 1.5*Interquartile range. Points falling outside of this are plotted individually. For the hatch plasticity experiment, survivorship to hatch was calculated based on the initial number of eggs in each aquarium and does not account for any that were deemed ‘missing’ at the end of the experiment. This is because if an egg was missing it is assumed that it was either consumed or died and decomposed. Data for the predation and substrate size experiment were analyzed using one-way ANOVAs. Shapiro-Wilk tests were used to confirm normalcy of data. Yolk-sac area data were not normally distributed so were log- transformed prior to the ANOVA. The predation rate data were also not normally distributed so a Kruskal - Wallis test was used in place of an ANOVA. For both the hatch plasticity and emergence timing experiments, two-way, type-three ANOVAs were used to analyze data. Linear mixed-effects models were attempted for the hatch 24 plasticity experiment with maternal family as the random factor, but often resulted in a singular fit. Some data were not normally distributed for the hatch plasticity and emergence timing experiments. When transformations were not sufficient to normalize the residuals and satisfy the assumptions of standard three-way ANOVAs, Sheirer-Ray Hare tests were used. 25 3 Results 3.I Hatch Plasticity Experiment 3.I.I Invertebrate Identification The predaceous invertebrates used were all identified using morphology to be from the family Perlidae, and the genera used were Doroneuria (9 individuals), Calineuria (8 individuals), and Claassenia (1 individual). 3.I.2 Survivorship Survivorship, the number of embryos that survived to hatch, was compared between treatment groups by a two-way, type-three ANOVA and found to not be significantly influenced by sand coating (χ21 = 0.012, p = 0.91), invertebrate presence (χ21 = 0.22, p = 0.64) or the interaction (χ21 = 0.10, p = 0.75), with a high dispersion parameter (244.88) (Figure 2). The effect of maternal family was found to be near zero, so was not included in this model for simplicity. The average survivorship within each treatment group was 29% ± 15 for control, 30% ± 8 for embryos coated in sand, 25% ± 14 for embryos exposed to an invertebrate only, and 30% ± 22 for embryos coated in sand and in the presence of an invertebrate (Figure 2). There were some eggs that were unaccounted for at the end of the experiment, on average 5.38 ± 2.91 for control, 5 ± 3.55 for sand coated, 6.11 ± 3.52 for invertebrate, and 6.38 ± 5.85 for combined. The number of eggs missing at the end of the experiment was found to not be significantly influenced by sand coating (F1,26.15 = 0.0031, p = 0.96), invertebrate presence (F1,26.15 = 0.90, p = 0.35), or the interaction between sand coating and invertebrate presence (F1,27.49 = 0.087, p = 0.77), with maternal family as a random factor. The lack of significant difference in the number of eggs missing between treatments with a predaceous invertebrate 26 present and no invertebrate present indicates that the predaceous invertebrates were not consuming whole eggs. Figure 2: Percent survivorship to hatch for each treatment group. Maternal family of each replicate is indicated by a shape. Unknown maternal family is either maternal family 1, 2, or 3, but is uncertain. N = 9 for all treatments. Combined refers to embryos coated in sand and exposed to a predaceous invertebrate, while in the control embryos were not coated in sand and no invertebrates were present. 27 3.I.3 Hatch Timing Mean date of hatch was calculated for each aquarium and compared among treatment groups. When considering the whole data set including aquaria with unknown maternal family, the mean date of hatch measured as a weighted mean of hatch on each DPF was 11.15 for both control and sand coated, 11.37 for invertebrate only, and 11.26 for combined (Figure 3). There was a significant positive effect of invertebrate presence (F 1,24.989 = 7.31, coefficient = 0.31, p = 0.012), but no significant effect of sand coating (F 1, 25.05 = 0.042, p = 0.84) and the interaction between sand coating and invertebrate presence (F 1, 25.34 = 0.44, p = 0.51) on the mean date of Figure 3: Mean proportion of hatch occurring on each day post fertilization. N = 9 for all treatments, and aquaria with unknown maternal family are included. Dashed red lines show the weighted mean date of hatch for each treatment group, measured in days post fertilization. Time of day 28 hatch according to a three-way, type-three ANOVA (REML = 13.3) with maternal family as a random factor (variance = 0.025). Maternal family could not be removed from this model since it had a significant effect when added as a fixed factor, so therefore only aquaria with known maternal family were tested. A post-hoc Tukey test revealed no significant difference in the mean date of hatch between any of the pairwise comparisons. On average, hatch was observed more at the morning checks for all treatment groups, and therefore inferred to have occurred overnight (Figure 4). When considering time of day as a factor in the mean proportion of total hatch equation, there was no effect of invertebrate presence or sand coating (χ21 = 0.00, p = 1 for both factors and the interaction), but a significant impact of time-of-day (χ21 = 38.95, p ≤ 0.0001). Maternal family was dropped from this model since the linear mixed-effects model that included it as a random factor had a singular fit and adding it to the model as a fixed-factor revealed that it did not have an effect. A post-hoc Tukey test revealed no significant difference in mean proportion of total hatch between treatment groups at one time of day, but night-day comparisons across all treatment groups showed significantly different means (Table 1). 29 Table 1: Results of post-hoc Tukey test comparing the proportion of embryos hatching overnight or during the day between treatment groups. Comparisons are denoted by dashes. Comparison p value Control Day – Control Night < 0.0001 Invertebrate Day – Invertebrate Night < 0.0001 Combined Day – Combined Night < 0.0001 Sand Coated Day – Sand Coated Night < 0.0001 Control Day – Combined Night, Control Night – Combined Day 0.015 Control Day – Sand Coated Night, Control Night – Sand Coated Day 0.012 Sand Coated Day – Invertebrate Night, Sand Coated Night – Invertebrate Day 0.012 Control Day – Invertebrate Night, Control Night – Invertebrate Day 0.012 Sand Coated Day – Combined Night, Sand Coated Night – Combined Day 0.015 Invertebrate Day – Combined Night, Invertebrate Night – Combined Day 0.015 Control Day – Combined Night, Control Night – Combined Day 1 Control Day – Invertebrate Night, Control Night – Invertebrate Day 1 Control Day – Sand Coated Night, Control Night, Sand Coated Day 1 Sand Coated Day – Invertebrate Night, Sand Coated Night – Invertebrate Day 1 Sand Coated Day – Combined Night, Sand Coated Night, Combined Day 1 Invertebrate Day – Combined Night, Invertebrate Night – Combined Day 1 30 Figure 4: Proportion of hatch occurring at each time of day across all treatment groups. N = 9 for all groups. Letters show results of a post-hoc Tukey test. 31 3.2 Predation and Substrate Size Experiment 3.2.I Invertebrate Identification Invertebrates used for this experiment were identified to the genus level and found to be from two families and three genera. Within the family Perlidae, there were Hesperoperla and Claassenia, while from the family Perlodidae there was one genus present: Isogenoides. The most common genera were Claassenia and Isogenoides. 3.2.2 Predation Rate Average predation rate, measured as the number of YSL unaccounted for at the end of the experiment, was highest in the sand treatment (3.4 ± 3.84 larvae missing), and decreased in a stepwise fashion by increasing substrate size for small gravel, mixed gravel, and coarse gravel (2.2 ± 2.7, 1.33 ± 1.73, and 1.2 ± 2.15 larvae missing, respectively). A Kruskal-Wallis test showed these differences were not statistically significant (χ23 = 3.71, p = 0.29, Figure 5). The number of YSL unaccounted for at the end of the experiment could also be influenced by factors such as counting error and death and subsequent decomposition unrelated to predation. However, given that aquaria were monitored closely throughout the experiment, I assume such differences were a result of predation. Regardless, across all substrate size treatments, the average number of YSL missing at the end of the experiment was only 4%. In two aquaria, there were more YSL retrieved and counted at the end of the experiment than expected, indicating that there must have been counting errors when initiating the experiment (Figure 5). 32 3.2.3 Larval Size and Condition Total larval length (cm), larval wet mass (mg), Fulton’s condition factor, and yolk sac area (mm2) were compared between substrate size treatment groups (Figure 6). There was a significant negative effect of increasing substrate size on total larval length (one-way ANOVA, adjusted R2 = 0.25, F3,34 = 5.02, p = 0.0055). Total larval length was highest on average for YSL reared over sand (1.55 cm ± 0.066), lowest for YSL reared over coarse gravel (1.43 cm ± 0.11), and intermediate for small gravel (1.52 cm ± 0.081) and mixed gravel (1.47 cm ± 0.12) Figure 5: Number of yolk-sac larvae missing at the end of the experiment (7 days post hatch). The number of YSL missing is presumed to be due to predation and all aquaria started with the same number of larvae (50). N = 9 for sand and mixed gravel, while N = 10 for coarse gravel and small gravel. Black points represent group means and error bars are the standard error. Arrow and number represent the value of an outlier above chart area. 33 treatments. Total larval length (cm) was significantly different between both sand and coarse gravel and small and coarse gravel treatments (p = 0.0055 and p = 0.033, respectively) according to a post-hoc Tukey test. When aquaria where the invertebrate was replaced during the Figure 6: A: Total larval length (cm) B: Larval weft mass (mg). C: Fulton’s Condition Factor (K). D: Yolk sac area (mm2). N = 9 for sand and mixed gravel, while N = 10 for coarse gravel and small gravel. The measurements for each replicate are an average for a sample of larvae from that aquarium. Substrate sizes are color-coded; sand is in purple, small gravel in blue, mixed gravel in green, and coarse gravel in yellow. Letters, where present, denote significant differences in group means that were determined by post-hoc Tukey tests. Arrows and numbers denote values of outliers above the chart area. 34 experiment are eliminated, there was no significant effect of substrate size on total length (adjusted R2 = 0.14, F3,25 = 2.48, p = 0.084). Condition factor was highest for YSL reared in coarse gravel (0.71 ± 0.13) while it was lowest for YSL reared in sand (0.61 ± 0.093). Condition factor was intermediate for both the small gravel (0.64 ± 0.098) and mixed gravel (0.67 ± 0.13) rearing conditions. The effect of substrate size on larval condition factor was found to be significant (positive with increasing substrate size) with a one-way ANOVA (Adjusted R2 = 0.2, F3, 34 = 4.11, p = 0.014). A post-hoc Tukey test showed that the difference in condition factor between sand and coarse gravel was significant (p = 0.011), while there was no significant difference in larval condition factor for other pairwise comparisons. However, when aquaria where the invertebrate was replaced during the experiment were excluded from the dataset, there was no significant effect of substrate size on larval condition factor (Adjusted R2 = 0.15, F 3,25 = 2.65, p = 0.071). Wet mass was highest for YSL reared over sand (22.4 mg ± 3.1) and decreased with increasing substrate size; 22.3 mg ± 3.2 for small gravel, 21.3 mg ± 3.3 for mixed gravel, and was lowest for coarse gravel at 20.7 mg ± 4.1. There was no significant effect of substrate size on wet mass according to a one-way ANOVA (adjusted R2 = -0.026, F 3, 34 = 0.7, p = 0.57). Yolk-sac area was highest for YSL reared over sand (5.66 mm2 ± 0.99) and decreased with increasing substrate size (5.63 mm2 ± 1.13 for small gravel, 5.61 mm2 ± 1.04 for mixed gravel, and 5.62 mm2 ± 1.40 for coarse gravel). A log-transformation was used prior to a one-way ANOVA to meet the assumptions of normality and yolk-sac area was not significantly affected by substrate size (Adjusted R2 = -0.08, F 3,34 = 0.039, p = 0.99). 35 3.3 Emergence Timing Experiment 3.3.I Invertebrate Identification Predaceous invertebrates were identified to the genus level and included Calineuria (13 individuals), Doroneuria (3 individuals), and Hesperoperla (2 individuals); all from the family Perlidae. 3.3.2 Survivorship Survivorship at the end of the experiment was highest for larvae in large gravel at 82% ± 30, lowest for larvae in large gravel with an invertebrate at 62% ± 26, and intermediate for both small gravel treatments. The mean survivorship was 69% ± 33 for small gravel and 72% ± 21 for small gravel with invertebrate (Figure 7). Survivorship was found to be significantly, negatively affected by invertebrate presence (square-transformed, two-way, type-three ANOVA, χ21 = 5.07, p = 0.024), but not by gravel size (χ21 = 1.90, p = 0.17) or the interaction (χ21 = 2.27, p = 0.13). However, this model appeared to have a poor fit, with a median deviance residual of 290.7. There were no significant differences between mean survivorship of treatment groups (post-hoc Tukey test). There were also some larvae that were unaccounted for at the end of the experiment. These losses are assumed to be due to predation, or mortality and decomposition. The number of larvae missing at the end of the experiment was lowest on average for large gravel at 2.2 ± 3.71, highest for large gravel and invertebrate at 6.78 ± 10.47, and 6 ± 9.81 for small gravel and 4.89 ± 3.76 for small gravel with invertebrate. There was no significant effect of invertebrate presence (square root-transformed, two-way, type-three ANOVA χ2 = 0.96, p = 0.33), gravel size (χ2 = 0.72, p = 0.4), or the interaction (χ2 = 0.54, p = 0.46). There were three aquaria with 36 negative values for the number of larvae missing (two from the large gravel treatment and one from small gravel), meaning that more larvae were recovered than expected, and became excluded from this test due to the square root transformation. Some eliminations were made to see how it would impact the results. I eliminated experimental units where the predaceous invertebrate was replaced or emerged during the experiment and eliminated those that had zero survival due to fungus. With these eliminations, the number of larvae missing (square root-transformed) was significantly affected by invertebrate presence (p = 0.03) but not gravel size (p = 0.5) or the interaction (p = 0.37). However, the residuals were not perfectly normal even with this transformation, with a Shapiro p-value of 0.02, perhaps causing the test to not be as powerful. Figure 7: A) Estimated survivorship (%) by treatment group. N = 8 for large gravel with invertebrate present, N = 9 for small gravel with invertebrate, and N = 10 for large gravel and small gravel. Invertebrate presence had a significant negative effect, but no significant effect of gravel size or the interaction. B) Number of larvae missing by treatment group. N = 9 for large gravel with invertebrate and small gravel with invertebrate, N = 10 for large gravel and small gravel. Arrows and corresponding values indicate the value of outliers above the chart area. There was no significant effect of invertebrate presence, gravel size, or the interaction. 37 Figure 8: A) the proportion of larvae that emerged by 21 days post hatch for all experimental treatments. Letters show the results of Tukey tests on square root-transformed data. N = 10 for large gravel and small gravel, N = 9 for large gravel with an invertebrate and small gravel with invertebrate. B) the proportion of surviving larvae that emerged for all treatment groups. N = 9 for large gravel, small gravel, and small gravel with invertebrate, N = 8 for large gravel with invertebrate. The y-axis can also be interpreted as the proportion of surviving larvae that emerged. 3.3.3 Proportion of Larvae That Emerged Overall, very few larvae emerged over the course of the experiment. The mean proportion of larvae that emerged by 21 DPH was 0.17 ± 0.13 for large gravel, 0.25 ± 0.16 for large gravel and invertebrate, 0.06 ± 0.063 for small gravel, and 0.071 ± 0.056 for small gravel and invertebrate. A two-way, type-three ANOVA on the sqrt transformed data showed a significant effect of gravel size (χ21 = 5.96, p = 0.015), but no significant effect of invertebrate presence (χ21 = 2.06, p = 0.15) or the interaction (χ21 = 0.46, p = 0.50). A post-hoc Tukey test showed that the mean number of larvae emerged was only significantly different between large gravel with an invertebrate compared to small gravel (p = 0.003) and compared to small gravel with invertebrate (p = 0.013) (Figure 8A). ab b a a ab b a a 38 Eliminations were made during analysis, and this reduced data set was analyzed to see if the eliminations had an effect. These eliminations were: aquaria where the predaceous invertebrate emerged (became an adult) throughout the experiment, aquaria where there was zero survival (which could be a husbandry issue), and aquaria where the predaceous invertebrate was dead at the end of the experiment. Following these eliminations, the results of the ANOVA changed slightly, and a transformation was no longer necessary. There was still a significant effect of gravel size on the proportion of larvae that emerged during the experiment (χ21 = 5.27, p = 0.022), but there was also a significant effect of invertebrate presence on the proportion of larvae that emerged over the course of the experiment (χ21 = 4.13, p = 0.042). There was still no significant effect of the interaction term (χ21 = 2.12, p = 0.15). A post-hoc Tukey test revealed a significant difference in the proportion of larvae emerged in small gravel with no invertebrate compared to large gravel with an invertebrate (p = 0.0018) and between small gravel with an invertebrate and large gravel with an invertebrate (p = 0.0027) for this reduced data frame, but no significant difference in other pairwise comparisons. The proportion of larvae that emerged during the experiment was scaled to survivorship (Figure 8B). A square root transformation was applied to the data to conform with assumptions of the general linear model. A two-way, type-three ANOVA revealed that there was a significant effect of invertebrate presence on the number of larvae emerged scaled to survival (χ21 =7.11, p = 0.0077) while there was no significant effect of gravel size or the interaction (χ21 = 2.58, p = 0.11 and χ21 = 3.29, p = 0.07 respectively). A post-hoc Tukey test revealed that the means were different between large gravel with invertebrate and small gravel, as well as between large gravel with invertebrate and small gravel with invertebrate (p = 0.0011 and p = 0.0016, 39 respectively), but that there was no significant difference in the means for the other comparisons. A reduced data set with the following eliminations was analyzed as well: eliminate aquaria where the predaceous invertebrate emerged (became an adult) during the experiment, aquaria where there was zero survival (which could be a husbandry issue), and if the predaceous invertebrate was dead at the end of the experiment). The results of the ANOVA on the square root-transformed data changed slightly following these eliminations. The two-way, type-three ANOVA on this reduced data frame showed a significant effect of invertebrate presence (χ21 = 12.36, p = 0.00044), and the interaction between invertebrate presence and gravel size (χ21 = 6.52, p = 0.011) on the proportion of surviving larvae that emerged. There was no significant effect of gravel size (χ21 = 2.83, p = 0.092). A post-hoc Tukey test was also done on this reduced data frame and showed a significant difference between large gravel with and invertebrate and large gravel only (p = 0.0078), as well as a significant difference between the large gravel with invertebrate and small gravel (p = 0.0001), and between large gravel with invertebrate and small gravel with invertebrate (p = 0.0002). The modeled mean proportion of surviving larvae that emerged is significantly greater in the large gravel with invertebrate treatment compared to all other treatment groups in this reduced data frame. 3.3.4 Time to Emergence The mean time to emergence was shortest for large gravel and large gravel with an invertebrate present with larvae emerging on average at 13.95 and 11.84 days post hatch, respectively. The mean time of emergence was much later for small gravel and small gravel with an invertebrate present with larvae emerging at 19.27 and 18.81 days post hatch, respectively 40 (Figure 9). A two-way, type-three ANOVA using ^4 transformed data showed that substrate size had a significant effect on time to emergence (χ21 = 28.90, p = 7.62 e -8), but no significant effect of invertebrate presence (χ21 = 0.001, p = 0.975) or the interaction (χ21 = 0.69, p = 0.4073). Small substrate size significantly increased the time to emergence. The model fit was very poor, with a median deviance residual of 9411. The time to emergence model had non-normally distributed residuals, making it necessary to do a transformation. A ^4 transformation brought the residuals closer to normal, but still not perfectly aligned to assumptions of normality with a Shapiro wilk p-value of p = 0.024. A Scheier-Ray Hare test on the untransformed data was used as a confirmation of the ANOVA on this transformed data. The results were similar to those of the two-way, type-three ANOVA, showing no significant effect of invertebrates or the interaction between gravel size and invertebrate on time to emergence (p = 0.38 and p = 0.55, respectively), but still a significant effect of gravel size on time to emergence (p < 0.0001). 41 Figure 9: Proportion of larvae that emerged on each day post hatch. Dashed red bars show the mean date of emergence within each treatment. N = 10 large gravel and small gravel, N = 9 for large gravel and invertebrate and for small gravel and invertebrate. 42 4 Discussion The goals of my thesis were to 1) determine if predatory stonefly nymphs are predators of White Sturgeon embryos and larvae, 2) determine if sand coating would impact embryo survival or predation rate by predatory stoneflies, 3) to see if hatch timing of White Sturgeon embryos is altered by the presence of a predatory stonefly, 4) to see if yolk-sac larvae would alter their emergence timing to avoid predatory stonefly nymphs, and 5) to determine if substrate size and in turn the size of interstitial spaces would impact the emergence timing of larvae in the presence of a predaceous invertebrate. I found that predatory stonefly nymphs did not appear to be predators of White Sturgeon embryos and that sand coating had no influence on this interaction. However, I did find that predatory stonefly nymphs had a significant effect on hatch timing of White Sturgeon embryos. Predatory stonefly nymphs also had a significant effect on survivorship for larvae in my emergence timing study, but based on the results of my predation and substrate size study, they did not seem to consume White Sturgeon larvae whole. Finally, predatory stonefly larvae did not have a significant effect on time to emergence for White Sturgeon larvae, whereas smaller gravel significantly delayed time to emergence. Many White Sturgeon populations are listed as endangered, with the most common reason for this status being recruitment failure. Despite evidence that recruitment failure is occurring sometime in the earliest life stages (embryo and YSL), these remain the least studied life stages of this species. There is a developing field of evidence to suggest that interstitial spaces are crucial to growth and evading predators for YSL and larvae (Boucher et al. 2014, McAdam 2011), but my research is the first study to examine predaceous invertebrates in relation to White Sturgeon embryos and larvae. 43 4.I Hatch Plasticity Experiment Hatch plasticity is a phenomenon which allows an organism to maximize its chances of survival by altering when it hatches. Due to evidence in other species that predator presence can alter hatch timing of embryos (Walquist et al. 2022, Ireland et al. 2007), and my own observations that small mechanical disturbances such as water changes can motivate hatch for White Sturgeon, I hypothesized that mechanical disturbance of the chorion by a predator could induce earlier hatch timing. I found that hatch timing was not significantly impacted by sand coating or the interaction between sand coating and invertebrate presence, but that invertebrate presence had a significant effect on hatch timing. However, my results also showed no significant difference between hatch timing for embryos reared in the presence of stonefly nymphs versus without (post-hoc Tukey test). My findings are similar to the findings of Walquist et al. (2022) who found that time to hatch for Lake Sturgeon embryos reared in the presence of perlids was not significantly different to timing to hatch for embryos reared in the absence of invertebrates. The exact mechanisms causing hatch plasticity are uncertain. I found that the mean date of hatch for embryos exposed to an invertebrate predator was approximately 0.2 days later (a ~1.5% delay) than for control and sand coated, and the mean date of hatch for the combined was only about 0.1 days later than for control and sand coated. This implies either variation or somewhat of an antagonistic interaction between sand coating and invertebrate predator presence. The delay in hatch appears as a shift in the proportion of embryos hatching overnight versus during the day. Instead of the greatest proportion of hatch happening overnight on Day 11 post fertilization, the treatments with an invertebrate present (invertebrate only and 44 combined) seem to have a much more even distribution of hatch occurring between Day 11 and Day 12 post fertilization. This could be a response to the periods of highest activity for the predatory stonefly nymphs. As an example, the predatory perlodid Megarcys signata is most active and visible on top of substrate during the night (McIntosh and Peckarsky 1999). If the adhesive coating of a White Sturgeon embryo acts as a deterrent for predation by stonefly nymphs and stonefly nymphs are only predators of White Sturgeon larvae and not embryos, then it could be reasoned that there would be an advantage for larvae to hatch during the day when these predators are least active. Moreover, Ireland et al. (2007) predicted that egg predators (leeches) would cause green frog embryos to hatch earlier and that larval predators (dragonfly nymphs) would cause green frog embryos to hatch later and at larger size. They ended up observing this experimentally, indicating that the green frog embryos were able to react to a predator cue by adjusting hatch timing to optimize chances of success (Ireland et al. 2007). The results of the hatch plasticity experiment suggest that perlid stonefly nymphs are not predators of White Sturgeon embryos whereas the results of the larval emergence experiment indicate that perlid stoneflies could be predators of White Sturgeon larvae. I did not measure the size of YSL at hatch in this experiment; however future experiments should investigate if size at hatch is significantly different for embryos reared in the presence of these presumed larval predators. If I follow the reasoning of Ireland et al. (2007), this could indicate that embryos are delaying hatch in order to attain larger larval size to maximize probability of survival in this next life stage. However, it is unlikely that 0.1 – 0.2 days longer in the embryo stage would lead to a large difference in size at hatch. 45 Interestingly, hatching was occurring mostly overnight or in the early morning in all treatment groups. This relationship was not affected by sand coating or invertebrate presence. This implies that there is some inherent mechanism which motivates White Sturgeon embryos to hatch overnight or in the morning. Alternatively, this could have resulted from different fertilization times, but given that embryos were hatching across multiple days and most commonly at night this seems unlikely. All embryos were fertilized on the same day. Unfortunately, I cannot say definitively if the embryos hatched overnight or within the first hour of the light phase since morning checks were conducted around 9 am, while the light phase began daily at 8 am. Other fish species have been observed to restrict their hatch timing to specific times of day. In Zebrafish (Danio rerio) reared under a similar 12 hour light: 12 hour dark light regime, hatching occurs in pulses during the light phase. In contrast, Senegalese Sole (Solea senegalensis) was observed to hatch in pulses during the dark phase in the same study (Villamizar et al. 2013). The authors note that these observed peaks in hatch timing correspond to the most active times of day for the two species (Villamizar et al. 2013). Moreover, the Somalian Cavefish (Phreatichthys andruzzii) observed in their study hatched in unevenly distributed pulses, but not restricted to one time of day which they hypothesize could be due to vision loss (Villamizar et al. 2013). I observed White Sturgeon hatching with a more similar pattern to the Somalian Cavefish, but the underlying mechanism is likely different between these two species. The observed tendency of White Sturgeon to hatch more frequently at night could be to avoid visual predators such as salmonids. What is interesting to note is that there was a more even distribution of hatch timing between night and day in treatments with an invertebrate present, with a far greater proportion of larvae hatching during the day in the 46 invertebrate and combined treatments. This relates back to the delay in hatch timing seen in the presence of an invertebrate and could be a response by the larvae to avoid predation. Future experiments should investigate the combined influence of salmonids and predaceous invertebrates on hatch timing. Sand coating and the presence of a Perlidae stonefly nymph did not significantly decrease the survivorship of embryos to hatch, suggesting that the species of Perlidae used are either not predators of White Sturgeon embryos, or do not prey on embryos at a rate high enough to be significant. Walquist et al. (2022) had similar findings with regards to survivorship with Lake Sturgeon embryos, where survivorship was not significantly different for embryos reared with perlids versus without; however, they report that the mean proportional survival of embryos reared with perlids was 0.27, while the mean proportional survival of embryos reared without was 0.53. I did not see such a large difference in proportional survival in my study. One feature that I can assume may impact predation by invertebrates is embryo size. Lake Sturgeon embryos are approximately 33% smaller in volume than White Sturgeon embryos (Wang et al. 1985). For invertebrates with a small gape perhaps this difference is enough to exclude White Sturgeon embryos from their diet. The relationship between perlid presence and embryo survival to hatch does not appear to be straightforward, due to the high variability in survivorship introduced by invertebrate presence. This should prompt future investigation into what the traits of predatory invertebrates are that would cause low survivorship, or if the high variability of survivorship observed in this study when perlids were present was due to chance. Walquist et al. (2022) appear to use the disappearance of embryos from their experimental units throughout incubation as justification that perlids were consuming entire 47 eggs. However, they make no mention of whether the number of embryos in the experimental units ever decreased for their control. Assuming that they only observed this decrease in the total number of embryos in their perlid treatment, their findings are different from mine in that I found that there was no significant difference in the number of eggs missing between treatments. This suggests that the missing eggs were due to counting errors, most likely caused by when an embryo would die and become coated in fungus and sometimes be difficult to count in their decomposed condition. The fact that sand coating did not cause a decrease in survival to hatch for White Sturgeon embryos in my study is consistent with unpublished findings from Jason McLellan (fish biologist for the Colville Tribe). The question still remains if sand coating could be having some other effect on embryos that is problematic later in life. Future experiments should incorporate post-hatch monitoring to observe if there are impacts of sand coating that may have gone unobserved by this study. 4.I.I Limitations The limitations of this study mainly come from the sand coating process. My personal observations are that embryos differ in their adhesiveness slightly based on maternal family, some embryos appeared more completely coated in sand than others. Controlling for this variability in adhesive property was not possible but embryos were repeatedly covered in sand during the sand coating process until there was ‘saturation’ and further additions of sand resulted in no change. Moreover, I used a grain size of sand that allowed me to visually determine if embryos were coated in sand, but this is not representative of the natural environment where sand is not sorted with sieves and includes smaller grain sizes. Therefore, 48 future experiments should be done utilizing smaller grain sizes of sand for coating to ensure that this does not have an effect. 4.2 Predation and Substrate Size Experiment Previous studies have established a connection between availability of interstitial spaces and the size of White Sturgeon larvae. I expected that larval total length would be greater for larvae reared in gravel versus over sand. However, I found that YSL from the sand and small gravel treatments were significantly longer than YSL from the coarse gravel treatment. This differs from results of previous studies with White Sturgeon, where larvae reared in gravel have been shown to be longer than larvae reared in bare tanks as early as 4 DPH, with the difference growing throughout development (Boucher 2012). It is generally accepted that increasing larval length is associated with decreasing predation risk in fishes. Increased larval length relative to predator length is related to decreased predation risk by medusae and crustaceans (Paradis et al. 1996). Furthermore, in marine fishes, larval length has been found to be positively correlated to escape speed (Bailey 1984). However, this is complicated by the relationship between rearing substrate and other traits involved in predation risk in White Sturgeon. Startle response time and maximal sustainable aerobic swimming speed have been demonstrated to be lower (5 – 10% lower and 20 - 30% lower, respectively) for White Sturgeon reared in a bare tank versus with Bioballs™ (Baker et al. 2014). Given this, one hypothesis to explain my results is that there could be higher selection for size in sand relative to coarse gravel, with the smallest larvae being most vulnerable to predation. However, this does not seem to be well supported by my results given that I did not observe a difference in the number of larvae missing between substrate sizes. 49 Therefore, further studies are required to determine how sand as a rearing substrate impacts traits involved in predation pressure for White Sturgeon. A higher Fulton’s condition factor is meant to indicate better health when comparing fish (Froese 2006). Consistent with other studies by Baker et al. (2014) and Boucher et al. (2014), I found that larvae reared in coarse gravel versus sand had significantly higher condition factor, and therefore improved health. However, my findings do provide a bit more information due to the range of substrate sizes used. Altogether, there is good evidence to suggest that White Sturgeon larvae achieve higher condition factor, and therefore better health when provided with interstitial spaces. However, the finding that larval length was higher for larvae reared in sand than in coarse gravel in my study indicates that larvae from the sand treatment may be less vulnerable to predation, indicating a trade-off between condition and predation risk. I predict that in coarse gravel larvae were less vulnerable to predation than in the sand treatment, therefore did not devote as many resources to growing in length, leading to their higher condition factor. With regards to wet mass, my findings are different to the findings of other studies. On the one hand, my finding that wet mass was not significantly different between treatment groups is similar to findings from Baker et al. (2014) who observed no significant difference in wet mass of White Sturgeon larvae reared in bare tanks versus with Bioballs™ until 11 DPH (I ended my experiment at 7 DPH). On the other hand, my findings seem to disagree with the findings of Bates et al. (2014) who found that when reared at 17.5 ˚C larvae reared in gravel had higher wet mass than larvae reared in bare tanks. The similar wet mass observed between treatments in my study indicates that the differences in condition factor were a result of the 50 differences in length alone. This furthers my prediction that in coarse gravel larvae devoted less energy to growing in length than in the sand treatment. I predicted that yolk sac area would be highest for larvae from gravel treatments, and smaller for larvae from the sand treatment, given that larvae cannot hide in sand and tend to remain in motion. I reasoned that this constant motion would lead to larvae using their yolk-reserves faster than if they were hiding in interstitial spaces (Boucher et al. 2018). In contrast, I did not find a significant difference in yolk sac area between treatment groups. Moreover, larvae from the sand treatment did not have the lowest yolk sac area on average, which was recorded in the mixed gravel treatment. My findings are consistent with those of Boucher et al. (2014) who report no significant relationship of substrate on yolk absorption rate. However, they also report that the efficiency of yolk absorption was significantly affected by the presence or absence of gravel, and the interaction between substrate and temperature (Boucher et al. 2014). Larvae reared in gravel had significantly higher yolk absorption efficiency than those reared without (Boucher et al. 2014). Moreover, gut development has been shown to occur faster for larvae reared in gravel relative to sand, leading to depletion of the yolk-sac earlier (Baker et al. 2014). It is possible that the larvae reared over sand in my study had a similar delay in gut development, thus explaining the higher observed yolk sac area since yolk-reserves were not used as quickly. The results of my experiment with regards to the number of larvae missing from each aquarium are surprising. I expected that predation would be highest in the sand treatment resulting in more larvae missing in these aquaria due to there being nowhere to hide. While I did find that the number of larvae missing was highest for the sand treatment, this was not 51 significant. I did not measure larval movement into the water column in this study but expect that larvae in sand treatments would have spent more time high in the water column instead of near the substrate to avoid predation. This inference is informed by a study by Crossman et al. (2018) who observed Lake Sturgeon larvae preferred the water column and spent ~40% less time on sand substrate in the presence of a crayfish. Additionally, Wassink et al. (2021) found significantly higher mortality of Lake Sturgeon larvae exposed to perlids relative to control after only three days, so the duration of my study is sufficient to observe predation. However, I did not determine survival beyond the number of larvae missing in this experiment, under the assumption that larva would be consumed whole. If larvae were not consumed whole and instead there were indirect predation effects occurring in my study, this may have gone unnoticed due to methodology. 4.2.I Limitations As noted in my results section, the number of larvae recovered was greater than expected in two aquaria which is indicative of counting errors at the start of the experiment. Therefore, the inability to detect differences between treatment groups is not definitive. I assumed that the number of larvae missing would be the predation rate but given this counting error it is difficult to say what is actually due to predation, or if predation was even occurring. It is unclear if or how predaceous invertebrates were impacting larval size in this experiment due to the lack of a true control. A control was not included because the primary question was whether predatory stonefly nymphs were predators of White Sturgeon larvae, and if their predation rates would be influenced by substrate size. However, in hindsight, including a true control with no predaceous invertebrates present would improve the experimental design. 52 Further experiments should be done including these controls and varying abundances of predatory invertebrates. Measuring yolk-sac area as early as seven days post hatch (as I did in this study) may not be reliable, perhaps explaining why I found no significant difference in yolk-sac area between treatments. A study by Boucher et al. (2014) found that differences in yolk sac area increased throughout development between White Sturgeon larvae reared with and without gravel. In terms of reliability, it is unlikely that the yolk was completely depleted by 7 DPH. In Siberian Sturgeon (Acipenser baeri) reared at 17.8 ˚C the yolk sac has been observed to be depleted at 9 DPH (Gisbert 1999). Furthermore, Siberian Sturgeon reared at 18.3 ˚C have been observed to have completely depleted their yolk reserves by 9 - 10 days post hatch (Gisbert and Williot 1997). Therefore, there was likely still yolk remaining in my larvae. However, it remains uncertain without histological analysis if what externally appeared to be yolk sac was not disguising organs that had begun to develop internally. 4.3 Emergence Timing Experiment My findings with regards to time to emergence are surprising since I expected that most larvae would emerge around 14 DPH, while in the small substrate treatments larvae emerged around 19 DPH. However, my findings with regards to the time of emergence for larvae in the larger gravel do appear consistent with those of other studies who observed emergence around 13-15 DPH (Boucher et al 2014, Coulter et al. 2023). There are a few possibilities to explain why larvae in the small gravel treatments emerged so late (19 DPH) in my study. The first is that this could have been a methodological error where a lack of flowing water and drift nets resulted in a failure to measure emergence as it occurred. Alternatively, larvae in the small gravel 53 treatments could have depleted their yolk-sacs slower than larvae from the large gravel treatment, therefore leading to a delay in emergence timing. Very few larvae emerged over the course of my experiment, which lasted a total of 21 days. I question if so many larvae remained ‘unemerged’ in my study due to a lack of feed. A study by Bennett et al. (2007) did not quantify emergence and ended the experiment at the onset of exogenous feeding, but report that larvae emerged from the substrate when feeding began. This suggests that feed should be added as an extra parameter in this study, similar to protocol conducted by Coulter et al. (2023). Moreover, larvae denied food have been shown to emerge two days later than larvae who were given feed and emerged at 13 DPH (Coulter et al. 2023). Therefore, it is possible that the results of my experiment would change if food were provided. While I cannot make definitive statements about how these results could extend to natural environments, I can make some inferences. I observed that survivorship to 21 DPH was significantly affected by invertebrate presence. It has been suggested that lower water velocity because of flow modification could make White Sturgeon embryos more susceptible to predation by fish predators that could have historically been excluded from areas with White Sturgeon embryos due to higher flow velocity (Miller and Beckman 1996). In line with this logic, if flow regulation has brought predatory invertebrates into contact more frequently with White Sturgeon larvae, this could potentially contribute to high larval mortality and therefore recruitment failure. Downstream of dams, Ephemeroptera, Plecoptera, and Trichoptera richness has been shown to be lower, but increased depth and width are also associated with lower richness (Lessard and Hayes 2003). The White Sturgeon spawning reach of the Nechako River in 54 Vanderhoof has seen a reduction in wetted width post dam construction (Vasquez et al. 2008). Future field sampling is needed to clarify the abundance of perlid stonefly nymphs in White Sturgeon spawning reaches due to these confounding relationships. Regardless, while it is possible that predation on White Sturgeon larvae by stonefly nymphs is occurring, it is unlikely to be the sole driver of recruitment failure. 4.3.I Limitations The lack of flowing water is likely a major limitation for this study, meaning that the results are not necessarily representative of natural drift timing. Past work has found that larvae drift more pre-hiding at higher water velocities (McAdam 2012). If there were moving water and drift nets to capture larvae as they emerge, we could get a more accurate picture of when larvae are actually emerging because in this experiment, I only observed how many larvae had emerged at a certain point in time, twice during the day. It also seems unlikely that larvae would emerge and stay above the gravel line indefinitely, since we assume that larvae in the wild would be emerging and then drifting downstream. Due to experimental design, it is unlikely that my results can be extended to make inferences about the exact timing of drift in the natural environment. Survivorship was also difficult to estimate due to limited time and inability to investigate if each individual larva was alive and well or not (see methods). Survivorship was estimated as the number of larvae in each aquarium that did not have obvious malformations or fungal growths, but it was difficult to determine if some larvae were dead or rather immobile. Therefore, this adds a level of uncertainty into the results that is difficult to quantify. 55 Because so few larvae emerged overall, I am not able to answer my original question of how invertebrate presence, and the interaction with substrate, would influence drift timing in the wild. However, I believe that an interesting trend is appearing that should be subject to future investigation. I observed that the presence of a predaceous invertebrate caused larvae to emerge two days earlier in the presence of large gravel. The absence of a similar effect in the small gravel treatment supports my hypothesis that more porous substrate makes White Sturgeon larvae more vulnerable to predaceous invertebrates. Moreover, there is a marked decrease in survivorship for larvae in large gravel with an invertebrate present relative to large gravel without (20% lower). This contrasts with the ~ 3% higher survivorship observed for larvae in small gravel with an invertebrate present relative to without. Therefore, larger gravel may make larvae more vulnerable to predatory stonefly nymphs, resulting in earlier emergence timing. This finding should be confirmed with future studies. 4.4 Synthesis and Future Directions Freshwaters are extremely biodiverse, but unfortunately this biodiversity is declining at a rapid rate, with the vast majority of monitored freshwater species seeing population declines in the past fifty years (World Wildlife Fund 2022). In light of the biodiversity crisis, understanding what causes population and species declines is crucial to effective conservation measures. Populations can decline if offspring consistently do not survive to maturity. When survivorship of young life stages is consistently lower than what is needed to replace dying adults, it is called recruitment failure. For White Sturgeon specifically, recruitment failure is caused by near zero survival of the embryo and larval stages. 56 In the next five years, Fisheries and Oceans Canada has included in the White Sturgeon Action Plan a goal to perform ‘pilot studies’ to achieve natural recruitment for white sturgeon populations (Fisheries and Oceans Canada 2023). Among the strategies listed in this updated action plan, is one to ‘clarify’ the threat of predation to white sturgeon (Fisheries and Oceans Canada 2023). My research provides insight useful both into the planning of these pilot studies and into the predation experienced by early life stages of white sturgeon. First, I provide information about what substrates would be most effective for facilitating natural recruitment through substrate restoration. Specifically, larger substrates with similar interstitial spaces to those provided by my coarse gravel treatments would appear to be optimal substrate for addition into spawning reaches. Wherein, I specify ‘larger substrates’ because the grain size of substrates used in my experiments are likely too small to remain stationary within a river. Second, I demonstrated that predation by stonefly nymphs is unlikely to be a driver of recruitment failure. Future experiments should be done with more frequent checks for hatch and or/video monitoring of hatching to examine the exact times at which embryos are hatching to narrow down the exact shift that predatory invertebrate presence is having on hatch timing. Furthermore, future studies should also provide higher densities of predators, and perhaps combine predaceous stoneflies with other predators, such as fish, to observe how White Sturgeon embryos and larvae respond to multiple simultaneous predation cues. Since I did not provide alternative prey to the predatory stonefly nymphs, future studies should incorporate other prey items to observe whether predatory stonefly nymphs would choose alternative prey over White Sturgeon larvae. If White Sturgeon larvae are not the preferred prey for stonefly 57 nymphs, survivorship for White Sturgeon larvae in the wild where other prey are present may not be as reduced as what was observed in my study in the presence of predatory stonefly nymphs. I propose future work to clarify the effects of sand as a rearing substrate on embryos and larvae in a natural environment. In my experiments, as a sole rearing substrate sand resulted in lower condition factor for larvae relative to coarse gravel, but embryos coated in sand did not have lower survivorship than embryos not coated. Therefore, if a White Sturgeon embryo becomes coated in sand before coming to rest in an interstitial space (a likely event in the Nechako River), this sand coating is unlikely to alter its chances at success. However, the vegetal pole of the White Sturgeon embryo is more adhesive than the animal pole, and this has been hypothesized to be important for maintaining orientation during development (the more adhesive vegetal pole would ideally be oriented downwards and attached to the substrate and the less adhesive animal pole oriented upwards) (Cherr and Clark 1985). Due to the inability of white sturgeon embryos to adhere to sandy substrates (Parsley and Kofoot 2013) and knowledge that continuous tumbling in Macdonald Jars is bad for eggs (Earhart et al. 2020). The sandy bottom of the Nechako River within the White Sturgeon spawning reach is contributing to low recruitment and studies should be done to address the potential effects of continuously rolling along a sandy river bottom on White Sturgeon embryo development and subsequent survival. Restoration is an expensive undertaking, making it critical that restoration efforts are specific and well-targeted to their goals. In the United States alone, approximately a billion dollars has been spent on river restoration every year since 1990 (Bernhardt et al. 2005). 58 Substrate restoration in rivers is a method used by conservation practitioners for a variety of species. For example, in Northern California gravel has been added to Chinook salmon spawning reaches to observe what material is preferential for redd building (Merz et al. 2019). In the Nechako River, while substrate restoration can be effective in the short term, it has been unsuccessful as a long term means for successful White Sturgeon recruitment since added substrate is filled in with fines within a short time period (sometimes less than a week) (Steve McAdam personal communication). My results support the current understanding that sand leads to lower condition for White Sturgeon larvae. Therefore, the primary goal should be to remove fine substrates from the riverbed and prevent them from settling within the spawning reach. This is underway in the sense that there is a push to create sand traps upstream of the spawning reach within the river (Northwest Hydraulics Consultants 2023). In addition to the implications for gravel additions, the conservation applications of my work are rather limited due to feasibility. I found that predatory stonefly nymphs significantly reduced survival for White Sturgeon larvae, but removing stoneflies from White Sturgeon larval habitat is both unrealistic and ill-advised. Another option would be to somehow exclude predatory stonefly nymphs from certain gravel patches for White Sturgeon larvae, but this is difficult to implement and past efforts to perform exclusion studies in the Nechako River have been unsuccessful (Steve McAdam personal communication). However, I still believe that predator and fine sediment exclusion boxes/netting with proper substrate (large gravel) could be successful in rearing White Sturgeon embryos and YSL in the wild. Further efforts would be needed to troubleshoot previous designs. 59 5 Conclusion Many White Sturgeon populations in Canada are endangered due to a lack of survival of embryo and larval stages. My experiments are the first known observations of the interaction between stonefly nymphs and early life stages of White Sturgeon. I studied whether predatory stonefly nymphs are predators of White Sturgeon embryos and yolk-sac larvae, and if their presence has any impact on key early life history behaviours, namely timing of hatch and emergence. I found no evidence to suggest that perlids consume White Sturgeon embryos, and conflicting evidence about whether they consume White Sturgeon larvae. However, I did find that the presence of predatory stonefly nymphs had a significant influence on hatch timing, shifting hatch timing slightly later. I also observed a much higher proportion of hatch overnight than during the day across all treatment groups. In terms of emergence timing, I found that larvae emerged later when reared in smaller gravel relative to larger gravel regardless of predaceous invertebrate presence. However, survivorship of larvae was significantly lowered by predaceous invertebrate presence. I also validate previous findings that interstitial spaces lead to improved larval condition. 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Methods Measurements This measurement was taken as part of the hatch plasticity experiment. Refer to the methods section of my thesis for detailed husbandry and experimental setup procedures. Glass jars measuring 65 mL were each affixed with an oxygen sensor spot (Pre sens, SP- PSt3-NAU-D5-YOP). Pre Sens OXY 4 SMA fiber optic probes were used for measuring dissolved oxygen concentration and measurements were conducted inside of a single incubator set to 14 ˚C. Sensors were first calibrated by using a standard aquarium air pump and air stone to bubble oxygen into dechlorinated water. This was the standard for 100% oxygen. To calibrate for 0% oxygen, nitrogen gas was bubbled into dechlorinated water with a bubble bar. Oxygen levels were measured in these jars filled with this de-oxygenated water for 25 minutes to ensure the absence of leaks. 73 Embryo respiration rate experiments were conducted 6 and 7 calendar days post fertilization. Two trials/replicates were conducted in total, each within 24 hours of each other. For each trial the glass respirometry chamber was rinsed with 70% ethanol to reduce bacterial growth. Two embryos were taken from each aquarium and pooled by treatment group before being separated into two groups with nine embryos each. Nine embryos, all from the same treatment group, were added to each of the eight respirometry chambers (jars) and left unsealed for 25 minutes to allow the embryos to acclimate to the chambers before sealing the chambers with parafilm and starting the measurements. Probes were set to take measurements every ten seconds. A total of eight measurements were taken at once. This added up to four replicates being conducted per treatment group across the two MO2 trials. Upon completing the respiration rate measurements for the embryos, embryos were removed from the chambers and the chambers were refilled with water and resealed and the background respiration rate was measured for an hour. Embryos were weighed together in their groups of nine using a Sartorius BP211D (accuracy 0.01 mg) balance (wet mass). The embryos from the first trial were weighed on calendar day 7 post fertilization, but within 24 hours of the respiration rate measurement. For the second trial, embryos were weighed at 8 calendar days post fertilization, but again within 24 hours of the respiration rate measurements. To weigh the embryos, they were blotted dry with Kimwipes®, but it was not possible to remove the grains of sand from the exterior of the embryos from sand-coated treatments without damaging the chorion. Therefore, to calculate water displacement in the sand-coated vials, the total mass of the embryos including sand 74 grains is used, but an average mass of all non-sand-coated embryos from each chamber is used for respiration rates for all replicates. Data Analysis The first 1.3 hours of measurements were used for analysis and an R2 value of 0.9 was used as a threshold for ensuring linear model fit. Chambers where the linear model calculating respiration rate from the change in oxygen concentration over time had an R2 lower than 0.9 were excluded from analysis, similar to Earhart et al. (2023). Results Mean embryo respiration rates (± standard deviation) were highest for the combined group (0.68 µmol O2 / g*hour ± 0.19), and were lower from invertebrate to sand coated to control (0.66 µmol O2 / g*hour ± 0.13, 0.61 µmol O2 / g*hour ± 0.20, 0.59 µmol O2 / g*hour ± 0.15, respectively) (Figure A1). Respiration rates were not significantly affected by sand coating (χ21 = 0.019, p = 0.89), invertebrate presence (χ21 = 0.32, p = 0.57), or the interaction (χ21 = 0.0005, p = 0.98) (two-way, type-three ANOVA). Model fit appears to be quite good with a low dispersion parameter (0.0289). Background respiration rates were negligible in all chambers so were not incorporated into measurements. 75 Figure A1: Respiration rate per embryo from each treatment group. N = 3 for control and sand coated, N = 4 for invertebrate and combined. Combined refers to embryos being reared both coated in sand and in the presence of a predatory invertebrate. Discussion Despite not finding a statistically significant difference in respiration rate between treatments, there does still appear to be a trend appearing in the data (Figure A1). The average respiration rate of embryos from treatments where an invertebrate was present (Invertebrate and Combined) are higher than those for embryos from the control and sand coated group. This is surprising, given findings from Bjornson and Anderson (2017, personal communication). Respiration rate µmol/(g*h) 76 When exposing Lake Sturgeon embryos to alarm cue (from conspecifics) and predatory cue (from crayfish) they found no difference in measured heart rates of embryos at 12 DPH. My results showed lower oxygen consumption rates than observed in a study on White Sturgeon embryos by Earhart et al. (2023). My results showed that mean respiration rate of embryos in the sand coated treatment was slightly higher than from the control treatment. This was unexpected given my hypothesis and I believe warrants further investigation. Greig et al. (2005a,b) found that Atlantic Salmon embryos had 41% lower rates of oxygen consumption when coated in clay. They proposed as a mechanism to explain this that either the clay was blocking oxygen channels on the chorion, or that the clay formed a complete sheath with low oxygen permeability around the whole embryo (Greig et al. 2005a, Greig et al. 2005b). If either of these mechanisms are indeed responsible for the decrease in oxygen consumption that they measured for embryos coated in clay it is possible that the grain size of sand used for my experiments was not fine enough to restrict oxygen uptake. The accuracy of these measurements was limited by a few factors. A last-minute error of incompatibility of sensors and probes resulted in using larger jars than previously planned that did not have stir bars with false bottoms. The lack of a stir bar means that water was not being mixed throughout the measurement which could potentially cause uneven distribution of dissolved oxygen and therefore an inaccurate measurement. Respiration rate measurements were conducted at which point embryos had decreased in adhesiveness so embryos from the sand coating treatments were not as coated in sand. Second, due to being limited by the number of oxygen probes available, there are very low sample sizes. Future studies should be done conducting these measurements for a shorter period of time so that more can be done in 77 the same day when the number of probes is limiting. Overall, I am not able to conclude definitively whether or not sand coating and predaceous invertebrates alter the respiration rate of White Sturgeon embryos. However, my results indicate that this relationship should be explored further with increased sample size.
Thesis/Dissertation
eng
Forestry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Richardson, John Stuart
McAdam, Steve
Early life history interactions of White Sturgeon (Acipenser transmontanus) with substrates and predaceous invertebrates
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