FACTORS AFFECTING THE LIFE HISTORY AND ESTABLISHMENT SUCCESS OF THE INVASIVE SPINY WATER FLEA, BYTHOTREPHES LONGIMANUS, IN CANADIAN SHIELD LAKES
NATALIE KIM
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GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO
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The Ponto-Caspian invader Bythotrephes longimanus (commonly known as the spiny water flea; herein Bythotrephes), continues to expand throughout North America, having been identified in > 170 water bodies. As a voracious predator, Bythotrephes has had profound impacts on native zooplankton abundance, species richness, diversity, and community size structure. Yet the open water habitats of the Canadian Shield, where the majority of new invasions are occurring, are in a state of flux. Among the many changes are rising summer water temperatures, declining waterborne calcium (Ca) levels, decreasing total phosphorous (which will likely impact primary productivity and herbivorous zooplankton, Bythotrephes' primary prey), and deteriorating algal food quality with respect to fatty acids.
What remains unclear is how these factors, singly or in combination, might affect the continuing establishment of Bythotrephes populations. It was my goal to investigate these knowledge gaps through controlled laboratory studies with reinforcing arguments from field observations, where available. I began by devising a culturing technique for
Bythotrephes, a species that is notoriously difficult to rear in the laboratory, and finding its temperature optima (Chapter 2). Next, I examined the potential impacts of low Ca on
Bythotrephes by determining its somatic Ca content, conducting a life table response experiment in which a cohort of Bythotrephes was reared at differing Ca concentrations, and by examining its distribution in relation to Ca in Norway, where it has been established for millennia (Chapter 3). After deducing that Bythotrephes will likely not be directly affected by falling Ca concentrations, 1 go on to explore the possible joint impacts of low Ca and Bythotrephes presence on native Daphnia, which have relatively high Ca demands. It became evident that Bythotrephes'' ability to survive and reproduce is highly influenced by prey availability. Therefore, the effects of prey quantity and quality (in terms of the omega-3 fatty acid eicosapentaenoic acid, or EPA) on
Bythotrephes life history and demography were investigated (Chapters 4 and 5, respectively). Not surprisingly, increasing prey quantity not only improved Bythotrephes population growth rates, it resulted in larger offspring and shorter generation times. Prey quality plays a paradoxical role; while supplementing daphniid prey with EPA made predation by juvenile Bythotrephes a challenge, the addition of this compound fosters increased clutch size in adult Bythotrephes. Finally, the results of these studies along with additional observations on the autecology of this species are summarized, and approaches for management are discussed (Chapter 6).
Bythotrephes' unintentional introduction and subsequent spread have served as a prime opportunity for an in-depth analysis of the invasion process. This study is one of the very few examining in detail a suite of factors governing the survival, growth, and reproduction of this species. Much of the information presented here will be useful for modeling and management efforts.
V DEDICATION
An ode to the spiny water flea
For this is the story of your life, One of success, but initially strife. Confined to a ship ballast1, it's lipids you lack, An immigrant from Russia, only a broodsac on your back. Straining your eye in the dark, you swim feebly forward, Spot a glimmer of movement, flex your spine like a sword. Looping and spiraling with renewed anticipation, You grasp your spent sister and proceed to the decapitation. With the elixir of life flowing dark through your gut, Naturally selected to endure—no if s, and's, or but's!
When at long last, is that sunlight you see? Discharged into the New World...yet it seems habitable, seriously. Your feverish palate delights at the unsuspecting buffet, And you feast on bosminids 'till your broodsac ruptures away. Moonlight is glistening on the warm surface waters, As you rise and release (in your image) two beautiful daughters. On you persist while another broodsac blisters, A week later, it's a boy, and his hefty twin sisters.
You're senescent and tired, but now it's okay, You sink to the sediments where just your soft parts decay. To be found decades later by keen scientists, Intrigued by your life story, a plot thick with twists. It's here I digress, and feel the need to pay tribute, Without the sacrifice of your descendants, this story would stay mute. You've left a legacy far longer than your spine, Been the star of countless chapters, six of them mine2.
Spaseeba.
1 Bythotrephes was most likely transported to North America in ballast tanks as diapausing resting eggs, not as live organisms. Dramatization for creative purposes only. 2 And for that 1 thank the chubby shrimp, the ones hatched in brine.
VI ACKNOWLEDGEMENTS
First and foremost, I'd like to express my utmost gratitude to my advisor Dr.
Norman Yan for taking me on, granting me independence to explore new ideas, and providing the raw materials needed to develop as a scientist. Thank you for your sage insights and infinite patience over the years. I appreciate the amazing opportunities you've given me to travel, present my work, and cultivate new connections. I'm honoured to have been a part of your outstanding crew, and admire your boundless energy and big-picture optimism, despite the gravity of threats facing our freshwaters.
To my collaborators—tusen takk to Bjorn Walseng from the Norwegian Institute for Nature Research, for your helpful suggestions which no doubt strengthened Chapter 3.
A huge thank you to Dr. Michael Arts for allowing me the opportunity to work with you for Chapter 5. Your expertise (especially when it comes to fatty acids), meticulous attention to detail, kindness, and top-notch technical support were truly appreciated.
To my supervisory committee—thank you to Drs. Bridget Stutchbury and
Roberto Quinlan, two scientists I admired before I even came to York. The comments imparted at progress evaluations most certainly helped steer the course of this work.
This entire body of work couldn't have been completed without the aid of many, who are specifically acknowledged at the end of each chapter.
I'm grateful to the entire staff at the Ontario Ministry of the Environment's Dorset
Environmental Science Centre for being so accommodating and willing to impart advice or join me in troubleshooting as needed. Thanks especially to Don Evans et al. for analyzing countless Ca samples. To the best and brightest assistants anyone could ask
vii for—Stephanie Hung, Melanie Shapiera, and Marthe Haarr—thank you a million times over for your tireless work and unending enthusiasm during those exceedingly long days.
I wish you the best in all of your prospective endeavours.
To my York and FLAMES colleagues—you have been a mighty fme group to work with. Thanks to Christine Gibson and Dr. Martha Celis-Salgado for showing me the ropes around FLAMES, and Dallas Linley for technical support. To Jen Petruniak and
Allegra Cairns, you've assumed countless roles over the years: lab and housemates, teachers, willing assistants, cut-throat competitors in Catan-land, and of course, friends.
Stephie, thanks for being a consistent ally in both academics and life, and for always seeing the brighter side of things. Thanks to Anneli Jokela of Queen's for many
Bythotrephes-related chats.
I'm lucky to have some of the most fantastic Homo sapiens on this planet as friends—my oldest amigo Ashleigh Allen, who introduced me to the wonders of the wild and instilled in me the need to approach everything with a critical yet comical eye; Jay
'illinit' Choi, hands-down best 114 ever; and Dr. Mike Mak, who urged me to take that first step toward a scientific career.
To my family, the most resilient support network one could ask for—gamsa hamnida, Appa and Umma for always giving me the freedom to explore and trusting that
I'll make sound decisions. Thanks to my fabulous five sisters (Jeanne, Tammy, Sylvia,
Sandra, Stephanie) and brothers by law (Rich, Martin, D) for generously supplementing my student income with complimentary food/drink, clothing, and paying odd jobs. I'm forever in debt, and couldn't have pulled through without each and every one of you!
viii To my partner Jungmin Choi—thank you for making the trek all the way to
Canada only to have to put up with my long "working vacations" in Muskoka. Kudos to you for holding down the fort while I was gone, and for encouraging me to keep at it despite a barrage of setbacks. Onward we march to destinations unknown!
To my traveling zoo—Atom, Nabi, Key (no doubt you're thriving on the shores of Crown Lake), Ms. Guppy (RIP)—thanks for the companionship during countless hours spent in un-ergonomic positions before LCD screens. Also a quick shout out to the late great Carl Sagan...when nothing made sense, your words never failed to put everything back into perspective3. We are all, after all, drifters on this pale blue dot.
3 Sagan, C. 1994. Pale Blue Dot: A Vision of the Human Future in Space. Random House, New York, U.S.A. 429 pp. See: http://www.youtube.com/wateh?v=p86BPMlGV8M
1Y TABLE OF CONTENTS
ABSTRACT iv DEDICATION vi ACKNOWLEDGEMENTS vii LIST OF TABLES xii LIST OF FIGURES xv LIST OF APPENDICES xvii
CHAPTER 1 Introduction
A brief introduction to invasion biology 1 Bythotrephes as a case study 6 Focus of dissertation 10 References 12
CHAPTER 2 Methods for rearing the invasive zooplankter Bythotrephes in the laboratory
Abstract 24 Introduction 25 Materials and Procedures 28 Assessment 37 Discussion 39 Acknowledgements 46 References 46
CHAPTER 3 Will environmental calcium declines hinder Bythotrephes establishment success in Canadian Shield lakes?
Abstract 56 Introduction 57 Materials and Methods 61 Results 69 Discussion 71 Acknowledgements 79 References 79
Y CHAPTER 4 Prey quantity impacts life history of the invasive predatory cladoceran, Bythotrephes longimanus
Abstract 91 Introduction 92 Materials and Methods 95 Results 99 Discussion 102 Acknowledgements 108 References 108
CHAPTER 5 Effects of eicosapentaenoic acid (EPA) limitation and rising water temperature on the aquatic invader Bythotrephes
Abstract 122 Introduction 123 Materials and Methods 129 Results 136 Discussion 142 Acknowledgements 151 References 151
CHAPTER 6 Concluding remarks
Summary and implications for establishment 171 Importance of autecological studies 175 Prognosis 177 References 179
Yl LIST OF TABLES
Table 2.1. Comparison of Bythotrephes development time to maturity, % surviving to parturition, mean number of neonates per female, and total neonate production in FLAMES soft-water medium, and lake water filtered to 20 and 80 (am. Animals were reared at 16°C in a 17 day bioassay. There were initially 13 test animals per treatment. Values with the same letter grouping are not significantly different from each other (a = 0.05).
Table 2.2. Comparison of Bythotrephes survival, neonate production, generation times and population growth rates in FLAMES soft-water medium at 16°C, 19°C, 22°C and 25°C.
Table 2.3. Life history data for Bythotrephes (n = 8) reared in FLAMES medium at 21°C, offered a diet of Artemia, B.freyii, D. ambigua, D. pulex.
Table 3.1. Effects of [Ca] (measured values) of bioassay media on neonate Bythotrephes reared over 23 d. Numbers of initial replicates, mean times to Instar 3, % survival to parturition, mean times to parturition, cohort generation times (Tc), net replacement rates (Ro), and intrinsic rates of natural increase (r) are presented, ± 1 SD. There were no significant effects of [Ca] on time to Instar 3 or time to parturition (one-way ANOVA, a = 0.05).
Table 4.1. Dry mass estimates of prey used to calculate biomass of Bythotrephes' predation rates. Only mid-size, barren prey were used for feeding. Sample replicates used in the estimations of dry mass are noted, where available. References:a Witty 2004; b Dumont et al. 1975's value for B. longirostris (O.F.M.);c Hoff & Snell 2007; d This study.
Table 4.2. Intrinsic rates of natural increase, net reproductive rates, and cohort generation times (± SE) for Bythotrephes (n = 9 • food treatment"1) reared on a mixed-prey assemblage of differing densities in 400 ml over 22 days. For each demographic metric, values not connected by the same letter indicate significant differences as identified by post hoc Tukey HSD tests (a = 0.05).
Table 4.3. Mean growth (estimated by the length of the intercalary tailspine segment between barb pairs, which are accrued at each moult) and adult lengths (± SE) of Bythotrephes reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. Lengths of Bythotrephes attaining the third instar stage are represented. Body sizes were corrected by a factor of 1.2 to account for shrinkage in sucrose formalin (following Yan & Pawson 1998), but statistical tests were not applied due to unacceptably high variability. For each size metric, values not connected by the same letter indicate significant differences as identified by post
Yll hoc Tukey HSD tests (a = 0.05). Values in parentheses indicate numbers of Bythotrephes included in analyses.
Table 4.4. Mean (± SE) and sample size (in parentheses) of brood and offspring sizes resulting from Bythotrephes reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. For each metric, values not connected by the same letter indicate significant differences (a = 0.05). Only one offspring from each brood was randomly selected to be represented for all length measurements. Time between broods was not assessed statistically due to unacceptably high variability.
Table 4.5. Bythotrephes' mean predation rates and estimates of mean biomass consumed (Hg DM) over two-day intervals (± SE). Bythotrephes (n indicated in parentheses) were reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. Mean feeding efficiencies ranged from 49% for both the medium and high food densities to 60% for the low food density.
Table 5.1. List of fatty acid (FA) abbreviations commonly used in this chapter, and where applicable, the corresponding structural nomenclature.
Table 5.2. Mean proportions (as % of FAs) of selected FAs in Bythotrephes reared without and with EPA addition at 21°C, ± 1 SD (n.d. = not detected). Also shown are mean proportions of fatty acids in Bythotrephes retrieved from Mary Lake, Huntsville, Ontario, Canada. There were 2 sample replicates of lab-reared Bythotrephes consisting of 7 - 8 individuals each, and 5 sample replicates of Mary Lake-caught Bythotrephes, consisting of 10 - 19 individuals each. Also included for reference are data from Persson and Vrede (2006) for Bythotrephes collected from four oligotrophic lakes in northwestern Sweden (n = 2 - 4 replicates total). There were no significant effects of EPA enrichment on any of the FAs in the laboratory-reared Bythotrephes (a = 0.05).
Table 5.3. Mean proportions of FA functional groups (as % of FAs) considered in this analysis, and X(d3:Xcd6 for 11-day-old Bythotrephes reared at 21°C in the laboratory and fed prey cultured on S. obliquus without and with EPA additions, ± SD. For reference purposes, data are also shown for wild-caught Bythotrephes from Mary Lake.There were 2 sample replicates of lab-reared Bythotrephes consisting of 7 - 8 individuals each, and 5 sample replicates of wild-caught Bythotrephes, consisting of 10 - 19 individuals each. There were no significant differences between treatments for Bythotrephes reared in the laboratory.
Table 5.4. Mean proportions (as % of total FAs) of selected FAs in D. ambigua fed the chlorophyte S. obliquus treated without and with EPA, at 21°C and 26°C, ± SD (n.d. = not detected). There were 5 sample replicates for each treatment.
Y111 Asterisks (*) indicate significantly different treatment means within a given temperature (a = 0.05).
Table 5.5. Mean proportions of FA functional groups (as % of total FAs) considered in this analysis, and £<»3:Xg)6 for D. ambigua fed the chlorophyte S. obliquus treated without and with EPA, at 21°C and 26°C, ± SD. There were 5 sample replicates in each treatment. Asterisks (*) indicate significantly different treatment means within a given temperature (a = 0.05).
Table 5.6. Mean proportions (as % of total FAs) of selected FAs in S. obliquus without and with EPA addition, at 21°C and 26°C, ± SD (n.d. = not detected). There were 5 sample replicates for each treatment. A single asterisk (*) indicates significantly different treatment means within a given temperature, while double asterisks (**) denote significantly different means for control S. obliquus between temperatures (a = 0.05).
Table 5.7. Mean proportions of FA functional groups (as % of FAs) considered in this analysis, and Xcq3:Xcq6 for S. obliquus cultured at 21°C and 26°C in the laboratory without or with EPA addition, ± SD. There were 5 sample replicates in each treatment. A single asterisk (*) indicates significantly different treatment means within a given temperature, while double asterisks (**) denote significantly different means for control S. obliquus between temperatures (a - 0.05).
YIV LIST OF FIGURES
Figure 1.1. A third instar specimen of the invasive spiny water flea, Bythotrephes longimanus Leydig (1860), with enlarged unpigmented broodsac, collected from Lake of Bays, Ontario, Canada. Parthenogenically-produced Bythotrephes typically develop through three instar stages prior to reproductive maturity, whereas sexually-produced animals undergo four instar stages.
Figure 1.2. Bythotrephes' life cycle, consisting of parthenogenic and gametogenic reproduction. It should be noted that gametogenically-produced females (i.e., from diapausing eggs) lack the 'kink' in the caudal process characteristic of parthenogenically-produced Bythotrephes.
Figure 2.1. Daily cumulative mortality of Bythotrephes (n = 13 Bythotrephes' treatment"1) plotted over time in a 96 h range-finding temperature bioassay conducted at 16°C (•), 20°C (•), 24°C (•), 28°C (x) and 32°C (•).
Figure 2.2. Mean numbers of prey items consumed or killed per day (n - 30 observations • species"1), shown with standard error bars. Abbreviations for prey species: Amb. (£>. ambigua), Bos. (B. freyii), Art. (Artemia nauplii), Hoi. (H. gibberum), Pul. (D. pulex). Values not represented by the same letter indicate significant differences (a = 0.05).
Figure 3.1. Relative proportions of first (light grey), second (open), and third broods (black) produced by Bythotrephes reared at different [Ca] intervals.
Figure 3.2. Mean first (open) and second brood (closed) clutch sizes for Bythotrephes reared at different [Ca] over one parthenogenic life cycle, ± SE. Numbers of Bythotrephes, n, ranged from 4-11, with the exception of the second brood clutch size at 0.1 mg • L"1, for which n = 2.
Figure 3.3. Proportions of circumneutral Canadian Shield lakes (pH 6-7) with Bythotrephes (n = 22 of 183 total lakes; open bars) and Norwegian lakes containing Bythotrephes (n = 116 of 499 total lakes; closed bars). Each interval represents a [Ca] bin width of 0.5 mg L"1.
Figure 4.1. Survivorship for Bythotrephes reared over 22 days in the laboratory at 21°C, and offered low (dotted line; n = 8), medium (dashed line; n = 9) and high (solid line; n- 9) food densities. One female was still alive after 22 days in the low food treatment.
Figure 4.2. Predation rates by Bythotrephes (n = 7 - 8) at low, medium, and high prey densities (15, 30, and 45 prey"1' 0.4 L"1' d"1, respectively) over six days, separated
YV in order of overall prey preference: a) A. franciscana nauplii, b) B.freyii, c) D. mendotae, d) D. pulex, and e) D. ambigua. Lines intersecting graphs represent grand means.
Figure 5.1. Example of laboratory-reared, parthenogenic, 11-day-old Bythotrephes female with two embryos in the early stages of resorption (e) visible through the broodsac. This particular female had been fed EPA-enriched daphniids. The smaller spacing between the articular spines (a - b) on the caudal process, compared to (b - c), indicates poor growth at the first instar stage.
Figure 5.2. Mean predation rates of Bythotrephes < 24 hour old on D. ambigua, ± SE. D. ambigua were offered at rates of 30 prey • d 1 or 60 prey • d"1 at 21°C and 26°C, respectively. There were 14-15 incubations • treatment"1. Bars not connected by the same letter within each temperature treatment indicate means with statistically significant differences at the (a = 0.05).
Figure 5.3. Mean clutch sizes of Bythotrephes reared at 21 °C on day 11 of the assay, ± SE. There were 11 and 9 fecund Bythotrephes in the control and EPA-enriched treatments, respectively. Bars not connected by the same letter indicate means with statistically significant differences (a = 0.05).
Y VI LIST OF APPENDICES
Appendix A. Results of curve-by-curve survival analyses for Bythotrephes reared at five different temperatures (16°C, 20°C, 24°C, 28°C, 32°C). P-values marked with asterisks (*) denote significant differences among pairs.
Appendix B. Results of Tukey-Kramer HSD multiple comparisons of Bythotrephes mean daily feeding rates on different prey species. P-values marked with an asterisk (*) denote significantly different predation rates by Bythotrephes (df= 1 for all comparisons).
Appendix C. Mean predation rates (± SE) of second instar Bythotrephes (n = 5 individuals • treatment"1) over 24 h when offered 6, 9, and 12 each of barren, mid-size D. ambigua and D. pulex in 175 ml at 21°C and 26°C. Prey offered were doubled for the higher temperature, as Bythotrephes' predation rates increase with this temperature increase (N. Kim personal observation).
Appendix D. Bythotrephes (n = 5 individuals ' treatment"1) exhibits significantly higher (t = 2.73, df= 7.9, one-tailed p = 0.013) predation rates on mid-size (500 - 850 fxm) Daphnia pulex in light (100 [imol' m"2' s"1) compared with dark (5 fxmol' m"2' s"1) conditions in the laboratory. Initial prey densities were 50 D. pulex' 175 ml"1.
Appendix E. Description of dewar malfunction during transport of lipid samples.
Appendix F. Temperature data for Lynch Lake (45.2°N, 79.2°W) in Huntsville, Ontario, Canada. TidbiT v2 UTBI-001 temperature loggers (Onset Computer Corporation, Pocasset, MA, USA) were deployed at the deepest point of the lake (maximum depth = 3.8 m). Upper line indicates temperature at 0.1 m from the surface and the lower line indicates temperature at 3.5 m depth.
Appendix G. Percent survival of first and second instar Bythotrephes (n = 6 individuals ' treatment"1) in a 96 hour pH range-finding bioassay conducted at 22°C.
Appendix H. Mean ages (in days) of first, second, and third instar Bythotrephes reproducing under differing conditions in the laboratory, alongside age ranges of third instar animals reproducing. Sample sizes are reported in parentheses. All Bythotrephes were hatched out in the laboratory and observed daily.
Appendix I. Summary of body, tailspine, and total lengths of third instar Bythotrephes observed throughout this study at 21°C. Values for test conditions 1 - 2 indicate results of Chapter 5 (n = 15 and 14 Bythotrephes, for each respective
YVtl condition); conditions 3,4, and 6 are presented in Chapter 4 (n = 4, 7, and 6, respectively); and condition 6 is taken from Chapter 3 (n = 7).
Appendix J. Growth at first and second instar for all third instar Bythotrephes observed throughout this study at 21°C. Values for test conditions 3, 4, and 6 are presented in Chapter 4 (n = 4, 7, and 6, respectively); and condition 6 is from Chapter 3 (n = 7).
YV111 CHAPTER 1
INTRODUCTION
A brief introduction to invasion biology
The impact of non-indigenous species has been recognized as a leading cause of global biodiversity loss, second only to human population growth and related anthropogenic activities (Pimental 2011). Those species deemed 'invasive' contribute to biodiversity loss via parasitism, predation, hybridization and competition with native species, resulting in their displacement (Pimental 2011). In addition, they can drastically alter habitats and modify ecosystem processes (e.g., nutrient cycling, productivity, hydrology), particularly if they differ in their functional role from native species
(Vitousek 1990; Mack et al. 2000). Although the transport, introduction, and establishment of non-indigenous species precedes the advent of humans, the rate at which these processes occur is now orders of magnitude greater than naturally-occurring background rates, owing to increases in international trade (Lodge & Shrader-Frechette
2003). The damage caused by invading species is expected to intensify in the future (Sala et al. 2000), and humans will continue to exacerbate the rates at which invasions occur.
Moreover, aquatic ecosystems are more prone to invasions than terrestrial ecosystems for various reasons related to the strong attraction humans have to water (Lodge et al. 1998).
Aquatic invasive species (AIS) have been widely cited as a major stressor to marine and freshwater systems (Catford et al. 2011). There are > 180 AIS established in the Laurentian Great Lakes alone (Holeck et al. 2004; Ricciardi 2006), acting as source
1 populations for secondary spread inland (Vander Zanden and Olden 2008). Zebra mussels (Dreissena polymorpha) are perhaps the best-known example of AIS in North
American freshwaters. The economic impacts of zebra mussels are staggering; in the
Great Lakes region at least $150 million is spent annually to clear clogged water intake pipes in power plants, municipal water supplies and industrial buildings (O'Neill 1997).
Despite the lag period that often precedes an invasion "disaster", the financial burdens associated with invasions are high and only increase as an invader continues to spread; moreover, once established, invasions are usually difficult to completely reverse (Lodge et al. 2009). Fortuitously perhaps, it is much less costly to prevent AIS than to manage them following establishment (Leung et al. 2002; Lodge et al. 2006). Examples of successful eradication following early detection include pampas grass (Cortaderia selloana), ragwort (Senecio jacobaea), and prickly hakea (Hakea sericea), all on islands in New Zealand (Timmins & Braithwaite 2002). These observations have culminated in strong scientific advocacy for early detection of potential invaders via careful monitoring programs (Lodge et al. 2006; Vander Zanden et al. 2010). It has even been suggested that managers treat invasions as natural disasters, with a parallel focus on early detection and rapid response; while both are relatively rare events, emergency contingency plans are much more commonplace for the latter (Ricciardi et al. 2011).
Due to the obvious practical implications of invasions, invasion biology has grown rapidly over the past two decades (Blackburn et al. 2011). As a scientific discipline, the field was spurred by the publication of ecologist Charles Sutherland
Elton's (1900-1991) seminal work The Ecology of Invasion by Animals and Plants in
1 1958. In this volume, Elton summarized the literature on the spread and impacts of non- indigenous species (Southwood & Clarke 1999). Interestingly however, he did not provide an explicit definition for the phenomenon (Richardson et al. 2000). Since then, the field has been beleaguered by inconsistencies in terminology (Richardson et al. 2000; see Colautti & Maclsaac 2004 for an extensive review), likely owing to a lack of consensus regarding what actually constitutes a biological invasion (Valery et al. 2008).
This ambiguity may have arisen because invasion biologists working with different taxa and in diverse environments have autonomously developed their own interpretations of the phenomenon (Blackburn et al. 2011).
I consider an invasive species to be a non-indigenous (or non-native) species that has spread and established at least one population in an area outside of its endemic range, to the extent that it exerts an undesirable impact. The latter point is in recognition of the reality that invasive species are generally known for their negative impacts. Since impacts are assessed from an anthropocentric viewpoint, however, individuals may disagree on what qualifies as an undesirable impact (Davis 2009). Perhaps because of this human factor, there are still no standard, universally-accepted definitions for even the most commonly used terms in invasion biology.
Given the often severe ecological and economic impacts of biological invasions, these ongoing divisions in the field may seem surprising. Davis and 18 other researchers
(2011), for example, recently argued that managers should evaluate organisms based on their environmental impacts rather than on whether they are native or non-native, citing a lack of data to support the idea that an introduced species presents an "apocalyptic threat to biodiversity". Their statements were met with swift opposition from Simberloff and
141 other scientists (2011; see http://go.nature.com/fl eqjn for list) who advocate a more precautionary approach, particularly since the impacts of invasive species may not be apparent for decades following their introduction.
Invasion biologists generally agree, however, that the invasion process consists of the following stages: 1) introduction, 2) establishment, 3) spread, and 4) impacts
(Lockwood et al. 2007). Williamson (1996) was the first to conceptualize invasions as a phenomenon in which spreading populations overcome a sequence of barriers, with the majority failing to cross all of them. Plant ecologists typically subscribe to the framework developed by Richardson and co-workers (2000), which also considers invasions as a set of barriers that a non-native species must overcome to become invasive, but focuses more on the fate of the individual colonist. Blackburn and co-workers (2011) recently proposed a unified framework for biological invasions by merging the ideas of the two. Despite these slightly different approaches, envisioning invasions as a series of sequential steps is conducive to classifying human actions as either facilitative or inhibitive to a non-native species' ability to transition a stage (Kolar & Lodge 2001).
To initiate the invasion process, organisms transported by a vector (along a pathway) must first be introduced (intentionally or accidentally) alive somewhere outside of their native range, with survival ability depending on the species' traits and prevailing environmental conditions (Lodge et al. 2009). Next, a potential invader must either establish in the new environment or go extinct (Tobin et al. 2011). Many workers have examined various factors contributing to establishment success and/or failure. Not
A surprisingly, propagule pressure—both propagule size (the number of individuals in a propagule), and propagule number (the rate at which propagules or colonists arrive)—is positively associated with establishment success (Simberloff 2009)Propagule pressure also interacts with environmental stochasticity, however, as a large propagule can be entirely decimated should it arrive to unfavourable conditions (Simberloff 2009).
Nevertheless, if individual colonists are capable of surviving and reproducing in the new environment, establishment will only occur when the long-term intrinsic rate of increase
(r) is positive (i.e., above 0; Blackburn et al. 2011). Crawley (1986) showed that insects with the highest r-values were more likely to establish, and that they exhibited smaller body size and higher fecundity, allowing them to produce several generations over the year. Careful examination of life history and demography may therefore be particularly important for determining the life history stages where management strategies may be the most efficient (Sakai et al. 2001). Allee effects or "a positive relationship between any component of fitness of a species and either numbers or density of conspecifics"
(Stephens et al. 1999) often apply at the establishment and spread stages and, if operational, may lead to decreased establishment success (Taylor & Hastings 2005;
Liebhold & Tobin 2008). Commonly-cited explanations for establishment failure include unsuitable climate, disturbance, and/or predation from native species, along with competition and disease (reviewed in Sakai et al. 2001). Still, up to 50% of vertebrates released into a novel habitat will succeed in establishing self-sufficient populations
(Jeschke & Strayer 2005). From these founding populations, up to 50% of introduced
1 It should be noted however, that large-scale invasions could result from very small propagules. The North American population of European bees, for example, probably originated from a singly mated female (Zayed et al. 2007). species will spread and establish in new locales, and of these satellite populations, up to
25% will eventually be categorized as "invasive" (Lodge et al. 2009), at which point the impacts of the invader will have been recognized.
Biological invasions in progress offer unique opportunities to examine the inner workings of population processes (Sakai et al. 2001), and much more research is needed in this constantly evolving field to inform policy makers dealing with the threat of incoming or already-established invasive species.
Bythotrephes as a case study
Bythotrephes longimanus (Leydig 1860; Crustacea: Onychopoda: Cercopagidae)
(Figure 1.1) is a predatory cladoceran zooplankter originating from the Ponto-Caspian region, where it inhabits large, deep, and nutrient-poor lakes (Grigorovich et al. 1998;
Maclsaac et al. 2000). Each individual possesses a caudal process with paired articular spines (commonly called "barbs", with a new pair typically added at each moult; Yurista
1992) that serves as a morphological deterrent to potential predators (Barnhisel 1991) and spans approximately 70% of its entire length (Rivier 1998). Mature Bythotrephes sometimes reach lengths upward of 15 mm (Schulz & Yurista 1998). Aided by its large eye consisting of approximately 200 ommatidia (Martin & Cash-Clark 1995),
Bythotrephes is primarily a visually oriented hunter (Muirhead & Sprules 2003; Pangle &
Peacor 2009). It consumes mostly small crustaceans, capturing prey using prehensile thoracic appendages, or thoracopods (Branstrator 2005). Bythotrephes' mode of feeding has been described as the shredding and discarding exoskeletal matter, followed by the selective intake of soft parts (Lehman 1993). Bythotrephes tends to aggregate in patches relatively high in the water column and may exhibit weak diel vertical migration patterns for reasons related to predator avoidance, feeding, and reproduction (Rivier 1998; Young & Yan 2008). As a conspicuous zooplankter owing to its large, pigmented eye (Zaret 1972), Bythotrephes is frequently selected by planktivorous fish (reviewed in Maclsaac et al. 2000). Known predators of Bythotrephes in North America include lake herring (Coregonus artedi), lake whitefish (C. clupeaformis), rainbow smelt (Osmerus mordax), alewife (Alosa pseudoharengus), chinook salmon (Oncorhynchus tshawytscha), pink salmon (O. gorbuscha), yellow perch (Perca flavescens), white bass (Morone chrysops), and walleye
(Stizostedion vitreum), among others (review and references in Grigorovich et al. 1998).
Bythotrephes' recent range expansion to North America has made it the world's most intensively studied planktonic invader (Bollens et al. 2002) and the model invader for a recent special publication of Biological Invasions (see Yan et al. 2011, and references therein). Likely introduced via the ballast water of transoceanic ships (Sprules et al. 1990) from Lake Ladoga, Russia (Berg et al. 2002), it was found in all of the
Laurentian Great Lakes throughout the 1980s (Bur et al. 1986; Lange & Cap 1986;
Lehman 1987; Evans 1988; Cullis & Johnson 1988). By the 1990s, it had moved inland to lakes in the cottage-heavy district of Muskoka, north of Toronto, Ontario, Canada (Yan et al. 1992), probably mediated by human migration (Weisz & Yan 2010). Bythotrephes' current North American range extends to > 170 lakes in south-central and northwestern
Ontario, Canada, the northeastern United States (Kerfoot et al. 2011; Strecker et al. 2011;
Yan et al. 2011), and most recently to Lake Winnipeg, Manitoba (Justin Shead, Manitoba
7 Ministry of the Environment, personal communication) from where it is apt to further spread and establish.
Of the potential biological invaders, predators typically exert the heaviest impacts on native communities (e.g., Case & Bolger 1991), and Bythotrephes is no exception. In lakes, planktivorous fish usually have high establishment rates and thus the most impact on native ecosystems (Moyle & Light 1996), yet Bythotrephes may fulfill a similar functional role. In Lake Huron, for example, Bythotrephes was estimated to have consumed more zooplankton than did fish, at rates exceeding that of zooplankton production (Bunnell et al. 2011). Bythotrephes has been associated with changes in native zooplankton communities in Lake Michigan (Makarewicz et al. 1995; Lehman 1991;
Lehman & Caceres 1993; Barbiero & Tuchman 2004), as well as in inland lakes of the
Canadian Shield (Yan & Pawson 1997; Boudreau & Yan 2003; Strecker et al. 2006).
Crustacean zooplankton species richness typically declines by -20% following invasion by Bythotrephes (Lehman & Caceres 1993; Schulz & Yurista 1999; Yan et al. 2002;
Barbiero & Tuchman 2004; Strecker et al. 2006). Bythotrephes also has indirect and non- consumptive effects on zooplankton. Daphniid growth rates are suppressed as they migrate deeper down the water column to avoid predation by Bythotrephes (Pangle &
Peacor 2006; Pangle et al. 2007). Bythotrephes also affects other macroinvertebrate predators through competitive interactions, and is steadily displacing Leptodora kindtii in its invaded range, most likely because the two species consume similar prey items (Foster
& Sprules 2009; Weisz & Yan 2011). Bythotrephes subsists on a much broader diet than
Leptodora, however (reviewed in Grigorovich et al. 1998), which may make it a superior
8 competitor. The diet of My sis relicta has also been altered by Bythotrephes (Nordin et al.
2008). Finally, pelagic rotifers and phytoplankton are indirectly impacted by
Bythotrephes (Hovius et al. 2007; Strecker et al. 2011).
Bythotrephes' establishment and interannual persistence in a range of habitats and conditions is possible due to its life cycle which consists of both parthenogenic and gametogenic modes of reproduction (Figure 1.2). In temperate locales when conditions are favourable, summer populations tend to be composed mostly of female parthenogens.
Later in the season, male production will be stimulated by one or more external cues (e.g., overcrowding, temperature decreases; Brown & Branstrator 2005). Males will mate with a gametogenic female by attaching to her via hooks on its first thoracopod, and copulating for 30 - 60 seconds (Zozulya 1979, in Rivier 1998). This results in a clutch of
2-5 "naked" (i.e., not contained in an ephippium; Herzig 1985) resting eggs (Yan and
Pawson 1998), which are resistant to freezing and desiccation, and remain viable for up to two years (Yurista 1997). These resting eggs accumulate on the bottom sediments from where they may hatch out later in the season or overwinter to emerge the following spring when water temperatures reach 10°C - 12°C (Rivier 1998), although hatching has been directly observed at just over 4°C (Herzig 1985; Yurista 1997).
Establishment success for an invader such as Bythotrephes is therefore contingent on the variability of resting egg mortality during winter and phenology from year to year, and population persistence over the first2 growing season, which is critical for founding a sexually reproducing population (Drake 2004). Brown and Branstrator (2011)
2 This likely extends to subsequent growing seasons, as populations may perish following an unusually hot summer (Norman Yan, York University, Biology Department, personal communication).
Q demonstrated high mortality rates of Bythotrephes resting eggs, embryos, and neonates extracted from a U.S. reservoir, suggesting that very high propagule pressure in terms of resting eggs is needed for initial establishment, followed by substantial resting egg production for the year-to-year persistence of established populations. Windows of opportunity for Bythotrephes establishment occur from early to mid-summer (Drake 2004;
Drake et al. 2006), which is primarily when parthenogenic propagation of Bythotrephes occurs (although there is no definite "safe" time for colonists to arrive; Gertzen et al.
2011). Allee effects exist for Bythotrephes, however (Gertzen et al. 2011), and may be largely dependent on temperature (Wittmann et al. 2011). Recent field-based evidence also suggests that spring prey availability may be the primary factor determining
Bythotrephes population size (Young et al. 2011).
Focus of dissertation
The Natural Sciences and Engineering Research Council of Canada (NSERC)- funded Canadian Aquatic Invasive Species Network's (CAISN) strategic five-year plan starting in 2006 was to identify and quantify the vectors by which AIS enter Canada, to investigate factors affecting establishment, and to develop risk assessment models. As part of this network, my task was to investigate factors (mostly abiotic) affecting
Bythotrephes' establishment, particularly on the Canadian Shield where it continues to expand its range.
Despite its high profile as an aquatic invader (281 Web of ScienceSM records include 'Bythotrephes' as a search term; Web of ScienceSM, retrieved 14 February 2012), little is known about its basic life history and specific tolerances to various abiotic and
in biotic conditions, besides inferences from field-based studies. This is because rearing
Bythotrephes in a laboratory setting from birth through ontogenesis until parturition is difficult, and past efforts have been mostly unsuccessful (Barnhisel 1991; Yurista 1992;
Ketelaars et al. 1995; Schulz & Yurista 1999). In-depth studies are needed to proceed with risk assessments and appropriate management decisions, however.
In this dissertation, I will address these knowledge gaps by examining the impacts of various abiotic factors on the survival, growth, and parthenogenic reproduction of
Bythotrephes under controlled laboratory settings—with a secondary aim to identify and quantify some of the key constraints imposed on its establishment success in nature.
Environmental stressors affecting the Canadian Shield include increasing temperature
(Hostetler & Small 1999), declining calcium (Ca) concentrations (Jeziorski et al. 2008), and possible future declines in prey quantity and quality, which may arise in response to observed decreases in total phosphorus (Quinlan et al. 2008) and shifts in algal community composition (Paterson et al. 2008), respectively. In Chapter 2,1 describe the development of a laboratory culturing protocol for parthenogenic Bythotrephes by exploring how it responds to different culture media, temperatures, and prey types. Since there is little evidence to support Bythotrephes' physiological limitation by colder temperatures (the last individuals disappear when water temperatures drop down to ~5°C;
Zozulya 1979, in Rivier 1998), I conduct a range-finding test to determine its temperature threshold for survival over 96 hours. Next, with the application of new culturing techniques, I report on Bythotrephes' development times to parturition in response to four temperatures. The culturing methods outlined here may be useful for other researchers
11 needing to maintain Bythotrephes (or other carnivorous zooplankton, such as the related and also invasive fishhook waterflea, Cercopagis pengoi; although C. pengoi is likely restricted to smaller prey) in a laboratory setting. In Chapter 3,1 investigate the effects of low Ca on Bythotrephes by quantifying its somatic Ca content, examining the effects of differing Ca concentrations on Bythotrephes' population growth in the laboratory, and by aligning Bythotrephes' distribution in relation to Ca on the Shield where is it relatively new, alongside its distribution in Norway, where it is endemic. I also explore the collective impacts that Ca decline and Bythotrephes invasions may have on native
Daphnia, since daphniids are generally less tolerant of low Ca. In Chapter 4,1 examine the impacts of prey quantity on Bythotrephes, given its reputation as a highly voracious predator. In Chapter 5,1 go on to consider the influence of prey quality on Bythotrephes size, survival and reproduction in a laboratory-based food chain at two water temperatures. In the final chapter, I conclude that the most important factors governing
Bythotrephes population dynamics—and hence, establishment success—stem from water temperature and prey availability, as well as factors that may influence prey capture such as light intensity and water clarity. I also discuss the value of autecological studies and potential management strategies for this invader, based in part on the information gleaned from these laboratory studies.
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91 FIGURES
Figure 1.1. A third instar specimen of the invasive spiny water flea, Bythotrephes longimanus Leydig (1860), with enlarged unpigmented broodsac, collected from Lake of Bays, Ontario, Canada. Parthenogenically-produced Bythotrephes typically develop through three instar stages prior to reproductive maturity, whereas sexually-produced animals undergo four instar stages.
3 The pigmentation characteristic of late stages of developing eggs and embryos is easily observed through the clear broodsac.
99 Figure 1.2. Bythotrephes' life cycle, consisting of parthenogenic and gametogenic reproduction. It should be noted that gametogenically-produced females (i.e., from diapausing eggs) lack the 'kink' in the caudal process characteristic of parthenogenically- produced Bythotrephes.
71 CHAPTER 24
METHODS FOR REARING THE INVASIVE ZOOPLANKTER
BYTHOTREPHES IN THE LABORATORY
ABSTRACT
The invasive spiny water flea's (Bythotrephes longimanus) current North
American distribution encompasses the Laurentian Great Lakes as well as a number of inland lakes, particularly on the Canadian Shield. In the past, poor survival in the laboratory has precluded controlled long-term studies on Bythotrephes. Here I investigated field collection techniques and choices of culture media, temperature, and diet that led to the successful maintenance of Bythotrephes from birth to reproduction.
Gravid parthenogenic females were collected from invaded lakes. Resulting offspring were reared in source lake water filtered through 20 or 80 [j.m, or a fully defined artificial culture medium, FLAMES. Individuals raised in FLAMES produced significantly larger broods than those in lake water, indicating that it is an appropriate culture medium. I next conducted a 96 h temperature bioassay on juvenile Bythotrephes. Survival was comparable at 16°C, 20°C, and 24°C but decreased after 48 h at 28°C, and most animals died after 24 h at 32°C. I also reared Bythotrephes at 16°C, 19°C, 22°C, and 25°C.
Corresponding intrinsic rates of natural increase (r) for animals maintained to first brood release were 0.02, 0.05, 0.06, and 0.03 d suggesting that Bythotrephes should be reared
4 Copyright, 2010 by the American Society of Limnology and Oceanography, Inc., Kim, N., and N.D. Yan. 2010. Methods for rearing the invasive zooplankter Bythotrephes in the laboratory. Limnology and Oceanography: Methods 8:552-561. Included here with permission from the publisher (http://aslo.org/lomethods/information/copyright.htmn and my co-author, N.D. Yan.
94 at ~22°C to benefit from maximum population increases. Feeding trials confirmed that young Bythotrephes prefer small, slow-moving prey. Finally, I devised a protocol for rearing Bythotrephes that yielded 100% survival to reproduction and r = 0.10 d 1 (for animals maintained to first brood release). Given these results, it is now possible to conduct long-term laboratory studies on this invader.
INTRODUCTION
Bythotrephes longimanus (Leydig 1860) (Arthropoda: Cercopagidae)— hereinafter Bythotrephes—is an exotic predatory invertebrate from Eurasia that was first discovered in the Great Lakes watershed in the early 1980s (Johannsson et al. 1991). It has since been detected in at least 129 water bodies in Ontario, Canada (Ontario
Federation of Anglers and Hunters unpubl. data), as well as in several lakes of the northeastern United States (Branstrator et al. 2006). Bythotrephes has been implicated as the primary cause for changes in native zooplankton abundance, diversity, and species richness in invaded lakes (Lehman 1991; Dumitru et al. 2001; Yan et al. 2001; Barbiero and Tuchman 2004; Strecker et al. 2006). Moreover, Bythotrephes' long caudal process inhibits ingestion by young-of-the-year fishes, possibly impacting growth rates and recruitment (Hoffman et al. 2001). Although Bythotrephes is readily eaten by some fishes such as lake herring (Coulas et al. 1998), its development times are rapid (Yurista 1992) and populations can grow more quickly than those of predator fishes (Lehman 1987).
To better assess the possible severity of impacts by Bythotrephes in a novel habitat and be able to forecast its future spread and impacts, long-term laboratory studies are needed to complement the growing body of field-based research. Unfortunately, few controlled laboratory studies have been conducted on this obligate zooplanktivore due to the difficulty of captive rearing. Attempts at Bythotrephes culture were initiated in the former Soviet Union in the late 1950s (e.g., Mordukhai-Boltovskaya 1959a, cited in
Grigorovich et al.1998), but the protocols used are not widely accessible, the trials are short and limited in scope (Berg and Garton 1988), and estimates of development times are "sketchy" (Yurista 1992). Yurista (1992) offers the most detailed insights into how one might embark upon culturing these animals, but even he admits the challenges involved in raising them from birth to reproduction. The lack of a standard culturing methodology has hindered progress in Bythotrephes research. Information on environmental limits, preferential habitat conditions, and age-specific life history characteristics remains scarce (Berg and Garton 1988; Branstrator 2005). For instance, limited empirical data exist on postembryonic development times at various temperatures.
If metrics such as Bythotrephes' basic reproductive rate in response to temperature could be obtained however, Bythotrephes' optimal thermal habitat may be deduced. These data would be useful for modeling potential establishment success and conducting risk assessments for potential invasion.
Herein, I have determined the environmental and dietary conditions necessary to maintain Bythotrephes under captive conditions. First, I compared Bythotrephes survival and reproduction in FLAMES soft-water culture medium, a fully-defined medium for
Cladocera that was devised in-house (Celis-Salgado et al. 2008), to water from the source population lake. The major advantage of using an artificial culture medium as opposed to
7f> lake water is that the risk of confounding effects arising from biological and chemical variations in culture conditions is minimized. Second, I conducted a 96 h range-finding temperature bioassay to determine suitable ranges for subsequent experiments, as well as to validate the upper threshold of 30°C reported by Garton and colleagues (1990) for field-collected Bythotrephes. Third, I examined the effects of four different water temperatures on Bythotrephes life history. This allowed me to determine the temperature(s) at which these animals exhibit maximum population increases, likely the ideal temperature for maintaining lab cultures. In its native range, Bythotrephes has been recorded in waters from 4 to 30°C, but it is believed to prefer 10 to 24°C (Grigorovich et al. 1998). Yurista (1999) provided evidence that Bythotrephes is well suited to 14 to 23°C, but likely hindered by the inactivation of respiratory enzymes above this range. To my knowledge, postembryonic parthenogenic development has been tracked only at 12.7°C
(Yurista 1992) and 21°C (Lehman and Branstrator 1995). Fourth, I carried out a five-day feeding experiment in which animals were offered satiating levels of diverse prey types, including four naturally encountered species from my study region. My objective was to determine what quantities of which prey species young Bythotrephes most frequently selected. As an active swimmer, Bythotrephes expends a high amount of energy relative to its assimilation efficiency (< 60% of ingested prey; Yurista and Schulz 1995), and the delineation of an adequate diet is crucial to the maintenance of Bythotrephes cultures.
Lipid-rich Artemia nauplii have been the prey of choice in the past (Yurista 1992;
Lehman and Branstrator 1995), but this diet is likely not nutritionally complete for all life stages, and it certainly does not reflect natural diets.
77 Finally, I combined the information from all four trials to initiate a Bythotrephes culture. The results indicate that Bythotrephes can be cultured successfully in the laboratory using my protocols, and I offer suggestions for future laboratory-based studies that are now possible on this invasive species.
MATERIALS AND PROCEDURES
Bythotrephes field collection
Animals for the culture medium and both temperature experiments were collected in June and July 2007, respectively, from Peninsula Lake east of Huntsville, Ontario,
Canada. Bythotrephes was first detected in the lake in 1991 (Yan et al. 1992), and has persisted ever since (Young 2008; N. Kim, personal observation). Animals were collected via slow vertical hauls (i.e., < 1 m • s ') at randomly selected pelagic sampling stations using a conical zooplankton net with 64 (im mesh (1 m diameter, 2 m length). In my field work, I strove to minimize exposure of the animals to air, physical damage from handling,
UV radiation, and cannibalistic conspecifics. Upon net retrieval, the mesh-bottomed cod end was immediately deposited into a deep white plastic tray filled halfway with filtered lake water to ensure the animals remained submerged, and then the animals were tipped into the tray. A hand-held umbrella shielded the animals from ultraviolet radiation during sorting. Employing a 5 ml pipettor with the plastic tip cut to a 9 mm diameter, actively swimming females with well-developed, pigmented brood sacs (Yurista 1992) were transferred into their own clear 500 ml polyethylene terephthalate (PET) jars (7.6 cm maximum diameter, 23.4 cm height), previously filled with lake water filtered through 80 (am Nitex mesh. Once contained, animals were placed in coolers with ice packs for transport to York University's Field Laboratory for the Assessment of Multiple
Environmental Stressors (FLAMES) located at the Ministry of the Environment's Dorset
Environmental Science Centre. All experiments were conducted in this facility.
Animals for the feeding experiment and final culturing trial were collected on two occasions in August 2009 from Fletcher Lake (45.294°N, 78.949°W), north of Dorset,
Ontario, where Bythotrephes was discovered in 2006 (Cairns et al. 2007). For these collections, I used a net composed of 300 f^m mesh (1 m diameter, 3 m length) and a fully closed, 1.5 L capacity PVC cod end. Following deployment and retrieval of the net, the cod end contents were carefully poured into the tray, but this time only actively swimming females with unpigmented broodsacs were selected. This latter measure was undertaken to allow for a longer period of laboratory acclimation before offspring were bom. Animals were transported to the laboratory in 50 ml Fisherbrand® PET centrifuge tubes filled with FLAMES medium, to reduce space requirements as well as possible contamination from bringing lake water back to the clean laboratory. Container size during transport does not appear to affect Bythotrephes survival (N. Kim, personal observation). During transport, the tubes were placed in Styrofoam trays and stacked in coolers with ice packs. The latter was a precautionary measure but ice packs are likely not necessary if the possibility of overheating the animals is minimal. Both Peninsula and
Fletcher lakes are located within a 30 min drive of the FLAMES laboratory.
?o Maintenance of prey cultures
For all experiments, cladoceran species (all Daphnia spp. and Bosmina) were kept in FLAMES medium contained in 1 L, steep-sided glass jars and fed a 1:1 ratio of the green alga Pseudokirchneriella subcapitata (UTCC 37) and Chlorella kesslerii (UTCC
266) ad libitum. Algae were grown in batch cultures from pure stocks obtained from the former University of Toronto Culture Collection (now the Canadian Phycological Culture
Centre). Animals were transferred via disposable pipettes into fresh FLAMES once a week. For the culture medium and temperature studies, Artemia salina nauplii were hatched out at room temperature every few days from commercial cysts (Ward's Natural
Science) in shallow containers. For the feeding experiment and culturing trial, Artemia francisccma nauplii were hatched out every 24 h from premium grade cysts (Brine
Shrimp Direct) cultured in conical aerated hatcheries (Aquatic Ecosystems Inc.) and housed in a growth chamber set at 28°C and continuous 100 (imol m 2 • s 1 lighting. All
Artemia were cultured using Instant Ocean artificial sea water (Spectrum Brands).
Culture medium experiment
Upon arrival at the laboratory, the PET jars containing individual Bythotrephes (n
= 43) were rinsed on the outside and placed directly into Conviron E7/2 growth chambers set at 16°C, a temperature within the range typically experienced by Bythotrephes during early summer in Peninsula Lake (Joelle Young, Department of Biology, York University, personal communication). A photoperiod of 14 light and 10 dark hours at a light intensity of 100 |amol' m"2 • s ' (Pangle and Peacor 2009) with 30 min dawn and dusk ramping periods was implemented to reflect in situ conditions and employed for all experiments. Once daily, animals were fed Artemia ad libitum, along with 5 individual laboratory- cultured Daphnia from a mix of D. pulex, D. pulicaria, D. ambigua, and D. catawba.
Following overnight acclimation, 93% {n = 40) of field-collected Bythotrephes survived.
Of these survivors, 85% (n = 34) attained parturition over the next few days, producing an average of 2.9 ± 0.80 (± SD) neonates. These results indicate that my chosen collection and acclimation regime was satisfactory. To determine if I could successfully culture Bythotrephes in a fully defined, softwater medium, a total of 39 Fi offspring < 24 h old were randomly assigned and placed individually into 250 ml of one of the three treatment media: FLAMES medium, 20 (im filtered lake water (FLW), or 80 |im FLW from Peninsula Lake (n = 13 Bythotrephes • treatment"1). FLAMES (Celis-Salgado et al.
2008) was designed with its chemistry based on two nearby Ontario Ministry of the
Environment lakes that support diverse and stable zooplankton assemblages (Yan et al.
2008). Lake water was collected every second day in opaque 20 L plastic carboys and filtered in situ through 20 or 80 nm Nitex mesh. Prior to use in experiments, the lake water was incubated at 16°C and filtered a second time. The FLAMES medium contained no phytoplankton, but the lake water treatments allowed for the inclusion of some rotifers and phytoplankton as possible supplemental food sources. Animals were transferred via wide-bore pipette to fresh media, with a small grain of dry cetyl alcohol added to discourage entrapment of animals in the surface tension (Desmarais 1997). Bythotrephes were fed twice daily with numbers of prey and species consistent with the earlier description of feeding of their field-collected mothers. Only barren Daphnia (without eggs, embryos, or ephippia) were used, so that prey consumption estimates would not be biased by prey natality. All prey were rinsed on a mesh screen with the appropriate culture medium prior to inoculation of the jars containing Bythotrephes. Before the second daily feeding, any dead or immobile Artemia from the first feeding were pipetted out and replaced. Bythotrephes survival was scored daily by examination over a light table. For this and all subsequent experiments, mortality was defined as immobilization for 10 seconds, even when prodded. The test endpoint was reproduction, at which point the timing of brood release and clutch sizes were recorded.
96 hour upper temperature range-finding experiment
Test animals for the temperature range-finding bioassay were comprised of F| and
F2 (i.e., juveniles of lab-reared Fi) individuals raised in 80 ^m FLW and maintained under the same conditions as described for the preceding experiment. Bythotrephes were transferred to fresh 80 jim FLW once daily. Each chamber initially held 17 randomly selected laboratory-born specimens ranging in age from 1 to 8 days. Before commencement of the assay, I implemented a 24 h acclimation schedule (Garton et al.
1990) with maximum increases of 0.5°C • IT1 (from 16°C to 20°C, 24°C, 28°C, and 32°C, respectively; the 16°C treatment remained unchanged). Only 13 of the original 17 animals survived the acclimation period at 32°C. As this treatment exhibited the lowest survival and I wanted a balanced design with equal numbers of test animals in each temperature treatment, 13 individuals were randomly selected within each of the other treatments to be included in the experiment. Survival was scored once every 24 h and differences in survival curves between temperature treatments were evaluated. Bythotrephes life history at four temperatures
Approximately equal numbers (-25) of field-collected Bythotrephes females were placed directly into one of four growth chambers set at 16°C, 19°C, 22°C, or 25°C.
Following overnight acclimation, half of the lake water in each container was replaced with FLAMES medium. After 4 h, animals were transferred to PET jars filled with pure
FLAMES medium. Both field-collected animals and their offspring were fed twice a day as described for previous experiments, with transfers to new media conducted every second day. Within each of the treatment chambers, 20 Fi neonates were transferred to their own PET jars filled with 375 ml of fresh FLAMES medium, which was pre-warmed to the appropriate temperature. Animals were observed daily for survival and reproduction, and the test endpoint was first brood release. Data were omitted in the rare instances that males were unintentionally included as test specimens. Possible differences in mean clutch sizes were assessed with a one-way ANOVA following application of the square root transformation. Life table analyses were also conducted to evaluate
Bythotrephes overall performance among temperature treatments. Net reproductive rates
(R0), cohort generation times (Fc), and intrinsic rates of natural increase (r) were calculated for each treatment group using the PopTools add-in for Microsoft Excel
(http://www.cse.csiro.au/poptools). The parameter r serves as a theoretical link between the outcomes of life cycle bioassays on individuals and effects at the population level. A positive value indicates that a population is increasing, whereas a negative value denotes a population decrease; an r-value of zero means there is no change in the population. Feeding experiments
Field-collected females (n = 42) were immediately transferred to 175 ml of
FLAMES medium upon arrival at the laboratory and kept in a growth chamber set at
21°C to mimic in situ lake temperature. Ten of the resulting Bythotrephes neonates were offered a mixture of variably sized prey items. Prey consisted of 5 specimens each of
Artemia nauplii, Bosmina freyii, D, ambigua, D. pulex, and Holopedium gibberum to total
25 prey items. This prey concentration was chosen because preliminary trials indicated that second and third instar Bythotrephes are capable of consuming between 7 and 22 small (< 850 ^m) D. pulex • d~' when offered at highly satiating levels (i.e., 50 D. pulex • 250 ml"1; N. Kim, unpublished data). All prey were cultured in the laboratory, with the exception of H. gibberum, which was collected from Fletcher Lake every second day. H. gibberum was included in this experiment because there is some support that
Bythotrephes may consume this jelly-clad cladoceran (Makarewicz et al.
1995; Schulz and Yurista 1998; Wahlstrom and Westman 1999), although it is one of the species that thrives in the invaded lakes of our study region (Yan and Pawson 1997;
Boudreau and Yan 2003). All prey provided were barren to the naked eye, and only mid size B. freyii were selected on the assumption that they would not release neonates within
24 h. I elected not to starve Bythotrephes before experimentation, as this would likely result in overestimates of feeding rates. Each day, I inoculated fresh prey into 175 ml clean FLAMES medium contained in 200 ml capacity glass Mason jars. Bythotrephes were then transferred to these Mason jars, and all live prey remaining in the preceding jars were identified and enumerated. Mason jars were employed instead of the PET jars
•\A used previously, as there is no difference in Bythotrephes survival when reared in this container type with reduced water volume (N. Kim, unpublished data), and glass jars are more easily cleaned than PET jars.
Bythotrephes culture
Drawing on the collective results of the culture medium, temperature, and food experiments, I attempted to close the life cycle of Bythotrephes in the laboratory with minimum mortality and maximum reproduction. Bythotrephes females (n = 18) were collected from Fletcher Lake in August 2009 and acclimated to culture conditions as described previously. Temperature was set at 21°C, and light conditions were maintained at 100 jjmol • m 2 • s ', on a continuous cycle of 14 light and 10 dark hours, with 30 min dawn and dusk ramping periods. Once daily, Bythotrephes were offered a mixed diet of 1 ml Artemia nauplii (estimated to contain approximately 50 - 100 nauplii), along with 10,
7, and 5 each of mid-sized B.freyii, D. ambigua, and D. pulex. These food levels were presumed to be saturating because prey were always left over the following day. Ten
Bythotrephes offspring born within 24 h of each other were then randomly selected and moved to individual glass Mason jars filled with 175 ml FLAMES medium. The feeding regime was maintained as described for the mothers, at which time animals were also transferred to clean FLAMES medium. Survival and reproduction data were recorded daily until first brood release. Hence, the reported r-values are likely underestimates of actual rates because Bythotrephes is able to produce multiple broods (see Chapter 2); still, direct comparisons may be made among experimental treatments. Life table parameters were calculated as described for the life cycle experiment at four temperatures. Statistical analyses
For the culture medium experiment, product-limit (Kaplan-Meier) survival curves were used to depict survivorship, and Wilcoxon-Gehan tests were conducted to compare the resulting curves. Wilcoxon, as opposed to log-rank X2 test statistics are reported, as our data are right-censored, and this test is more sensitive to early mortality (Newman
1995). I performed one-way ANOVAs to compare mean development times to maturity
(which we define as the third instar stage) and mean clutch sizes among treatments.
Clutch size data were first square root transformed using Bartlett's equation, X' = Vx +
0.5 (Zar 1999) to meet test assumptions. Post hoc Tukey-Kramer HSD tests were used for pairwise comparisons. Statistical analyses for this and all other experiments were completed in JMP 8 (SAS Institute). For the computation of feeding rates, only data acquired from vigorously swimming animals were used (n = 6). Differences between
Bythotrephes were assessed using a one-way ANOVA. Mean daily feeding rates on each prey species were then compared via a repeated-measures ANOVA, with post-hoc
Tukey-Kramer HSD tests for pairwise comparisons. Count data (i.e., numbers of prey eaten or killed) were first arcsine transformed to meet assumptions of homogeneous variance (Zar 1999).
ASSESSMENT
Culture medium experiment
There were no differences in survival among the culture media treatment groups
(Wilcoxon X2 = 0.353, df~ 2,p = 0.838). However, the type of culture medium significantly influenced the mean time to third instar (F2,2o = 3.823, p = 0.039) (Table 1).
Bythotrephes reared in FLAMES reached maturity in approximately 8.7 d, compared with their counterparts in the 20 |im FLW treatment, which developed over the span of about 10.6 d (p = 0.0309). There were also significant differences in clutch size among treatments (F2.20 = 10.422,/? = 0.0008), with mothers reared in FLAMES producing significantly larger broods when compared with those in either 80 jam FLW {p = 0.011) or 20 (xm FLW treatments (p = 0.001). There were no differences in clutch size between lake water treatments, however (p = 0.592). Overall, Bythotrephes raised in FLAMES produced a total of 30 neonates—the sum of neonates in the two lake water treatments combined. Because Bythotrephes exhibited increased survival and produced more offspring when reared in FLAMES compared with either lake water treatment, I elected
FLAMES as the culture medium of choice. The chemistry of this medium is thoroughly defined such that for future assays, I can be confident that the variability in my observed data will likely not be attributable to the culture medium itself.
96 hour upper temperature range-finding experiment
Bythotrephes survival times differed among temperature treatments (Wilcoxon X2
= 72.617, df- 4 ,p< 0.0001), namely between the three lowest and two highest treatments. Whereas no differences in survival were discerned for animals reared at 16°C,
20°C, and 24°C, mortality increased rapidly after 48 h at 28°C, and all but one test animal died after 24 h at 32°C (Figure 2.1). These results indicate that Bythotrephes' upper threshold temperature for survival must lie above 24°C and below 28°C. Bythotrephes life history at four temperatures
As to be expected, generation times for Bythotrephes became shorter with increasing temperature (Table 2.2). Mean brood sizes—ranging from approximately 2.2 to 2.5 neonates—did not differ significantly among temperature treatments (F3 43 = 0.395, p = 0.757). Given that r is a function of survivorship and fecundity, the increased proportion of animals surviving to reproduce at 22°C must have contributed to the relatively high r-value obtained for this group.
Feeding experiments
Each Bythotrephes ate or killed an average of 12.8 ± 0.33 (± SE) prey animals daily, with no significant differences in overall feeding rates between Bythotrephes (F$j4
= 0.469, p = 0.795). The test animals most frequently consumed or killed were £>. ambigua (4.2 ±0.18 d '), followed by B.freyii (3.2 ± 0.22 d !), Artemia nauplii (3.2 ±
0.28 d"1), H. gibherum (1.2 ± 0.21 d"1), and D. pulex (1.00 ± 0.20 d ') (Figure 2.2).
Within-subjects analysis revealed significant differences among prey species selected by
Bythotrephes (F4JS = 45.640, p < 0.0001). These results clearly indicate that juvenile
Bythotrephes are limited to smaller, somewhat slower-moving prey.
Culturing Bythotrephes
Life history data were obtained for 8 of the 11 Bythotrephes that I attempted to culture under the best possible conditions. Two of the test animals turned out to be male because I collected mothers from the field in the fall when water temperatures had dropped substantially. One animal with black-eyed embryos visible in her broodsac went
^8 missing near the end of the assay. I retained a high survival rate, with all of the remaining
8 females surviving to reproduce, and an overall rate of increase of 0.10 d"1 (Table 2.3), the highest r-value observed in any of the trials thus far. Thus I am confident in my ability to provide an optimal setting at which to culture these animals. Utilizing the same protocols described for this culturing trial, I have recently completed experiments on minimum calcium thresholds for Bythotrephes and achieved up to 100% survival to reproduction, as well as the production of second and occasionally third broods (see
Chapter 3).
DISCUSSION
My success with Bythotrephes culture may be attributed to several factors. First of all, as a relatively soft-bodied cladoceran with a delicate exoskeleton (Yurista and Schulz
1995), Bythotrephes is liable to injury and stress from excessive handling, as seen with
Leptodora (Pichlova et al. 2004), and high mortality rates are often encountered within
24 h of collection (Schulz and Yurista 1999). The high survival rates of field-collected animals in my study were likely a result of the initial in situ screening of animals for vigor as well as careful handling and efforts to minimize exposure to air, UV, and cannibalistic conspecifics.
The use of FLAMES artificial culture medium also proved to be valuable for keeping Bythotrephes alive and reproducing. This may be due to the absence of bacteria, viruses, parasites, fish kairomones, and chemical contaminants that we presume are present in coarsely filtered lake water. As such, the smaller clutch sizes of lake water animals when compared with those reared in FLAMES may not necessarily be an indication of reduced fitness, but simply a normal response to biochemical signals. It is likely that Bythotrephes can be cultured quite effectively in lake water if it is bacteria filtered and properly purified. Regardless, I advocate the use of an artificial culture medium to avoid these types of confounding effects in controlled studies.
Bythotrephes' ideal temperature appears to be in the region of 22°C. The observed high death rate of Bythotrephes at 32°C is in agreement with the observations of Garton et al. (1990), and is likely attributable to the inactivation of respiratory enzymes at elevated temperatures (Yurista 1999). Even though generation times were fastest at 25°C, test animals had to be excluded because two of the neonates born into this temperature treatment were identified as male. Mothers for the temperature assay were collected in early July, a time when males are infrequently encountered in field populations; however field-collected mothers were subject to this increased temperature in the laboratory while carrying embryos. This observation lends some support to the theory that temperature stresses may be responsible for the "switching on" of male production. Life history metrics were similar for the 19°C and 22°C groups, but for the purposes of maximum reproductive output, I recommend that Bythotrephes be cultured at closer to 22°C to take advantage of quicker generation times.
Yurista (1992) constructed a model to predict postembryonic development times along a temperature gradient, based on his observation of Bythotrephes' mean parthenogenic development time of 14 ± 1.63 (± SD) days at 12.7°C. His projections appear to be slightly faster than my observed times, particularly at the lower temperatures.
40 This is likely because the test animals used for his study were restricted to 2 ml culture wells, which does not permit natural swimming and prey capture behavior. My data are strikingly consistent with Mordukhai-Boltovskaya's (1959b) observations, who reported development times to be 15, 11,9, and 7 d at 16°C, 19°C, 22°C, and 25°C, respectively.
My results are also in line with an early study by Zaffagnini (1964 cited in Sprules et al.
1990) who determined age at first reproduction to be 8 - 9 d at 20°C, and with Lehman and Branstrator (1995) who reported 9 d at 21°C.
With the application of feeding rate and prey selection information imparted by the diet trial, large and healthy-looking Bythotrephes exhibiting excellent survival and a relatively high r-value of 0.10 d"1 were produced in the final culturing run. I must reiterate that all r-values reported here are underestimates of actual rates because experiments were only carried out until first brood release. The success of this culturing protocol thus appears to be mainly attributable to the provision of a high-quality diet, which is no surprise given Bythotrephes' voracious appetite. The availability of ample amounts of attainable prey (i.e., smaller and slower) seems particularly critical for stimulating feeding and the survival of younger life stage Bythotrephes. Despite the report that first instars can handle prey as large as 1.5 mm (Burkhardt and Lehman 1994), the results of this study along with my numerous failed attempts at Bythotrephes culture on a strict diet of small (< 850 |i.m) Daphnia (N. Kim, personal observation), leads me to think that a mixed-species diet incorporating diverse prey types is key. Although I did not observe any appreciable change in mean daily feeding rates over the 5 d trial, I did note that not a single D. pulex, one of the largest and fastest prey species, was killed or
di consumed within the first 24 h. In fact, the large but slow-moving H. gibberum was just as likely to be selected, even though it is enveloped in a thick protective jelly sheath.
Early investigations on the diet of Bythotrephes also suggested that it prefers small, soft, slow-moving prey including Bosmina and Polyphemus (Mordukhai-Boltovskaya 1958, cited in Schulz and Yurista 1999; Vanderploeg et al. 1993). Copepod nauplii— particularly cyclopoid nauplii—also appear to be a major component of Bythotrephes' diet (Schulz and Yurista 1995; Schulz and Yurista 1998; Dumitru et al. 2001), although
Bythotrephes is probably too "clumsy" a swimmer to catch rapidly accelerating adult copepods (Vanderploeg et al. 1993). Finally, there is an overall consensus that
Bythotrephes feeds heavily on crustacean zooplankton such as Daphnia (Lehman and
Caceres 1993; Lehman and Branstrator 1995; Dumitru et al. 2001). Taken together, these observations imply that Bythotrephes is a nonselective feeder with respect to species.
Different life stages appear to have different dietary needs, but this is probably more a reflection of prey capture proficiency. Adult Bythotrephes are known to consume a wider range of prey than juveniles because they are capable of handling a diverse range of prey sizes (Muirhead and Sprules 2003). Large prey must be included when culturing for the benefit of adult Bythotrephes, which favor bigger prey (> 2.0 mm D. pulicaria; Schulz and Yurista 1998), although prey > 2.5 mm may be too large for Bythotrephes (Barbiero and Rockwell 2008).
The ability to reliably rear Bythotrephes in the laboratory affords an array of research possibilities. Further studies on Bythotrephes are urgently required, as this invader occupies a pivotal mid-trophic position in pelagic food webs from which it
Al profoundly influences plankton, as well as fish communities (Lehman and Branstrator
1995). Information derived from controlled studies can then be aligned with field data for a more comprehensive illustration of a species' ecology. For example, my temperature results suggest that in shallow and/or unstratified lakes or reservoirs, Bythotrephes populations will peak when water temperatures reach about 22°C. Conversely I expect populations to be limited in shallow water bodies lacking a cooler thermal refuge if temperatures exceed 25°C, up to 28°C. In Russia, population maxima of Bythotrephes in situ are reported to occur regularly at 20 to 24°C (Grigorovich et al. 1998), with greatest abundances at 20 to 22°C in the Rybinsk Reservoir (Mordukhai-Boltovskaya 1956, cited in Berg and Garton 1988; Zozulya and Mordukhai-Boltovskoi 1977). In Lake Erie,
Bythotrephes' highest abundance was also observed when temperatures reached approximately 20°C (Berg and Garton 1988). Population declines, on the other hand, may be the result of reduced food intake and decreased reproduction in warmer waters
(Grigorovich et al. 1998), which occurs when temperatures surpass 25°C (Mordukhai-
Boltovskaya 1959b, cited in Grigorovich et al. 1998). These field observations thus concur with my laboratory findings, and may explain why Bythotrephes prevalence is much lower in smaller lakes that may lack cool, profundal refuges from summer heat waves. Future work on temperature in combination with factors such as food levels may help to elucidate what conditions trigger changes in population growth, and the production of males. Research on Bythotrephes' ability to acclimate to different temperatures would also be useful, and are now possible.
4^ Closer examination of Bythotrephes feeding to further elucidate prey capture abilities of different instars would aid in the study of predation impacts. To forecast colonization success in the field, studying the impacts of multiple environmental stressors on Bythotrephes life history will be strengthened by the ability to rear Bythotrephes in a fully defined medium. Physical and chemical factors of interest may be adjusted, facilitating experimentation on the acute and chronic response thresholds of Bythotrephes to a range of water quality variables. Comparisons of seasonal changes in Bythotrephes condition in the field versus captive individuals are now possible. The quantification of the proportion of animals that survive and reproduce after release of their first brood should also be investigated further, as this may have significant repercussions for population viability analyses. Finally, examining Bythotrephes' interactions with potential competitors and predators is also feasible. Much of this work can now be carried out over the life cycle of the animal, and all such work in multiple laboratories would be greatly facilitated by the adoption of proven standardized culturing methods.
Although rearing each Bythotrephes individually in its own 175 ml volume of water proved labor-intensive, this set-up was chosen and is recommended for two main reasons. First of all, results of preliminary trials conducted at 13°C - 17°C showed that
Bythotrephes exhibits poor long-term survival in 40 ml culture tubes (i.e., < 10% surviving to reproduce; N. Kim, personal observation), probably because the animals swim rapidly and tend to bump into container walls (Vanderploeg et al. 1993; N. Kim, personal observation). Another explanation could be that Bythotrephes requires a certain minimum volume of water for effective prey capture. Small vessels would be even more
44 stress inducing for animals reared at higher temperatures because swimming and feeding rates increase. The second reason for maintaining Bythotrephes individually is that my attempts at mass-culture have been largely unsuccessful, as this species is highly cannibalistic. In my experience, a maximum of 3 to 4 animals could be kept in approximately of 1 L culture medium but food levels must be satiating (i.e., 3-4 times the diet provided to an individual Bythotrephes) and the risk of cannibalism is pervasive.
Therefore the advantages normally associated with bulk culture would be negligible, while many of the life history metrics used in this study could not be tracked individually.
For the purposes of obtaining sufficient numbers of test animals for these studies,
I opted to collect large numbers of mothers from the field and conduct experiments on their laboratory-born offspring. However, this practice likely does not completely eliminate the presence of lingering maternal effects. As such, neonate development and overall quality may be pre-determined by maternal nutrition and body weight (Schulz and
Yurista 1999; Branstrator 2005). I attempted to lessen the magnitude of these effects by experimenting only on neonates produced by mothers kept in the laboratory for as long as possible and for a minimum of 2 d. If possible, experiments should be carried out on F2 or later generations of animals. It may also be worthwhile to establish a clonal line for future experimentation. Due to time and resource constraints, I did not continue to rear the animals on a mass scale over multiple generations, but I have been able to keep four successive generations initiated from a few animals using similar methods but offering a less diverse diet (i.e., no Artemia or large daphniids). Cultures were voluntarily terminated after four generations. The resulting individuals appeared healthy and
4S continued to reproduce, although they were not as active and pigmented as the animals in this study (N. Kim, personal observation), an observation I attribute to the less varied diet.
ACKNOWLEDGEMENTS
I am grateful for the financial support of the Canadian Aquatic Invasive Species
Network (CAISN) and the Natural Sciences and Engineering Research Council of
Canada (NSERC). Thank you to M.E. Palmer and two anonymous reviewers for helpful comments on an earlier version of this manuscript, to M.L. Haarr, M.B. Shapiera, S.
Hung, J. Petruniak for technical assistance, to H.P. Riessen for scientific advice, and to the staff of the Ministry of the Environment Dorset Environmental Science Centre for technical and logistic support.
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so TABLES
Table 2.1. Comparison of Bythotrephes development time to maturity, % surviving to parturition, mean number of neonates per female, and total neonate production in FLAMES soft-water medium, and lake water filtered to 20 and 80 jxm. Animals were reared at 16°C in a 17 day bioassay. There were initially 13 test animals per treatment. Values with the same letter grouping are not significantly different from each other (a = 0.05).
FLW FLW FLAMES (20 nm) (80 |im)
8.9 ± 0.46a 10.6 ± 0.52 b 9.4 ± 0.52 a'b Mean days to 3rd instar (± SE) (n = 9) (n = 7) (n = 7)
% Surviving to parturition 69 54 54
Mean number of neonates per 3.3 ± 0.71 a 2.0 ± 0.58 b 2.3 ± 0.49b female (± SD) (n = 9) (» = 7) (n = 1)
Total number of neonates produced 30 14 16 Table 2.2. Comparison of Bythotrephes survival, neonate production, generation times and population growth rates in FLAMES soft-water medium at 16°C, 19°C, 22°C and 25°C.
Temperature 16°C 19°C 22°C 25°C Replicates 20 20 20 18
% Surviving to parturition 50 55 75 61
Mean number of neonates 2.5 ± 0.79a 2.5 ± 0.80a 2.2 ±0.81a 2.3 ± 0.80a per female (± SD) (n = 10) (/i =11) («= 15) («=11)
Cohort generation time, T c 14.6 10.6 8.9 7.0 (days)
Replacement rate, R„ 1.25 1.62 1.65 1.25
Intrinsic rate of increase, r 0.02 0.05 0.06 0.03
Total number of neonates 25 27 33 25 produced Table 2.3. Life history data for Bythotrephes (n = 8) reared in FLAMES medium at 21°C, offered a diet of Artemia, B.freyii, D. ambigua, D. pulex.
Metric
% Surviving to parturition 100 Mean number of neonates per female (± SD) 2.5 ± 1.07
Total number of neonates produced 20
Cohort generation time, Tc (days) 9.6
Replacement rate, R0 2.5 Intrinsic rate of increase, r 0.10 FIGURES
120 -i
100 -
f t! 80 - o E
« 60- >
3 40 - E 3 o 20 -
0 24 48 72 96 Time (h)
Figure 2.1. Daily cumulative mortality of Bythotrephes {n - 13 Bythotrephes' treatment"1) plotted over time in a 96 h range-finding temperature bioassay conducted at 16°C (•), 20°C (•), 24°C (A), 28°C (x) and 32°C (•).
S4 B B
5.0 -i
5 3.0
Amb. Bos. Art. Hoi. Pul. Prey species
Figure 2.2. Mean numbers of prey items consumed or killed per day (n = 30 observations • species"1), shown with standard error bars. Abbreviations for prey species: Amb. (D. ambigua), Bos. {B.freyii), Art. (Artemia nauplii), Hoi. (H. gibberum), Pul. (D. pulex). Values not represented by the same letter indicate significant differences (a = 0.05). CHAPTER 3s
WILL ENVIRONMENTAL CALCIUM DECLINES HINDER BYTHOTREPHES
ESTABLISHMENT SUCCESS IN CANADIAN SHIELD LAKES?
ABSTRACT
Recently, calcium-rich daphniids have declined on the Canadian Shield in response to falling lake-water calcium concentrations, or [Ca]. Meanwhile the invader
Bythotrephes longimanus, a predator that feeds on Daphnia, continues to spread. My goal was to determine if ongoing calcium declines might directly or indirectly affect
Bythotrephes' establishment success. To address direct effects, I provide the first quantification of Bythotrephes' calcium content, which is very low (0.03% as dry mass) compared with other Cladocera. I also examined the effects of differing [Ca] (0.1 - 2.6 mg • L*1) on Bythotrephes' performance in the laboratory. For all [Ca], population growth rates remained positive, indicating that Bythotrephes has great tolerance of low [Ca],
Finally, I examined Bythotrephes' distribution in relation to calcium on the Shield where is it relatively new, alongside its distribution in Norway where it is endemic and found that Bythotrephes inhabits very low calcium environments in Norway (minimum = 0.2 mg • L"1). These results suggest that Bythotrephes establishment in Canada is currently
5 Copyright, 2012 by NRC Press. Kim, N., Walseng, B., and Yan, N.D. In press. Will environmental calcium declines hinder Bythotrephes establishment in Canadian Shield lakes? Canadian Journal of Fisheries and Aquatic Sciences. Included here with permission from the publisher (http://www.nrcresearchpress.com/page/authors/information/rights) and my co-authors, B. Walseng and N.D. Yan. not—and in the future will likely not—be limited by falling calcium. Rather, as
Bythotrephes is more tolerant of low [Ca] than are its daphniid prey, I propose that both calcium decline and Bythotrephes invasions may contribute to Daphnia decline.
INTRODUCTION
Most Canadian Precambrian Shield lakes have nutrient-poor and soft waters, and recently their calcium concentrations, or [Ca], have been falling over broad geographic areas (Keller et al. 2001; Jeziorski et al. 2008). After decades of increased atmospheric acid deposition, calcium ions were leached from the thin soils that overlay weathering- resistant bedrock (Likens et al. 1996; Watmough et al. 2005), resulting in an initial rise in
[Ca]. This was succeeded by the paradoxical effect of reduced acid deposition, whereby the remaining calcium in the base cation pool was removed at a slower rate (reviewed in
Jeziorski et al. 2008). Further losses of net calcium have been attributed to a number of factors that vary by region, and include the logging of watersheds and subsequent forest regrowth (Watmough and Dillon 2003), decreased atmospheric deposition of calcium
(Hedin et al. 1994), and reduced water load attributable to recent declines in precipitation
(Yao et al. 2011). The median [Ca] in non-acidic south-central Ontario Shield lakes was
2.34 mg • L"1 in 2005 and 2006 (Canadian Aquatic Invasive Species Network (CAISN),
Great Lakes Institute for Environmental Research, University of Windsor, Windor,
Ontario, unpublished data), but [Ca] is projected to decrease by 10 - 40% in the future
(Watmough and Aherne 2008). Calcium availability is a critical factor affecting the competitive interactions of species and the composition of crustacean zooplankton communities in regions where
[Ca] ranges broadly (Hessen et al. 2000; Waervagen et al. 2002). Calcium uptake by organisms from the surrounding environment is active (Alstad et al. 1999), with aqueous rather than dietary calcium acting as the major source for aquatic crustaceans (Cowgill et al. 1986; Wheatly 1999). Environmental uptake accounts for 97 - 100% of Ca acquired by Daphnia magna (Tan and Wang 2009), for example. The impacts of low [Ca] on the calcium-rich (~2 - 8% calcium as dry mass; Jeziorski and Yan 2006) zooplankter
Daphnia pulex have been demonstrated in both the laboratory and field. For well-fed D. pulex reared across a [Ca] gradient, reduced population growth rates resulting from delayed growth and maturation become discernable at [Ca] < 1.5 mg • L"1 (Ashforth and
Yan 2008). Paleolimnological field surveys document reductions of calcium-rich daphniids at [Ca] as high as 2 mg • L'1 (Jeziorski et al. 2008; DeSellas et al. 2011). Not only does calcium serve a necessary role as an intracellular secondary messenger of hormone activity, it is also critical for the biomineralization of the crustacean carapace following ecdysis (Ahearn et al. 2004). Calcium deficiency in zooplankton can thus be manifested as incomplete calcification and diminished carapace hardness, which may increase an afflicted individual's susceptibility to predators, pathogens and physical damage (reviewed in Rukke 2002).
Alongside calcium declines, the Palearctic invader Bythotrephes longimanus
(commonly known as the spiny water flea, and hereafter Bythotrephes) continues to spread across the Shield. The first North American record of this species was in the Laurentian Great Lakes in the early 1980s (Johannsson et al. 1991), and populations have spread inland to lakes in eastern Canada and the northeastern United States. This voracious invertebrate predator has since been found in 150 water bodies in Ontario,
Canada (Yan et al. 2011) and in many U.S. states (Kerfoot et al. 2011; Strecker 2011). It has most recently been recorded outside of the Great Lakes watershed in the Winnipeg
River, and it was recently collected in Lake Winnipeg (Justin Shead, Manitoba Ministry of the Environment, personal communication), from where it will most likely continue to spread. In south-central Ontario, lakes with high boater traffic supply propagules in large enough numbers to permit the establishment of Bythotrephes (Gertzen and Leung 2011), and the dilute, nutrient-poor habitats of the Shield appear to be particularly amenable to this invader (Weisz and Yan 2010). Nutrient-poor lakes are also characteristic of lakes in
Scandinavia, the northernmost part of Bythotrephes'' endemic range (Rivier 1998).
There is broad consensus that Bythotrephes is directly responsible for declines in the species diversity and richness of native planktonic food webs in Canada, along with changes in zooplankton abundance and shifts in community size structure (Dumitru et al.
2001; Yan et al. 2001; Boudreau and Yan 2003). Bythotrephes has also been linked to declines of the native predatory macroinvertebrate Leptodora kindtii, which shares the same prey base (Branstrator 1995; Foster and Sprules 2009; Weisz and Yan 2011).
Cladoceran zooplankton, including members of the important genus Daphnia, have been severely affected by Bythotrephes (e.g., Lehman and Caceres 1993; Yan et al. 2001;
Barbiero and Tuchman 2004). Daphnia, as mentioned earlier, is also vulnerable to low
[Ca].
SQ What remains unknown is if falling Ca may limit—or alternatively, facilitate—
Bythotrephes establishment on the Shield through direct (Ca tolerance) and concomitant indirect (food web effects) interactions. Weisz and Yan (2010) found that Bythotrephes are more likely to occur in large, higher-calcium lakes on the Shield, a correlation they hypothesized was spurious, and perhaps instead linked to the higher [Ca] of lower elevation lakes that had increased recreational boater traffic, the best predictor of
Bythotrephes presence (Potapov et al. 2011). The relationship between Bythotrephes presence and aqueous [Ca] has not been examined experimentally in the laboratory, however, nor explicitly in the field in regions where prevalence of the predator in relation to water quality can be assumed to have stabilized.
To address these knowledge gaps, I assessed whether the ongoing establishment of Bythotrephes on the Shield may be hindered by current declines in lake-water [Ca], and if it will be limited in the future as [Ca] continues to fall. To fulfill my objective in a thorough and definitive manner, I employed three approaches utilizing observations from both the laboratory and the field: i) I measured the body calcium content in wild-caught
Bythotrephes to determine its inherent calcium requirements; ii) I examined the effect of a [Ca] gradient on Bythotrephes survival, reproduction, and adult as well as offspring body size in the laboratory; and iii) I compared Bythotrephes distributions with respect to
[Ca] in Canadian Shield versus Norwegian lakes. Bythotrephes has been established in
Norway for millennia, while it is relatively new to the Shield (Yan et al. 1992). Because the impacts of low calcium on Bythotrephes have never been directly examined, and its body calcium requirements are unknown, I did not construct specific hypotheses regarding expected outcomes.
MATERIALS AND METHODS
Body Ca content of Bythotrephes
To determine the body calcium content of Bythotrephes, I adapted the methods described in Jeziorski and Yan (2006). On 22 July 2010,1 retrieved Bythotrephes from
Fletcher Lake (45.294°N, 78.949°W), a lake north of Dorset, ON, Canada, that has supported the invader since at least 2006 (Cairns et al. 2007a). The mixed layer [Ca] of this lake was 2.1 mg • L"1 in 2006 (CAISN, Great Lakes Insitute for Environmental
Research, University of Windsor, Windsor, Ontario, unpublished data), close to the median [Ca] in south-central Ontario Shield lakes. I collected animals using a conical zooplankton tow net constructed of 80 jam mesh. While in the field, I picked 85 barren, actively swimming Bythotrephes and transferred each individual via wide-bore pipet to its own 50 ml capacity Fisherbrand centrifuge tube filled with filtered lake water. The tubes were kept cool and dark for transport to the Field Laboratory for the Assessment of
Multiple Environmental Stressors (FLAMES), located at the Ministry of the
Environment's Dorset Environmental Science Centre (DESC). FLAMES is a restricted- access laboratory that employs strict protocols to reduce chemical and biological contamination. All samples were handled wearing powder-free gloves and clean laboratory attire. Prior to use, all equipment including Teflon strips, forceps, Petri dishes,
fs 1 centrifuge tubes, and pipet tips were soaked in a 1% nitric acid bath for 30 - 60 minutes and rinsed at least seven times with deionized water.
Within 6 h of collection, Bythotrephes were separated from the bulk solution using a 300 ^m sieve, rinsed with distilled water, then transferred via plastic forceps to preweighed Teflon boats measuring 15 x 10 x 0.25 mm. The animals were dried overnight at 65°C, held over desiccant until weighing, and weighed on a Mettler-Toledo
GmbH AG 104 balance (± 0.1 mg accuracy; n = 5 replicates of 17 Bythotrephes, mean dry mass (DM) per replicate = 2.28 ± 0.086 mg SE). Each sample of dried animals was then transferred to a polypropylene centrifuge tube and digested in 50% nitric acid at 90°C for
60 min. The resulting clear solutions were allowed to cool and then diluted to 3 ml with distilled water. Two blanks were prepared in the same manner to identify any procedural sources of calcium contamination. [Ca] of the final solutions were analyzed by atomic absorption spectrophotometry (AAS; Evans 2007). Negligible amounts of calcium were detected in the blanks (maximum 0.056 mg • L"1, mean sample to blank ratio of the digests = -5:1). I do not report blank-corrected values here as this does not alter the main result when figures are rounded; had I done so, the reported mean value for the percent calcium as DM in Bythotrephes (below, in Results) would decrease by only 0.001%.
Potential Ca limitation of Bythotrephes growth and reproduction
Field collection and rearing of maternal line
Bythotrephes were collected on 20 August 2009 from Fletcher Lake following the methods outlined in Kim and Yan (2010). Animals were returned to the FLAMES laboratory, and each individual was transferred to separate glass mason jars. The 250 ml
fo capacity jars were filled with 175 ml of FLAMES culture medium (Celis-Salgado et al.
2008), and they were then incubated at 21°C, the temperature of the upper strata of
Fletcher Lake at the time of collection. Approximately 50 Artemia franciscana nauplii, along with seven barren, laboratory-cultured D. pulex were offered as prey to each
Bythotrephes. A small grain of dry cetyl alcohol (CH3(CH2)i4CH20H) was added to each container to discourage entrapment of Bythotrephes and prey by the surface tension
(Desmarais 1997). The jars containing the wild-caught Bythotrephes were then placed into a Conviron CMP4030 E7/2 growth chamber (Conviron, Winnipeg, MB, Canada), in which ambient temperature and cool white fluorescent light intensity were set at 21°C
•Y i and 100 nmol ' m • s~ (7400 lux), respectively. The photoperiod consisted of 14 h light to 10 h dark, with 30 minute dawn and dusk ramping periods. A TidbiT v2 UTBI-001 temperature logger (Onset Computer Corporation, Pocasset, MA, USA) was placed in the experimental chamber in 175 ml of water to monitor actual in situ temperature, and was observed to average 21.5°C (± 0.005°C SE) over the experimental duration of 23 days.
The wild-caught Bythotrephes were left to acclimate to container and chamber conditions overnight. Over the next two days, I transferred only vigorously swimming animals to fresh medium with prey.
Prey cultures for Bythotrephes (Bosmina freyii, D. ambigua, and D. pulex) were housed in 1 L glass jars filled with FLAMES medium and fed a 1:1 mixture of the green algae Pseudokirchneriella subcapitata and Scenedesmus obliquus ad libitum, grown in batch culture and harvested during the log phase of growth. Pure starter cultures of both had been obtained from the Canadian Phycological Culture Centre in Waterloo, Ontario, Canada. Daphnia ambigua and D. pulex reared in FLAMES were rinsed in 118 (xm sieves and suspended in FLAMES of the appropriate [Ca] before feeding. Bosmina freyii was not rinsed owing to its small size and susceptibility for retention in the sieves; instead I transferred individual animals to jars via glass capillary pipets. In addition,
Artemia franciscana nauplii were hatched out every 24 h from premium grade cysts
(Brine Shrimp Direct, Ogden, Utah, U.S.A.) in conical aerated hatcheries (Aquatic
Ecosystems Inc., Apopka, Florida, U.S.A.) inside a growth chamber set at 28°C with continuous 100 (imol • m2 • s"1 lighting. Artemia franciscana were cultured in Instant
Ocean® artificial sea water (Spectrum Brands Inc., Atlanta, Georgia). The nauplii, once harvested, were slowly poured into a 118 |im sieve and rinsed thoroughly with deionized water, followed by 3 submersions in FLAMES medium of the appropriate [Ca]. The nauplii were re-suspended in FLAMES of the corresponding [Ca] prior to being offered as food to the Bythotrephes.
Bioassay conditions
All culture media were prepared several days prior to the onset of the experiment and continuously aerated. [Ca] was adjusted by taking appropriate aliquots from a
CaSC>4 • 2H2O stock solution (0.547 g • L"1). Nominal [Ca] were set at 0.0, 0.5, 1.0, 1.5,
2.0, and 2.5 mg • L"1 to reflect real-world ranges of calcium in soft-water lakes in Canada and Norway and to centre around 1.5 mg • L"1, the identified performance threshold for D. pulex (Ashforth and Yan 2008). Actual mean [Ca] following the addition of food were determined by AAS to be 0.1 (± 0.019), 0.6 (± 0.013), 1.0 (± 0.008), 1.5 (± 0.010), 2.0 (±
fid 0.017) and 2.6 (± 0.012) mg • L"1 (n = 3 for each [Ca]). Hereafter, I use the experimentally determined [Ca], not the nominal values.
First generations of laboratory-born Bythotrephes were used in experiments.
When adequate numbers (i.e., 66) of neonates < 24 h old were obtained, 11 individuals were randomly assigned to one of the [Ca] treatments, transferred to individual Mason jars which had been filled with 150 ml of FLAMES adjusted to the appropriate [Ca], and inoculated with prey. Bythotrephes were fed laboratory-cultured prey of differing sizes, consisting of 10 B.freyii, 7 D. ambigua, 5 D. pulex and approximately 50 A. franciscana nauplii per day (as per Kim and Yan 2010). Each day, survival and reproduction of the
Bythotrephes were assessed.
I measured Bythotrephes because body size is an important contributor to overall fitness (reviewed in Hart and Bychek 2011), and low calcium limits daphniid growth
(Hessen et al. 2000). Immediately upon mortality or at the test endpoint of 23 days, all test animals were preserved in 4% sugar formalin. All offspring were preserved within the first 24 h of birth. The somatic growth of Bythotrephes ceases after maturation
(Branstrator 2005), allowing me to directly compare adult lengths among treatments. I only measured adults that had reproduced at least once. To ensure that data for offspring size measurements were independent of each other, I randomly selected one neonate from each brood to include in analyses. All animals were measured from the top of the head to the anus (designated the body length) and from the anus to the distal tip of the caudal process (denoted the tailspine length), with the sum of these two measurements equaling total length. Specimens were measured via a Leica MZ 12(5) dissecting microscope with attached Hitachi HV-C20M video camera and the ZEBRA zooplankton sample counting software (Allen et al. 1994). Bythotrephes body lengths have been noted to shrink by
20% following preservation in sugar formalin of the strength I used (Yan and Pawson
1998), thus I present corrected body length measurements (i.e., multiplied by 1.2).
Statistical analyses
Life table response experiments conceptually bridge the gap between an individual organismal response in the laboratory and a population-level response that may occur in situ. Here I reared a cohort of conspecific Bythotrephes across a [Ca] gradient over one lifespan. Then, based on observed survivorship and fecundity schedules,
I calculated life history parameters including cohort generation times (Tc), net reproductive rates (Ro) and intrinsic rates of natural increase (r). Ro is the replacement rate of a population per generation or the number of daughters expected per female in her lifetime, and r is the per capita instantaneous rate of increase in a population with a stable age distribution (Stearns 1992). Thus, when Ro is 1.0 and r is 0.0, the population is neither increasing nor decreasing. Life history parameters were calculated using the
PopTools add-in for Microsoft Excel (http://www.cse.csiro.au/poptools). Because these metrics assume no male production, data for any males unintentionally included in the assays were omitted (typically one to three males across all [Ca] and none at 1.5 mg • L"1).
To assess whether there were any effects of [Ca] on Bythotrephes growth, development and reproduction—as seen in daphniids—I also examined age at third instar
(determined by counting the number of paired articular spines on the caudal appendage;
Martin and Cash-Clark 1995), survival to reproduction, age at first reproduction, adult body and tailspine lengths, first and second brood clutch sizes, and offspring body and tailspine lengths. For all metrics, I excluded male data, as well as any Bythotrephes that were unintentionally lost or damaged during routine maintenance; thus there were an unequal number of replicates among treatments. Mean values of all parameters measured are presented ± standard errors of means, unless otherwise stated. To compare the effects of [Ca] on age at third instar, mean time to parturition, clutch size, and all size measurements, one-way ANOVAs were performed to discern differences among groups, followed by—where applicable—post hoc Tukey tests to pin-point significant pairs of treatments. Homogeneities of variances were assessed by Levene's tests. Clutch size data were square-root-transformed prior to statistical analyses using Bartlett's equation (X' =
Vx + 0.5). Statistical analyses were completed in JMP 8 (SAS Institute Inc., Cary, NC,
2009).
Bythotrephes distributions in relation to [Ca] in nature
To determine whether Bythotrephes occur at low [Ca] (i.e., < 2 mg • L"1) in situ, I examined lake [Ca] at which it is present in Norway (and where it has been established for millennia), alongside lake [Ca] in which Bythotrephes have so far been found on the
Shield (where they are relatively new). Complete methods for lake selection, biological and chemical sample collection, and analyses are described in detail in Cairns et al.
(2001b) and Hessen and Walseng (2008) for the Canadian and Norwegian datasets, respectively. In brief, the Canadian dataset was the outcome of a 311 -lake sampling effort by CAISN in 2005 - 2006 in the inland-lake region of south-central Ontario, where
Bythotrephes first appeared (Yan et al. 1992) and has subsequently spread rapidly (Weisz
67 and Yan 2010). Sampling was conducted from mid-June to late August, when
Bythotrephes abundances are typically highest (Yan and Pawson 1998). Samples were collected using a 63 jim conical sampling net (diameter = 30 cm, slant length = 1.4 m).
Two vertical hauls were collected and combined from 2 m above the lake bottom to the surface at each of five, evenly spaced sites located along the longest fetch of each lake. A sixth horizontal haul was taken from the downwind area of the lake where Bythotrephes might have been translocated by recent persistent winds. At the deepest sampling point on the lake, a mid-metalimnion to lake surface composite water sample was collected with a DESC "space age sampler" (Ingram et al. 2006). [Ca] was analyzed via AAS, and pH was measured using a pH meter and a combination glass electrode with a reference cell. In Norway, samples were collected from June to September across Norway's mainland. Of a total of 2467 lakes, 1736 were represented by pelagic samples. These lakes range widely in altitude, area, pH, and conductivity. Zooplankton were collected at the deepest point using a vertical haul with a 90 jim net (diameter = 27.5 - 30 cm) from the lake bottom to the surface. For data analyses, any lakes missing either pH or [Ca] values were rejected, and only those with pH at or between 6.0 and 7.0 were retained, reducing the number of lakes from 835 to 499 in Norway and from 311 to 183 in Canada.
This ensured I excluded acid lakes (assuming that pH < 6 would affect biota; see Holt et al. 2003) as well as lakes with high pH (> 7), which had very high [Ca] (20 - 80 mg • L"1; but I am only concerned with lower Ca thresholds). This narrow pH range also allowed me to better align the field distribution data with the laboratory bioassay results in which pH measured 6.4 - 6.8.
*8 RESULTS
The body calcium content of the Fletcher Lake Bythotrephes was very low, ranging from 0.0231% to 0.0416% as DM (mean = 0.03% ± 0.003 SE) for the five replicate samples.
Overall, Bythotrephes survival, growth and reproduction were not greatly impacted by low [Ca] in the laboratory assays, indicating that this is a species with low calcium demands and with great tolerance for very low waterborne calcium. Only at 0.1 mg • L"1 was Bythotrephes survival markedly decreased (56% survival to reproduction compared with the 80 - 100% survival observed in the other treatments; Table 3.1).
While 43% of first brood offspring in the 0.1 mg • L'1 treatment were aborted or non viable at birth, Bythotrephes was still able to produce a second brood (Figure 3.1), and offspring in this treatment were of comparable size to those at the higher [Ca].
Bythotrephes exhibited the highest Ro and r at 1.5 mg • L"1 because the majority of animals in this treatment survived to primiparity and a large proportion produced second
1 and third broods. Bythotrephes reared at 1.0 mg • L" exhibited the longest Tc (13.6 days compared with 11.6 - 12.9 days for the other treatments) and produced the largest second brood clutch sizes. Although there were no statistically significant differences in mean clutch sizes across treatments, 1 noted animals producing up to six second brood offspring
(Figure 3.2). The incidence of third brood production was also highest at 1.0 mg • L"1. There were no detectable effects of [Ca] on: time to third instar (F$^ = 0.741,/? =
0.597; overall mean = 6.5 d), age at first reproduction (7*5,45 = 0.574, p = 0.720; overall mean = 10.5 d), adult body length (^5,30= 1.844,/? = 0.134; overall mean = 2.35 ± 0.368 mm), adult tailspine length {F^ = 1.702,/? = 0.165; overall mean = 7.27 ± 0.560 mm), or adult total length (^5,30= 1.836,/? = 0.136; overall mean = 9.62 ± 0.732 mm).
Furthermore, there were no significant impacts of [Ca] on first (^5,43 = 1.310 ,p = 0.278) or second brood clutch size = 1 -222, p = 0.325), nor on first (Fsj7 = 1.084, p =
0.385) or second brood offspring body size (Fs,26= 0.433,/? = 0.821). Tailspine and total lengths could not be statistically assessed reliably, however, owing to small sample sizes and unacceptably high variability, even after data transformations.
Treatments were thus combined because they were not significantly different from each other. I found an effect of brood order on overall clutch size, which increased significantly from first (2.5 ± 0.84 offspring) to second brood (3.9 ± 0.86 offspring) (fo =
6.965, p < 0.0001; see also Figure 3.2). Overall offspring body size also increased significantly from first (1.25 ± 0.189 mm) to second brood (1.53 ± 0.192 mm; *73 = 6.284, p < 0.0001), as did offspring tailspine length from first (4.56 ± 0.470 mm) to second
(5.20 ± 0.411 mm) brood (/73 = 5.182,p < 0.0001) and offspring total length from first
(5.91 ± 0.592 mm) to second (6.73 ± 0.452 mm) brood (/73 = 6.532, /? < 0.0001).
Finally, Bythotrephes was observed at lower environmental [Ca] in Norway than they were on the Canadian Shield. Of the 183 sampled Shield lakes, overall mean and median [Ca] were 3.03 and 2.34 mg • L"1, respectively, while the respective mean and median [Ca] at which Bythotrephes was present (n = 22) were 3.22 and 2.47 mg • L"1.
70 The lowest [Ca] environment in which Bythotrephes was found on the Shield was 1.64 mg • L"1. In Norway, overall mean and median [Ca] for the 499 sampled lakes were 2.05 and 1.60 mg • L"1, respectively, while mean and median [Ca] for Norwegian lakes with
Bythotrephes {n = 116) were 1.90 and 1.79 mg • L"1, respectively, and animals were found in lakes with [Ca] as low as 0.20 mg • L"1 (Figure 3.3). To determine whether this discrepancy in minimum [Ca] arose from the relatively smaller number of lakes sampled on the Shield in comparison with Norway, I randomly selected 100,200, 300,400, and
499 Norwegian lakes and observed the spread of Bythotrephes distributions. I found no difference in the shapes of the curves except when w =100 lakes (in which case,
Bythotrephes sometimes does not appear to be present in lakes with [Ca] < 0.5 mg • L"1).
I included close to 200 Shield lakes in this analysis, however, so the fact that
Bythotrephes did not occur at < 1.6 mg • L"1 does not necessarily indicate that it is limited by low Ca on the Shield.
DISCUSSION
Clearly, Bythotrephes has minimal Ca requirements. Its body calcium content, at
0.03% as DM, is much less than the very low (relative to daphniids) 0.2 - 0.4% value established for non-Daphnia cladoceran taxa in Ontario by Jeziorski and Yan (2006), as well as recently measured values for littoral cladocerans in softwater (Shapiera et al.
2011). Bythotrephes is thus a very weakly calcified organism, and structures such as exoskeletons and rigid tailspines must be composed mostly of 'chitinous' (Mordukhai-
Boltovskoi 1968) cuticle (Martin and Cash-Clark 1995) with little mineralized hardening.
71 In future, microchemical analyses (via scanning electron microscope and energy dispersive spectrophotometry) could be used to determine mineral contents of
Bythotrephes tissues and to pinpoint what accounts for the rigidity of Bythotrephes' tailspine.
I observed only subtle effects of low [Ca] on Bythotrephes survival, reproduction and body size. Although survival and population growth (r) rates were lowest at 0.1 mg • L"1, r remained above zero, indicating positive population growth. Experimental duration was not a factor hindering my ability to detect an effect of low [Ca] on
Bythotrephes, as I adopted a 'time-to-death' test endpoint, which in the majority of cases extended throughout the entire life cycle of the animals. Neither was it likely that the test animals were obtaining much Ca from their food. Bosmina freyii and D. ambigua contain very little calcium (both < 1% as DM; Jeziorski and Yan 2006), as do A. franciscana nauplii (0.023% as DM; Watanabe et al. 1983). D. pulex has a relatively higher calcium content (Jeziorski and Yan 2006), but most of it is stored in the carapace (Alstad et al.
1999), and Bythotrephes typically ingests only the soft parts of prey (Schulz and Yurista
1995). Thus I presumed that the only difference among treatments was the waterborne— and not the food-supplied—calcium. Crustaceans in general are known to obtain the majority of their calcium from the environment via active branchial uptake (Wheatly
1999), and Bythotrephes should be no exception. As my primary objective was to quantify impacts of declines in waterborne calcium on this species while being fed natural-encountered prey {A. franciscana nauplii excepted), I was not overly concerned
7? with whether Bythotrephes could supplement waterborne calcium with a dietary source of calcium.
Bythotrephes simply appears inherently well equipped to tolerate low ambient [Ca] and adjusts to less-than-optimal [Ca] by shifting life history strategies. Ashforth and Yan
(2008) found that D. pulex did not survive to reproduce at 0.1 mg • L"1, which was not the case with Bythotrephes. This difference is likely due to the fact that Bythotrephes contains much less somatic calcium than do daphniids and hence requires less energy expenditure to obtain and replace body calcium upon moulting. Parthenogenically- produced Bythotrephes typically undergoes two moults during its life cycle (Yurista
1992), with an additional moult upon the release of each brood. One daphniid will moult
10-18 times throughout its lifespan (Hessen and Rukke 2000), however—much more frequently than Bythotrephes—and lose as much of 90% of its calcium at each moult
(Alstad et al. 1999). Rather, Bythotrephes' high reproductive rate at 1.5 mg • L"1 appears to indicate a shift in life history strategy in response to low [Ca], with animals reared at
1.0 mg • L"1 resorting to r strategy by producing larger numbers of offspring. Furthermore, while 1.5 mg • L"1 has been distinguished as the performance threshold of r for D. pulex
(Ashforth and Yan 2008), this [Ca] environment actually stimulates r for Bythotrephes.
As Bythotrephes' establishment success is constrained by Allee effects (Gertzen et al.
2011), this increase in propagule production at low calcium does not bode well for many non-acidic, as-of-yet non-invaded lakes with [Ca] at or near 1.5 mg • L"1.
My laboratory results provide reasonable explanations for the Norwegian distributional data, where low [Ca] was not a factor discouraging Bythotrephes presence.
IT, The calcium content of daphniids is a useful predictor for their occurrence along [Ca] gradients in nature (Hessen et al. 1995; Cairns 2010), and I can extend the same logic to
Bythotrephes. Bythotrephes is obviously capable of surviving and reproducing in waters with very low [Ca] in Norway. Assuming that there are no substantial physiological differences among endemic and invading populations, calcium declines likely do not limit Bythotrephes on the Shield. I thus concur with Weisz and Yan (2010) that the positive relationship they observed between lake-water [Ca] and Bythotrephes presence in Ontario may well be spurious. The fact that Bythotrephes has not been found at low
[Ca] (< 1.5 mg • L"1) may be attributable to a lag phase (i.e., Bythotrephes has not been introduced to enough low [Ca] lakes yet), but this may change in future as a result of continued anthropogenic activity.
The stoichiometric, bioassay and survey results support the hypothesis that
Bythotrephes establishment in North America will not be directly limited by ongoing calcium declines. However, daphniids are clearly being impacted by calcium decline in
Ontario (Jeziorski et al. 2008). Given that [Ca] has dropped below 1.5 mg • L"1 in as much as 50% of the lakes in this region (Jeziorski et al. 2008) and Bythotrephes is now common in many of these lakes (Weisz and Yan 2010; Kerfoot et al. 2011), I hypothesize that damage to Daphnia populations on the eastern Canadian Shield is already well underway, from each stressor individually and when mutually present additively. To date, no one has examined how daphniids and other calcium-rich species may be affected by the presence of Bythotrephes in calcium-deficient waters, although there is developing evidence for a synergistic negative impact of low calcium and Chaoborus on daphniids
74 (Howard Riessen, Department of Biology, SUNY College at Buffalo, 1300 Elmwood
Avenue, Buffalo, New York, USA, unpublished data). Impact assessments of
Bythotrephes and environmental calcium decline on native biota should be conducted and will contribute to a growing understanding of how abiotic factors affect the interactions of invasive species with native biota (e.g., Couillard et al. 2008).
Based on existing evidence, smaller species such as Ceriodaphnia spp., D. ambigua and D. catawba are readily consumed by Bythotrephes, but are relatively less likely to be affected by calcium declines. Ceriodaphnia spp. and D. ambigua are frequently selected when Bythotrephes are offered a choice of prey (Vanderploeg et al.
1993; Kim and Yan 2010), and lower mean abundances of D. catawba have been noted in invaded lakes when assessed alongside non-invaded lakes (Boudreau and Yan 2003).
Body calcium contents of these daphniids have been quantified at 0.6%, 2.8%, and 4.0% respectively for Ceriodaphnia quadrangula (Waervagen et al. 2002), D. ambigua, and D. catawba (Jeziorski and Yan 2006). Lower calcium thresholds for the prevalence of these latter two daphniid species in the field could not be defined through multiple regression models, which may indicate that they both have relatively high tolerances for low calcium (Cairns 2010).
Meanwhile, the reverse seems true for Daphnia longiremis and Daphnia mendotae, two relatively large daphniids that are less susceptible to Bythotrephes but likely to be negatively impacted by low calcium. Daphnia longiremis dwells in the hypolimnion of stratified lakes (Keller and Conlon 1994) where it is unlikely to encounter Bythotrephes, since the predator spends most of its time in the meta- and
7S epilimnion (Young 2008). Daphnia longiremis has a lower calcium prevalence threshold of 1.26 (± 0.69 SE) mg L"1 (Cairns 2010) however, which suggests that it may be sensitive to declines in environmental calcium. Daphnia mendotae on the other hand often co-exists with Bythotrephes in North America (e.g., Lehman 1991; Yan et al. 2001;
Strecker et al. 2006), and population increases have even been recorded following invasion (Dumitru et al. 2001). This may be because Bythotrephes is less adept at capturing D. mendotae, owing to the latter's rapid escape reflexes (Pichlova-Ptacnikova and Vanderploeg 2011). Still, D. mendotae is not completely impervious to the effects of low calcium, as it is a relatively large daphniid (1.2 - 2.8 mm length; Witty 2004), with a high calcium content (5.1% as DM) and [Ca] prevalence threshold of 1.63 mg L"1 (±
0.59 SE; Cairns 2010).
Finally, Daphnia retrocurva, Daphnia dubia, and Daphnia pulicaria may be particularly at risk from the combined effects of both stressors. Daphnia retrocurva is frequently selected when Bythotrephes are presented with a choice of prey (Vanderploeg et al. 1993), and populations of D. retrocurva declined following invasion by
Bythotrephes (Lehman and Caceres 1993; Yan et al. 2001; Barbiero and Rockwell 2008).
Lower mean abundances of D. retrocurva and D. dubia were also observed in invaded lakes when compared with non-invaded lakes (Boudreau and Yan 2003). Last, D. retrocurva and D. dubia have lower prevalence thresholds in the field of 1.69 mg L"1 (±
1.15 SE) and 1.58 mg L"1 (± 0.96 SE) respectively (Cairns 2010), which suggests that they respond negatively to low calcium. Daphnia pulicaria, being one of the largest (1.4
-3.2 mm; Witty 2004) native daphniid species, has an optimal aqueous [Ca] requirement
7* of 16.1 mg L"1 (Cairns 2010). The evidence for impacts of Bythotrephes predation on D. pulicaria is less straightforward. Daphnia pulicaria is readily consumed by adult
Bythotrephes in the laboratory, but juveniles appear to be limited to smaller specimens (N.
Kim, personal observation). While overall reductions in D. pulicaria abundance have been observed following Bythotrephes invasions (Lehman and Caceres 1993; Branstrator
1995; Barbiero and Tuchman 2004), some workers have noticed declines in only the smaller size range (0.8 - 1.8 mm) of this species and a shift toward larger (> 2.5 mm) individuals (Lehman 1991; Hoffman et al. 2001). Theoretically, this could translate to elevated calcium requirements for this species' persistence, since larger daphniids require proportionally more calcium than do smaller ones (Waervagen et al. 2002; Jeziorski and
Yan 2006).
The disappearance of large Daphnia species could have major ecological repercussions for pelagic food webs. Populations of Bosmina spp., Ceriodaphnia spp., D. ambigua, and D. catawba will likely increase through a release in competition as [Ca] negatively impacts other cladocerans, but all are preferred prey of Bythotrephes and their presence may initially help support establishment. In Norway, Bythotrephes occasionally occurs in the absence of daphniids, proving that populations can persist on other prey such as bosminids and copepod nauplii in the absence of, or after the depletion of, daphniids. Holopedium glacialis (the renamed H. gibberurtr, Rowe et al. 2007) and D. mendotae are the only two Cladocera that have prospered in Harp Lake, Ontario, after the
Bythotrephes invasion (Yan et al. 2001); [Ca] of Harp Lake is still well above critical thresholds for Daphnia, however (Yan et al. 2008). Holopedium glacialis, while edible, is
77 not often selected by Bythotrephes when presented with a choice of prey (Kim and Yan
2010), probably because it is well protected from invertebrate predators by its gelatinous capsule, a defense that daphniids lack. Holopedium glacialis may possess an additional competitive advantage over daphniids in low calcium lakes because they have low calcium demands (Hessen et al. 1995). Body calcium contents of//, glacialis have been reported to be as little as 0.1% (Wasrvagen et al. 2002) to a mean of 0.4% as dry mass
(range 0.2% - 0.5%; Yan et al. 1989). Should the relative importance of H. glacialis rise at the expense of daphniids, however, there may be additional major consequences that are beyond the scope of this study.
In summary, the lack of an effect of low [Ca] on Bythotrephes is important given that we are dealing with an invader with the proven potential to disrupt native pelagic food webs, which include species vulnerable to calcium declines. Given sufficient time, propagule pressure, and the absence of other major stressors such as limited prey availability and temperature extremes, Bythotrephes should readily establish populations in lakes with much lower [Ca], as it has in Norway. At the moment, calcium decline is more common in smaller lakes, while Bythotrephes has been more successfully invading larger lakes in the region (Weisz and Yan 2010), but convergence of the two stressors is likely over time. I further hypothesize that mid-size lakes may be at the most risk, as calcium decline will have a greater effect in small lakes, and Bythotrephes is more common in larger lakes. It is thus the mid size lakes with [Ca] near 1.5 mg • L"1 that should be targeted as areas for protection against Bythotrephes invasions. ACKNOWLEDGEMENTS
This project was funded by the Canadian Aquatic Invasive Species Network, the
Natural Sciences and Engineering Research Council of Canada, and the York University
Faculty of Graduate Studies. Field and laboratory assistance was provided by Melanie
Shapiera and Stephanie Hung.Technical and logistic support was provided by Ron
Ingram and Don Evans from the Ontario Ministry of the Environment in Dorset, ON. The
Norwegian dataset is based on a number of published reports and unpublished data, and thanks is given to all those who have contributed with data to this study. Thank you to two anonymous reviewers whose suggestions greatly improved the quality of this manuscript.
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8ft TABLES
Table 3.1. Effects of [Ca] (measured values) of bioassay media on neonate Bythotrephes reared over 23 d. Numbers of initial replicates, mean times to Instar 3, % survival to parturition, mean times to parturition, cohort generation times (Tc), net replacement rates (jRo), and intrinsic rates of natural increase (r) are presented, ± 1 SE. There were no significant effects of [Ca] on time to Instar 3 or time to parturition (one-way ANOVA, a = 0.05).
[Ca] (mg L') 0.1 0.6 1.0 1.5 2.0 2.6
Number of replicates 10 10 11 10 (excluding males)
Mean time to 6.5 ±0.71 6.6 ±0.99 6.8 ±0.87 6.3 ±0.60 6.4 ±0.57 6.3 ±0.41 Instar 3 (d) (n = 5) (n = 8) (n = 9) («=11) (n=10) (n = 6)
% Surviving to parturition 56 80 80 100 100 86
Mean time to 10.7 ± 10.6 ± 10.9 ± 10.4 ± 10.3 ± 10.5 ± parturition (d) 0.45 1.13 0.92 0.54 0.42 0.63 Cohort generation 12.5 11.6 13.6 11.9 12.4 12.9 time, Tc (d)
Net reproductive rate, Ro 1.9 3.8 6.4 6.6 5.6 4.3 (number of offspring)
Intrinsic rate of natural Q()5 0J1 0.13 0.15 0.13 0.11 increase, r (d )
87 FIGURES
1.0 1 0.9
0.8 - 0.7 inninnp' 0.6 - 0.5 " 0.4 - 0.3 -
0.2 *
0.1 -
0.0 0.1 0.6 1.0 1.5 2.0 2.6 1 , -i , 1^1^ , 1 , [CaJ mg-L
Figure 3.1. Relative proportions of first (light grey), second (open), and third broods (black) produced by Bythotrephes reared at different [Ca] intervals.
88 o> c ra
« £1 E 3 ® N '35 .c o 5 o 1.0 1.5 [Ca] mg-L-1
Figure 3.2. Mean first (open) and second brood (closed) clutch sizes for Bythotrephes reared at different [Ca] over one parthenogenic life cycle, ± SE. Numbers of Bythotrephes, n, ranged from 4-11, with the exception of the second brood clutch size at 0.1 mg • L"1, for which n = 2.
89 1.0 - 0.9 - 0.8 • 0.7 - 0.6 - 0.5 - 0.4 - 0.3 - 0.2 - 0.1 - 0.0 I i IIMl i I f i i i i r—i "i— i i r™' r"~ i i™ i II i i i 1 'i i i i i i OrNn^lfilDSBOidrCNiri^ [Ca] interval (mg-L~1)
Figure 3.3. Proportions of circumneutral Canadian Shield lakes (pH 6-7) with Bythotrephes (rt = 22 of 183 total lakes; open bars) and Norwegian lakes containing Bythotrephes (n= 116 of 499 total lakes; closed bars). Each interval represents a [Ca] bin width of 0.5 mg L"1.
90 CHAPTER 4
PREY QUANTITY IMPACTS LIFE HISTORY OF THE INVASIVE
PREDATORY CLADOCERAN, BYTHOTREPHES LONGIMANUS
ABSTRACT
Life history characteristics and demographic responses to three mixed-species prey densities (equivalent to 15, 30, and 45 prey organisms • day"1; designated 'low',
'medium', and 'high' food treatments, respectively) were examined for Bythotrephes longimanus reared over 22 days at 21°C in the laboratory. Predation rates at different prey densities were also quantified. Bythotrephes' intrinsic rates of natural increase and net reproductive rates were significantly diminished when offered low versus medium and high prey densities (r = 0.01 compared with r = 0.09 and 0.10, respectively). With increasing prey density, generation times were significantly faster and maximum life spans decreased. Prey density positively impacted Bythotrephes growth at instars 1 and 2, as well as first brood clutch and offspring sizes. Bythotrephes preyed on the daily equivalent of 9, 14, and 22 prey organisms (or 105, 176,249 as estimated dry mass, respectively) at the low, medium, and high food densities, representing a feeding efficiency of 50% - 60%. The smallest, slowest prey were most often selected. In the field, population maxima of Bythotrephes are positively correlated to population maxima of edible prey. I propose that this is likely due to improved growth, survival to reproduction, and fecundity; faster generation times; and larger numbers and sizes of progeny with increasing prey densities. 91 INTRODUCTION
The Cladocera represent a group of small crustaceans commonly called 'water fleas' that likely date back to at least the late Paleozoic era (Frey 1987). Cladocerans are widespread throughout freshwater communities worldwide, and may be grouped into approximately 4 orders, 15 families, 80 genera, and 400 species (Dodson & Frey 2001), although debates on cladoceran taxonomy continue to the present day. Herbivorous cladocerans (e.g., Daphnia) are important ecological regulators, as they feed directly on algae, and in turn represent an important food source for higher trophic levels. Predatory cladocerans include the families Podonidae, Polyphemidae, Cercopagidae, and
Leptodoridae (Rivier 1998). These and other invertebrate predators have become of increasing interest because they may be capable of strongly influencing plankton communities, especially in the absence of fish (Sprules 1972; Vanni 1988).
Bythotrephes longimanus (Leydig 1860; Crustacea: Onychopoda:
Cercopagidae)—herein Bythotrephes—is a predatory cladoceran that is widely distributed throughout the Palearctic region, where it typically occupies the open water habitats of large, clear and nutrient-poor lakes (Grigorovich et al. 1998; Rivier 1998;
Maclsaac et al. 2000). It has recently garnered attention as it has expanded its range into
North America, likely introduced to the Laurentian Great Lakes via contaminated ballast water (Sprules et al. 1990), from where it has spread inland. Bythotrephes poses a serious threat to pelagic biodiversity in its invaded range (e.g., Lehman & Caceres 1993; Yan et al. 2002; Barbiero & Tuchman 2004). Non-indigenous species are typically perceived as
"invasive" when impacts considered to be undesirable—ecological and/or economic— 92 can be detected (Lockwood et al. 2007). For Bythotrephes both consumptive (Lehman &
Caceres 1993; Yan & Pawson 1997; Bunnell et al. 2011) and non-consumptive impacts
(Pangle & Peacor 2006; Hovius et al. 2007; Pangle et al. 2007; Strecker et al. 2011) have been documented on the pelagic communities of its invaded range.
Bythotrephes' reputation as a highly voracious predator is supported by various literature accounts. The dominant prey items in Bythotrephes' diet include, but are not limited to, herbivorous cladocerans including Daphnia and Bosmina (Vanderploeg et al.
1993; Burkhardt & Lehman 1994; Wahlstrom & Westman 1999; Kim & Yan 2010).
Bythotrephes requires relatively high amounts of food because it swims constantly, loses
38% of its prey to "sloppy feeding", and has assimilation rates < 60% relative to what it ingests (Yurista & Schulz 1995; Yurista et al. 2010). Bythotrephes also invests a disproportionately large amount of resources in its offspring, with its broodsac—when occupied by several embryos—making up over 100% of its barren dry mass (DM)
(Yurista & Schulz 1995). In Long Lake, Michigan, Bythotrephes was found to ingest up to five times the zooplankton biomass that yellow perch did (Hoffman et al. 2001). In
Lake Huron, there is evidence that Bythotrephes is now the main planktivore (surpassing
Mysis relicta and fish), with estimated consumption greater than that of zooplankton production between July and October (Bunnell et al. 2011). As a visual predator without a fixed-size feeding basket, Schulz and Yurista (1998) likened Bythotrephes to an age-0 planktivorous fish in terms of its functional role in food webs.
Recently, Young et al. (2011) implicated spring prey availability as the key determinant of Bythotrephes population size in Ontario lakes. In lakes and reservoirs of 93 its endemic range, Bythotrephes has also been seen to attain its population maxima in the mouths of rivers when small crustacean quantity is highest (Rivier 1998). Cladocerans including Daphnia typically exhibit 100-fold fluctuations in population size over the year
(Dodson & Frey 2001) however, due to variations in edible algae as well as predation pressure (Lampert et al. 1986). Therefore, changes in prey availability most certainly affect Bythotrephes' population dynamics, and by extension, its potential for successful establishment in a new environment.
Given the presumed importance of prey for Bythotrephes, its consumption rates must be quantified to better assess impacts on invaded communities (Burkhardt &
Lehman 1994; Yan & Pawson 1998). Mordukhai-Boltovskaia (1958) noted early on that
Bythotrephes is able to consume 1-17 small-bodied (300 - 400 jim) crustaceans within
24 h of birth, and that this number increases to 21 prey by its fifth day. However, Schulz and Yurista (1998) found that adult Bythotrephes typically select large (> 2 mm) D. pulicaria as opposed to smaller specimens. Based on phosphorus budgets, Burkhardt and
Lehman (1994) calculated that Bythotrephes consumes an average of 14 Daphnia daily at
19°C. Through a bioenergetics model, Yurista and Schulz (1995) estimated that first, second, and third instar Bythotrephes optimally consume approximately 96,244, and 374 fig DM prey • d"1 at 16°C. Although some researchers have attempted to empirically quantify consumption rates (Sprules et al. 1990; Vanderploeg et al. 1993), their resulting estimates were much lower than the values predicted by bioenergetic analyses (Lehman
& Caceres 1993; Yurista & Schulz 1995), probably because Bythotrephes were not confined individually and cannibalism is common in this species (Schulz & Yurista 1998). 94 Still, there are no empirical, controlled, long-term studies directly examining
Bythotrephes predation rates, prey selection, and the impacts of prey availability on its life history. This is mainly because Bythotrephes is a difficult organism to rear under controlled conditions (Barnhisel 1991; Yurista 1992; Schulz & Yurista 1999), but recently developed techniques have improved Bythotrephes survival in the laboratory
(Kim & Yan 2010). The primary purpose of this study was thus to determine the effects of food quantity on Bythotrephes growth, survival, reproduction, and demography. A secondary aim was to quantify Bythotrephes' predation rates and prey selection at differing prey density.
MATERIALS & METHODS
Third instar Bythotrephes—identified by counting the number of paired articular barbs on the caudal process—with unpigmented broodsacs (Yurista 1992) were collected on 20 August 2009 from Fletcher Lake (45.3°N, 78.8°W) as per Kim and Yan (2010).
Bythotrephes were brought back to the Field Laboratory for the Assessment of Multiple
Enviromental Stressors (FLAMES laboratory), located at the Ministry of the
Environment's Dorset Environmental Science Centre in Dorset, Ontario, Canada. The maternal line was maintained as described in Chapter 3, with an ad libitum food regime consisting of Artemia franciscana nauplii and Daphniapulex.
Prey species offered to Bythotrephes were chosen to represent a diverse size range and to emulate what may be encountered by the predator in situ. Selected species included laboratory-cultured Bosmina freyii, A. franciscana nauplii (intended as a 95 substitute for copepod nauplii, which I did not have in culture), Daphnia ambigua,
Daphnia mendotae, and D. pulex. All prey cultures and Bythotrephes were kept in
FLAMES softwater medium (Celis-Salgado et al. 2008). Artemia franciscana were hatched out and prepared every 24 h as described in Kim and Yan (2010). Bosmina freyii and daphniids were fed ad libitum with a 1:1 mixture of the green algae
Pseudokirchneriella subcapitata and Scenedesmus obliquus batch-cultured at 21°C under
100 jimol • m"2 • s"1 (7400 lux) continuous fluorescent lighting, and harvested during the log phase of growth to ensure consistent quality. Pure starter cultures for both algal species had been obtained from the Canadian Phycological Culture Centre in Waterloo,
Ontario, Canada.
Once an adequate number of parthenogenically-produced Bythotrephes neonates
(< 24 h old) were available (i.e., 30), each individual was transferred to its own 500 ml glass mason jar which had been filled with 400 ml of FLAMES medium. Broodmates were segregated between each of the three food treatments to minimize potential maternal effects in the results. There were initially 10 Bythotrephes • treatment"1. These
Bythotrephes were held for 22 days at 21°C under 100 jimol • m"2 • s"1 (7400 lux) fluorescent lighting and a photoperiod of 14 dark to 10 light hours. Every other day, they were transferred via a wide-bore pipet to fresh medium with 6, 12, and 18 each of A. franciscana nauplii and mid-sized, barren B. freyii, D. ambigua, D. mendotae, and D. pulex. Total prey densities were 30, 60, and 90 prey organisms • 0.4 L"1 • 2 d"1, and will be designated the 'low', 'medium', and 'high' prey densities (or food treatments), respectively. These densities were chosen based on a 24 h pilot trial examining predation 96 rates of Bythotrephes when offered differing concentrations of small and large daphniids
(Appendix C), and they are levels that fall within ranges that occur in nature (Michelle
Palmer, York University, Biology Department, unpublished data). Upon each transfer of
Bythotrephes, the remaining contents of the previous container were slowly poured through 118 [xm mesh, which was then suspended in a shallow Petri dish. All sieved contents were carefully picked with a thin glass capillary pipet over a light table, and preserved in 4% sucrose formalin for later enumeration of consumption or kill rates
(denoted "predation rate" for brevity). Bythotrephes were observed once daily for survival and brood production. Upon brood release, offspring were preserved in 4% sucrose formalin so that length measurements could be made at a later time. Because
Bythotrephes body sizes have been noted to shrink by -20% following preservation in formalin (Yan and Pawson 1998), I present corrected body size measurements (i.e., multiplied by 1.2).
Demographic data (net reproductive rates, R0; cohort generation times, Tc\ and intrinsic rates of natural increase, r) for Bythotrephes were derived from life tables using the PopTools add-in for Microsoft Excel (http://www.cse.csiro.au/poptools). Since these metrics assume no male production, data for any males unintentionally included in the assays were omitted (i.e., one male per treatment). To estimate variance associated with these metrics, life table data were jackknifed (Meyer et al. 1986), and resulting pseudovalues were analyzed via one-way ANOVA. Significant test results (a = 0.05) were followed by Tukey HSD tests to isolate significant treatment pairs. Survivorship
97 was assessed via product-limit (Kaplan-Meier) survival curves, and log-rank ^ test statistics are reported.
All counting of remaining intact prey, growth and length measurements were completed under a Leica MZ 12(5) dissecting microscope with attached Hitachi HV-
C20M video camera and ZEBRA software package for enumerating and measuring zooplankton (Allen et al. 1994). Bythotrephes body size was considered to span from the distal rim of the eye to the anus. Tailspine length was from the anus to the distal tip of the caudal process. Total length was simply the sum of these two measurements. Because
Bythotrephes accrues a new intercalary segment and set of barbs to the tailspine following each moult (Branstrator 2005), the measure of space between adjacent lateral barbs sets was used as proxies for growth between instars 1 and 2, and instars 2 and 3.
To ensure that data for offspring size measurements were independent of each other, 1 randomly selected only one neonate from each brood to include in analyses. For the low food treatment in particular, this resulted in small sample sizes (reported). Prior to statistical testing, all count data (i.e., clutch size and predation rate) were square-root transformed using Bartlett's equation X' = Vx + 0.5 (Zar 1999) to meet test assumptions of homogeneous variances, as assessed by Levene's tests. All statistical analyses were completed in JMP 9 (SAS Institute Inc., Cary, NC).
Prey dry masses (DM) for A. franciscana nauplii, D. ambigua, D. mendotae and
D. pulex were estimated (Table 4.1) by drying representative specimens in an oven overnight at 60°C, then weighing on a Mettler-Toledo GmbH AG 104 balance (± 0.1 mg accuracy) and dividing by the number of individuals in the sample. Because accurate 98 estimates could not be obtained for B.freyii, literature values were used. From smallest/lightest to largest/heaviest are: B.freyii, A. franciscana nauplii, D. ambigua, D. mendotae, and D. pulex, representing individual biomasses of ~2 - 33 ng DM.
RESULTS
Demography
Available prey density clearly affects Bythotrephes demographics. The intrinsic rate of natural increase (r) for Bythotrephes reared on low food was significantly less than the values exhibited by Bythotrephes fed the medium and high food (0.01 compared with
0.09 - 0.10; p < 0.0001; Table 4.2). Correspondingly, the net reproductive rate (Ro) was also significantly lower for Bythotrephes reared on low food compared with those fed medium and high food (1.2 compared with 2.9 for both the medium and high food groups,
F2,24 = 51.4, p < 0.0001). Finally, with increasing food Bythotrephes' cohort generation time (Tt) was significantly faster by five days (for the high compared with low food treatments; p < 0.0001).
Survival
At all food densities, Bythotrephes were able to produce a second brood, which contributed to the positive r at low food despite only 33% survival to primiparity for
Bythotrephes in this treatment. In contrast, survival to primiparity was 67% for both the medium and high food treatments. While there were no significant differences in the survivorship curves (/2 = 2.56, p - 0.278; Figure 4.1), Bythotrephes' maximum life spans
99 decreased with increasing prey densities (> 22, 19, and 17 d for the low, medium, and high food treatments, respectively). Median life spans were 9, 17, and 12 d for the low, medium, and high food treatments, respectively. The relatively short median life span at low food can be attributed to high juvenile mortality but the few individuals surviving to reproduce were generally longer-lived.
Growth and adult size
Available prey density positively affected the lengths of the intercalary segments accrued by Bythotrephes at instar 1 (0.64 mm versus 0.88 mm for the low compared with high food treatments; p = 0.003) and instar 2 (0.64 mm versus 0.86 mm at the low compared with high food treatments; p = 0.009), resulting in longer adult tailspine lengths (a difference of 0.65 mm for low versus high food) and total lengths (a difference of 1.22 mm for low versus high food), although these differences were not statistically significant at the a = 0.05 level (p-values of 0.482 and 0.203 for tailspine and total lengths, respectively; Table 4.3). Larger adult body sizes were attained with increasing food (2.31 mm versus 2.87 mm for the low compared with high food treatments), but statistics were not conducted due to unacceptably high variances.
Fecundity and offspring size
Bythotrephes' first brood clutch sizes increased significantly with increasing food density (1.7 versus 3.0 offspring for the low versus high food treatments; p = 0.047;
Table 4.4). First brood neonate body, tailspine, and total lengths also increased by 0.18
100 mm, 0.82 mm, and 1.03 mm, respectively (p-values of 0.030, 0.003, and 0.003 respectively) for the low versus high food treatments.
There were no significant effects of prey density on second brood clutch sizes, which numbered from 2-3 offspring across all food treatments (p = 0.329), however.
While second brood neonate body, tailspine, and total lengths increased (differences of
0.24 mm, 0.58 mm, and 0.82 mm for the low compared to high food treatments respectively), these differences were not statistically significant (p-values of 0.233,0.406, and 0.220 respectively), which may be attributed to low statistical power resulting from small sample sizes (n = 2 second brood offspring in the low food treatment). Clutch sizes increased from first to second brood at the low and medium food levels by the equivalent of one offspring, however. There was no change in clutch size from first to second brood at the high food density, with Bythotrephes producing 3 offspring per clutch. Time between broods decreased by one day going from the low to the high food treatments.
Finally, total numbers of offspring produced were reduced at the low, compared with the medium and high food densities (10 compared with 26 and 25 offspring respectively).
Bythotrephes predation
Bythotrephes' predation rates increased positively with available prey density
(Table 4.5). Since there was no significant effect of predator age on the mean numbers of prey organisms or estimated mean biomass preyed upon within each of the food treatments (p > 0.05 in all cases, one-way ANOVA tests), values were pooled to obtain grand means. Thus, Bythotrephes consumed the daily equivalent of 9, 14, and 22 prey
101 organisms (or 105, 176, 249 DM) at the low, medium, and high prey densities, respectively. These predation rates represent a capture efficiency of 50 - 60% of available prey.
Across all food densities, Bythotrephes most frequently preyed on A. franciscana nauplii, followed closely by B.freyii, D. mendotae, D. pulex, and D. ambigua (Figure
4.2). There was, however, an overall lack of clear prey selection by Bythotrephes in this study. Despite D. mendotae's common co-occurrence with Bythotrephes, for example
(partly attributable to its rapid escape speeds; Pichlova-Ptacnikova & Vanderploeg 2011), it was not discriminated against.
DISCUSSION
In the field, population maxima of Bythotrephes are positively related to population maxima of edible prey (Young et al. 2011). I propose that this may be due to enhanced growth, survival to reproduction, and fecundity; faster generation times; and larger offspring with increasing prey densities. All of these factors contribute to higher population growth rates. Indeed, Bythotrephes' intrinsic rates of natural increase increased positively with increasing food. While Bythotrephes offered the high food density were the shortest-lived, they were more fecund than those reared with low food.
Decreased longevity with concomitant increased fecundity has been observed in other cladocerans such as Ceriodaphnia dubia, Simocephalus mixtus, D. pulex, and Daphnia magna when fed increasing concentrations of the green alga Scenedesmus (Munoz-Mejia
& Martinez-Jeronimo 2007; Pietrzak et al. 2010). Higher investments in first clutch 102 offspring were strongly correlated with a decreased lifespan in D. pulex, suggesting that there is a life history trade-off between diminished life span and increased allocation of resources toward early reproduction (Pietrzak et al. 2010). This may also be the case with
Bythotrephes. Overall, these observations are consistent with the highly conserved phenomenon across taxa (e.g., yeast, invertebrates, fish, mammals) that caloric restriction results in life extension (Lynch & Ennis 1983; Holliday 1989; see reviews and competing explanatory hypotheses in Masoro 2005 and Sinclair 2005).
Bythotrephes growth was positively affected by increasing prey densities. For
Bythotrephes reared at medium food, however, growth at the second instar decreased relative to the first instar, suggesting that Bythotrephes may have been diverting its mildly limited resources to reproduction at the later life stage. For Daphnia, maintenance is prioritized over both growth and reproduction when resources are limiting, but subsequent resource allocation to growth or reproduction takes precedence over the other depending on life stage (Glazier & Calow 1992).
While Bythotrephes were larger with increasing food, the lack of statistically significant differences in realized adult tailspine and total lengths probably reflects two points: i) I only included parthenogenic females surviving to the third instar stage for these measurements, thus any individuals not surviving may have been smaller but are not represented, and ii) Bythotrephes used in this study were derived from well-fed mothers. To elaborate on the latter point, embryos were not subject to low food conditions via their mothers prior to the start of the experiment. Had the mothers of the
103 test animals also been subject to similar feeding regimes, size differences would likely have been more pronounced.
The significantly smaller progeny produced by Bythotrephes reared at low food does have implications for establishment in the field, however, as larger progeny are generally better equipped to cope with periods of starvation (Ketelaars et al. 1995).
Although larger-bodied animals are more conspicuous to predators, they are also better competitors for necessary resources when limiting (Gliwicz 1990). In addition, the impacts of Bythotrephes on zooplankton communities may well be influenced by the mean body size of the invading population (Yurista & Schulz 1995). According to the size-efficiency hypothesis, body size is one of the key factors governing the relative abundances of zooplankton (Brooks & Dodson 1965). Should this experiment have proceeded for successive generations, the effects of low food on Bythotrephes would likely have been even more pronounced.
Clutch size is typically more variable than offspring size, however, and almost directly reflects investments in reproduction (Stearns 1992). Well-fed daphniids are known to have greater numbers of larger offspring as they age (Ebert 1991). While there was no increase in clutch size for the first versus second brood for Bythotrephes reared with high food, I noted a tendency toward larger clutch sizes as Bythotrephes aged in
Chapter 3. There was also the occasional production of third broods, which was not observed in this experiment. Maternal effects were likely minor, as the two experiments were run concurrently with mothers collected from the field on the same date and reared under the same conditions. For the bioassay in Chapter 3, Bythotrephes were offered 104 manifold larger amounts of A. franciscana nauplii than the Bythotrephes in this study, suggesting perhaps that additional factors (e.g., prey quality) may play a role in determining clutch size.
Bythotrephes is clearly a voracious predator, as it consumes more prey with increasing available densities. This is a similar result to that found for Cercopagis pengoi
(the fishhook waterflea, also a Ponto-Caspian onychopod invader now in North America) where predation rates also did not saturate at the highest concentrations examined (40 or
100 individuals' L1; Pichlova-Ptacnikova & Vanderploeg 2009). Bythotrephes also appears to exhibit a cyclic gluttonous type of feeding behaviour, in which it will consume very high amounts of prey one day, and much less the following day (N. Kim, personal observation). At 16°C, Yurista and Schulz (1995) predicted that consumption rates increase as Bythotrephes progresses through instar stages. I did not see this pattern for
Bythotrephes reared at 21°C. In a past feeding trial conducted at 22°C, however, I observed an increase in predation rate with age for Bythotrephes incubated with one prey type (500 - 850 |xm D. pulicaria), which I attributed to an increase in capture ability as the Bythotrephes grew larger (N. Kim, personal observation). A possible explanation for this discrepancy could be that because temperature greatly influences Bythotrephes' predation rates (Yurista et al. 2010; see also Appendix C) and activity levels, juvenile
Bythotrephes at 21°C may have higher physiological needs and improved capture abilities than those of their counterparts at 16°C. Further investigation on this topic is warranted.
Given the impacts of prey density on Bythotrephes described above, a daily predation rate of approximately 9 prey organisms (or 105 fig DM) is likely insufficient to 105 meet Bythotrephes' physiological demands at 21°C. It is plausible that the low food condition imposed here is actually below the required threshold for Bythotrephes in nature—though population growth metrics were positive, these animals appeared weak and less active than their medium and high food counterparts (N. Kim, personal observation). Malnourished Bythotrephes may thus be more likely to succumb to disease, parasites, and potential planktivorous predators. It should be restated, however, that my predation rates are slight overestimates of consumption because Bythotrephes does not consume the entire prey organism.
The lack of clear prey selection patterns (excepting A, franciscana, which was the most frequently selected across all prey densities) indicates that container effects were present (i.e., differential avoidance responses among prey species were dampened as they were constrained by the walls). Daphnia mendotae were consumed more often than expected, given this species' rapid escape response and frequent co-occurrence with
Bythotrephes in the latter's invaded range (Pichlova-Ptacnikova & Vanderploeg 2011). In future prey selection studies, a rotating jar system would likely produce more realistic results (Scott Peacor, Michigan State University, Department of Fisheries and Wildlife, personal communication). An additional explanation for the lack of clear selection is that
Bythtorephes is simply a true "generalist predator" (Schulz & Yurista 1995). After all,
Bythotrephes clearly thrive on a mixed-species diet (Kim & Yan 2010), and there have even been accounts of Bythotrephes consuming green algae and large diatoms (reviewed in Grigorovich et al. 1998; Dodson & Frey 2001). Curiously, Bythotrephes reared at low and medium prey densities did not select D. ambigua as often as other prey species 106 (contrary to Kim & Yan 2010), suggesting perhaps that if a choice is available,
Bythotrephes first exhaust the smaller and easily-caught prey before turning to larger prey.
In future studies, determining maximum rates of predation with even higher prey densities than those used here (but which are still ecologically relevant) would help delineate the maximum impact of Bythotrephes in invaded communities. Future efforts should include more work with temperature and light as experimental factors. After all,
Bythotrephes' consumption rates (Burkhardt 1994; Yurista et al. 2010) and generation times (Kim & Yan 2010) increase with increasing temperature. Increasing water temperatures result in increased edible prey availability, which may lead to increased body size (Burkhardt 1994). As a visual predator, Bythotrephes' predation rates are also affected by light intensity (Pangle & Peacor 2009; Anneli Jokela, Queen's University,
Department of Biology, personal communication; Appendix D). Additional studies should be conducted to better estimate the availability of edible prey in relation to
Bythotrephes abundances in the field. Young et al. (2011) provide abundances for herbivorous cladocerans and Bythotrephes in invaded Canadian Shield lakes, but do not include copepods. While Bythotrephes is unlikely to consume cyclopoid copepods
(Vanderploeg et al. 1993; Anneli Jokela, Queen's University, Department of Biology, personal communication), it is known to consume calanoid copepods, copepod nauplii, and copepodites (Vanderploeg et al. 1993; Grigorovich et al. 1998; Schulz & Yurista
1998). In addition, during periods of low cladoceran prey availability, Bythotrephes may resort to cannibalism (Rivier 1998; Schulz & Yurista 1998). I have frequently observed
Bythotrephes'' highly cannibalistic tendencies in the laboratory, even when contained 107 with very high densities of alternative edible prey. When population densities are high and suitable prey are lacking, cannibals are often rewarded with a nutritional advantage
(Via 1999). Bythotrephes retains and may have high requirements for the long-chain fatty acid eicosapentaenoic acid, for example, thus preying on conspecifics would help alleviate deficiencies. In conclusion, more research on the impacts of food quantity and quality on Bythotrephes should be conducted, as all such information would be highly valuable for risk and impact assessments.
ACKNOWLEDGEMENTS
I thank the Canadian Aquatic Invasive Species Network (CAISN), the Natural
Sciences and Engineering Council of Canada (NSERC) and the York Faculty of Graduate
Studies for funding this project. Field and lab assistance were provided by S. Hung and staff at the Ontario Ministry of the Environment's Dorset Environmental Science Centre.
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114 TABLES
Table 4.1. Dry mass estimates of prey used to calculate biomass of Bythotrephes' predation rates. Only mid-size, barren prey were used for feeding. Sample replicates used in the estimations of dry mass are noted, where available. References:a Witty 2004;b Dumont et al. 1975's value for B. longirostris (O.F.M.);c Hoff & Snell 2007;d This study.
Length DM Replicates, Prey species range (mm) estimate # * (ng) individuals - replicate'1
a b Bosmina freyii 0.4 - 0.6 2 — Artemia franciscana 0.4-0.5 c 3d 6, 300 nauplius Daphnia ambigua 1.3 (max) a yd 5,200
D. mendotae 1.2- 2.8 a 22 d 5,60
D. pulex 1.1 - 3.5 a 33 d 5,40
115 Table 4.2. Intrinsic rates of natural increase, net reproductive rates, and cohort generation times (± SE) for Bythotrephes (n - 9 • food treatment"1) reared on a mixed-prey assemblage of differing densities in 400 ml over 22 days. For each demographic metric, values not connected by the same letter indicate significant differences as identified by post hoc Tukey HSD tests (a = 0.05).
Prey density
Low Medium High Demographic metric (15 prey • d"') (30 prey • d"1) (45 prey • d"1)
Intrinsic rate of natural increase, r (d1) *** 0.01 ±0.008 a 0.09 ± 0.004 b 0.10 ± 0.004 b
a b b Net reproductive rate, R0 *** 1.2 ± 0.12 2.9 ± 0.14 2.9 ± 0.15
a b c Cohort generation time, Tc (d) *** 15.3 ± 0.14 12.4 ± 0.05 10.4 ± 0.05
* significant atp< 0.05; ** significant atp< 0.005; *** significant atp< 0.001
116 Table 4.3. Mean growth (estimated by the length of the intercalary tailspine segment between barb pairs, which are accrued at each moult) and adult lengths (± SE) of Bythotrephes reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. Lengths of Bythotrephes attaining the third instar stage are represented. Body sizes were corrected by a factor of 1.2 to account for shrinkage in sucrose formalin (following Yan & Pawson 1998), but statistical tests were not applied due to unacceptably high variability. For each size metric, values not connected by the same letter indicate significant differences as identified by post hoc Tukey HSD tests (a = 0.05). Values in parentheses indicate numbers of Bythotrephes included in analyses.
Prey density
Low Medium High Size metric (15 prey • d"1) (30 prey • d"1) (45 prey • d'1)
0.64 ± 0.064 a 0.72 ±0.043 a'b 0.88 ± 0.047 b Growth at instar 1 - 2 (mm) * (4) (8) (6)
0.64 ±0.061 ab 0.66 ± 0.040 b 0.86 ± 0.049 3 Growth at instar 2-3 (mm) * (4) (8) (6)
2.31 ±0.063 2.57 ±0.058 2.87 ±0.250 Adult body size (mm) (4) (8) (6)
6.50 ±0.107 6.89 ±0.285 7.15 ±0.354 Adult tailspine length (mm) (4) (8) (6)
8.81 ±0.158 9.45 ±0.355 10.03 ±0.504 Adult total length (mm) (4) (8) (6) significant atp< 0.05; ** significant atp< 0.005; *** significant atp< 0.001
117 Table 4.4. Mean (± SE) and sample size (in parentheses) of brood and offspring sizes resulting from Bythotrephes reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. For each metric, values not connected by the same letter indicate significant differences (a = 0.05). Only one offspring from each brood was randomly selected to be represented for all length measurements. Time between broods was not assessed statistically due to unacceptably high variability.
Prey density
Low Medium High Fecundity metric (15 prey • d') (30 prey • d"1) (45 prey • d"1)
1.7 ±0.33 a 2.3 ±0.21 a'b 3.0 ± 0.37 b Size of brood 1 * (3) (6) (6)
2.3 ± 0.66 3.3 ±0.25 3.0 ±0.58 Size of brood 2 (3) (4) (3)
Offspring body size 1.21 ±0.086 a 1.48 ±0.061 b i.42 ± o.o3 rb brood 1 (mm) * (3) (6) (6)
Offspring body size 1.24 ±0.030 1.31 ±0.081 1.48 ±0.133 brood 2 (mm) (2) (4) (3)
Offspring tailspine 3.42 ± 0.300a 4.37 ± 0.136 b 4.24 ±0.106" length brood 1 (mm) ** (3) (6) (6)
Offspring tailspine 4.11 ±0.406 4.25 ±0.161 4.69 ± 0.246 length brood 2 (mm) (2) (4) (3)
Offspring total length 4.63 ± 0.354 a 5.84 ± 0.173 b 5.66 ± 0.077 b brood 1 (mm) ** (3) (6) (6)
Offspring total length 5.35 ±0.197 5.56 ±0.322 6.17 ±0.177 brood 2 (mm) (2) (4) (3)
Time between broods 7.3 ±0.67 6.5 ± 0.29 6.0 ± 0.00 (d) (3) (4) (3)
Total # offspring 10 26 25 produced * significant at p< 0.05; ** significant atp< 0.005; *** significant atp< 0.001
118 Table 4.5. Bythotrephes' mean predation rates and estimates of mean biomass consumed (|Ag DM) over two-day intervals (± SE). Bythotrephes {n indicated in parentheses) were reared on a mixed-prey assemblage of differing densities in 400 ml at 21°C over 22 days. Mean feeding efficiencies ranged from 49% for both the medium and high food densities to 60% for the low food density.
Age of Mean predation rate (# prey organisms) Mean predation estimate (ng DM)
(d) Low Medium High Low Medium High (30 prey • 2 d"') (60 prey • 2 d'1) (90 prey • 2 d"1) (30 prey • 2 d"1) (60 prey • 2 d"1) (90 prey • 2 d"')
31.1 ±2.21 53.7 ± 2.16 208.1 ±31.45 400.0 ±31.84 643.8 ± 42.84 1-2 19.6 ± 1.29 (8) (9) (9) (8) (9) (9)
31.2 ± 2.19 40.2 ± 4.03 224.6 ± 18.02 377.3 ± 37.48 448.0 ±91.60 3-4 16.8 ±0.98 (8) (9) (9) (8) (9) (9)
25.9 ± 1.77 35.7 ±2.74 231.0 ±28.67 275.6 ±36.25 352.9 ±36.25 J D 18.6 ± 1.59(7) (9) (7) (7) (9) (7)
28.9 ±2.36 46.8 ± 6.00 149.5 ± 54.38 352.4 ±37.01 525.4 ± 62.06 7-8 15.3 ±2.36 (4) (8) (5) (4) (8) (5)
17.9 ±0.74 29.3 ± 1.09 44.3 ±2.15 210.3 ± 15.24 351.3 ±18.89 497.5 ± 37.40 Pooled mean (27) (35) (30) (27) (35) (30)
119 FIGURES
0.90 •
0.80 •
0.70 -
0.60 •
a 0.50 •
0.40 •
0.30 •
0.20 •
0.10 •
0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Day
Figure 4.1. Survivorship for Bythotrephes reared over 22 days in the laboratory at 21°C, and offered low (dotted line; n = 8), medium (dashed line; n- 9) and high (solid line; n = 9) food densities. One female was still alive after 22 days in the low food treatment.
120 a) A. franciscana naupiii b) a freyii
c) D. mendotae c) D. pulex
High
d) D. ambigua
Prey density
Figure 4.2. Predation rates by Bythotrephes (n = 7 - 8) at low, medium, and high prey densities (15, 30, and 45 prey"1' 0.4 L"1 ' d"1, respectively) over six days, separated in order of overall prey preference: a) A. franciscana naupiii, b) B. freyii, c) D. mendotae, d) D. pulex, and e) D. ambigua. Lines intersecting graphs represent grand means.
121 CHAPTER 5
EFFECTS OF EICOSAPENTAENOIC ACID (EPA) LIMITATION AND RISING
WATER TEMPERATURE ON THE AQUATIC INVADER BYTHOTREPHES
ABSTRACT
Bythotrephes longimanus is an invasive predatory cladoceran that has negatively affected indigenous biota in North American lakes, particularly on the Canadian Shield.
Food quality can have substantial impacts on consumers, but the impacts of prey quality on Bythotrephes population dynamics and establishment are currently unknown. Given recent changes in phytoplankton communities (and thus food quality) on the Shield and increasing summer water temperatures, I consider the influence of prey quality on
Bythotrephes size, survival and reproduction in a laboratory-based food chain at two temperatures. The chlorophyte Scenedesmus obliquus was cultured at 21°C and 26°C; in each case either with or without (controls) enrichment with the co3 fatty acid, eicosapentaenoic acid (EPA, 20:5co3). EPA-enriched or control S. obliquus was then fed to Daphnia ambigua, which were offered as prey to Bythotrephes. At 21°C, predation rates of young Bythotrephes on EPA-supplemented daphniids were much lower than those fed control daphniids, owing to the fact that daphniids reared on EPA-enriched S. obliquus were significantly larger (faster) than those fed control S. obliquus. Eleven-day- old Bythotrephes reared on the EPA-enriched daphniids had significantly larger clutch sizes than those consuming control daphniids, however. Both food treatments supported the ontogenesis, but not reproduction, of Bythotrephes. Comparisons of fatty acid profiles
122 suggest that the laboratory-reared Bythotrephes—even those consuming EPA- supplemented daphniids—were EPA-impoverished in comparison to field populations.
At 26°C, EPA enrichment did not appear to benefit daphniids or Bythotrephes, and there was no difference in predation rates by juvenile Bythotrephes. This is, to my knowledge, the first evidence that the availability of a single fatty acid may affect population growth, and hence establishment success, of an invasive species.
INTRODUCTION
The nutrient-poor softwater lakes of the Canadian Shield are currently in a state of flux due to manifold environmental stressors arising from climate warming (e.g.,
Hostetler & Small 1999), the spread and establishment of non-native species, and the interactions of these factors (Yan et al. 2008a). The zooplankton communities in these lakes have been influenced by a host of natural and human-induced abiotic and biotic drivers such as lake acidification (Yan et al. 20086), falling total phosphorus (TP) concentrations (Quinlan et al. 2008), changing phytoplankton assemblages (Paterson et al.
2008), and the spread of the Ponto-Caspian zooplanktivorous invader, Bythotrephes longimanus Leydig (1860; herein 'Bythotrephes', commonly known as the spiny water flea). Initially detected in Lake Ontario in the early 1980s (Johannsson et al. 1991),
Bythotrephes was soon after discovered in the remaining Laurentian Great Lakes (Bur et al. 1986; Lange & Cap 1986; Lehman 1987; Evans 1988; Cullis & Johnson 1988), with secondary invasions occurring inland in the Muskoka region north of Toronto, Ontario,
Canada, in the early 1990s (Yan et al. 1992). Bythotrephes was likely introduced to North 123 America from a source population in Lake Ladoga, Russia as a consequence of shipping activities (Berg et al. 2002). Its current distribution encompasses at least 170 lakes from south-central to north-western Ontario, and southern Manitoba, Canada and the north eastern United States (Yan et al. 2011; Kerfoot et al. 2011; Strecker et al. 2011; Justin
Shead, Manitoba Ministry of the Environment, personal communication). It was recently adopted as a model organism for studying the spread of pelagic invaders (see Yan et al.
2011, and references therein).
The direct, ecological repercussions of Bythotrephes introductions on native pelagic food webs are far-reaching. Documented impacts on indigenous zooplankton include declines in species diversity, plummeting species richness (30% lower in invaded versus non-invaded lakes), changes in abundance, and shifts in community size structure
(Dumitru et al. 2001; Yan et al. 2001; Boudreau & Yan 2003; Strecker et al. 2011).
Additionally, Bythotrephes appears to be displacing the native predatory macroinvertebrate Leptodora kindtii through competitive interactions (Branstrator 1995;
Foster & Sprules 2009; Weisz & Yan 2011). Avoidance of Bythotrephes by some predators such as young-of-the-year fishes (owing to Bythotrephes' morphological defenses; Compton & Kerfoot 2004) and competition for prey with planktivorous fishes may exacerbate Bythotrephes' negative impacts. Non-trivial indirect effects of
Bythotrephes on native food web dynamics have also been recognized (Pangle & Peacor
2006; Pangle et al. 2007; Strecker & Arnott 2008; Strecker et al. 2011). However, the research focus on Bythotrephes' direct, consumptive food web impacts emphasizes its reputation as a voracious predator. 124 Young et al. (2011) found that Bythotrephes abundances in Harp Lake, Ontario, may best be predicted by spring prey availability. Small, slow-moving prey with relatively slow escape speeds are at the highest risk of capture by Bythotrephes
(Muirhead & Sprules 2003; Pichlova-Ptachnikova & Vanderploeg 2011), and cladocerans such as Daphnia and Bosmina comprise Bythotrephes' principal prey items
(Vanderploeg et al. 1993; Burkhardt & Lehman 1994; Kim & Yan 2010). At 21°C under relatively high food conditions in the laboratory, Bythotrephes' mean predation rate is 22 prey organisms • d"1 (Chapter 4). Bythotrephes requires such a high consumption rate because it inhabits warm surface or metalimnetic waters (Young & Yan 2008), exhibits fast generation times (Kim & Yan 2010), swims constantly, is a wasteful feeder (i.e.,
38% of prey are lost to "sloppy feeding"), and has assimilation rates of < 60% relative to ingestion (Yurista & Schulz 1995; Yurista et al. 2010). Bythotrephes also allocates a large amount of food resources to offspring production, with its broodsac amassing more than 100% of its barren dry mass when occupied with several developing embryos
(Yurista & Schulz 1995). Cladocerans typically have high rates of reproduction (Allan &
Goulden 1980), but because of its high energetic allocation to offspring, Yurista and
Schulz (1995) suggested that Bythotrephes may well represent an "extreme" case of maternal investment. Thus, food availability is most certainly an important regulator of
Bythotrephes' population dynamics and its potential for successful establishment in novel habitats. This is partially due to the strong Allee effects which exist for this species; that is, a minimum number and sufficient quality of resting eggs are needed to guarantee persistence beyond initial colonist survival and reproduction (Wittmann et al. 2011). 125 All consumers must acquire, ingest, digest, and assimilate food items to obtain the requisite nutrients for the maintenance of vital metabolic processes, growth, and energy production and storage. While mineral limitation for zooplankton has been widely examined (e.g., phosphorus requirements for Daphnia', Sterner & Hessen 1994; Elser et al. 2001; Makino et al. 2002; Becker & Boersma 2003), lipids are quickly gaining recognition for their importance in the context of zooplankton nutrition. Lipids occur in various classes, but sterols and polyunsaturated fatty acids (PUFAs; refer to Table 5.1 for a complete list of abbreviations used in this article) have received the most attention.
One PUFA in particular, eicosapentaenoic acid (EPA, 20:5co3) is highly retained in freshwater cladocerans (Kainz et al. 2004; Persson & Vrede 2006; Ravet et al. 2010), a group that includes both Bythotrephes and Daphnia. EPA is found in the phospholipids
(PL)6 of cell membranes where it helps maintain fluidity, and from where it is continually mobilized to be incorporated into eggs, and serve as precursors for physiologically significant biochemicals such as eicosanoids, which include prostaglandins and leukotrienes (Smyntek et al. 2008). EPA availability has been strongly correlated with the somatic growth and fecundity of Daphnia in both field and laboratory settings (Muller-
Navarra et al. 2000; von Elert 2002; Becker & Boersma 2003; Wacker & Martin-
Creuzburg 2007). Wacker and Martin-Creuzburg (2007) observed that D. magna heavily incorporate EPA into its subitaneous eggs (2.4-fold higher than that invested in somatic tissues), suggesting that this FA is vital for reproduction. The incorporation of lipids in
6 EPA is also found in the triacylglyercols (TAG) but in lower proportions that the PL, depending on the diet of the animal. When diets are EPA-rich, there will be some EPA in TAG and even more in the PL. 126 eggs ensures that offspring will be equipped with the resources needed for initial growth and development, and buffers against starvation during the first day or two after parturition. This maternal lipid investment is important given that cladocerans provide no maternal care to their newly hatched offspring (Arts 1999).
Approximately 98% of FAs in Daphnia are derived from their algal diets rather than synthesized de novo (Goulden & Place 1990) and zooplankton lipid profiles tend to mirror that of their diets (Brett et al. 2006). Moreover, Fuschino et al. (2011) recently observed that the production of co3 FAs in Scenedesmus obliquus is inhibited by temperature increases consistent with global warming scenarios. Since the retention of long-chain PUFAs generally increases with trophic level (Persson & Vrede 2006), it is likely that Bythotrephes will also be impacted by differences in algal PUFA contents— benefiting if the quality and quantity of PUFA supplies are adequate, and suffering some negative effects if dietary PUFA supplies are inadequate.
There is very little information on the role of lipids for Bythotrephes population dynamics, including its establishment success. No studies have been conducted under controlled settings with a defined diet, and no attempt has yet been made to investigate whether a lack of essential fatty acids (EFAs) may be limiting for this species. What is known is that 10% - 19% of Bythotrephes dry mass (DM) in the Great Lakes is formed by total lipids (TL) (Bilkovic & Lehman 1997), and that TL concentrations fall from the first to third instars; a trend that reverses during the development of advanced-stage embryos. It has been hypothesized that Bythotrephes deplete lipids to promote rapid juvenile and young adult growth, and later allocate lipids to their developing embryos, 127 since the latter are nourished entirely from their mother's hemolymph and grow in broodsacs devoid of yolk (after Yurista 1992; Bilkovic & Lehman 1997). Bythotrephes accumulates relatively high proportions of EPA in TL. Smyntek and colleagues (2008) found that EPA was by far the most abundant PUFA in Bythotrephes collected from the
Great Lakes, comprising 38 |xg • mg organic C"1 (followed by arachidonic acid, ARA or
20:4co6 at 17 jAg EPA • mg org. C"1). In its native range, late summer Bythotrephes have been found to contain from 11% - 16% (in a Russian reservoir; Bychek & Gushina 2001) up to 23% EPA in TL (in oligotrophic, northwestern Swedish lakes; Persson & Vrede
2006). It is probable that Bythotrephes has very strong EPA requirements since these values are proportionally higher than those of Daphnia from the same lakes (Bychek &
Gushina 2001; Persson & Vrede 2006; Smyntek et al. 2008). As a predator, it is moreover likely that Bythotrephes is highly dependent on the FA contents of its prey to meet its needs (Brett et al. 2009).
The main focus of this study was to elucidate how EPA availability might impact
Bythotrephes' survival, size, and reproduction under a climate warming scenario.
Experiments were conducted under controlled laboratory conditions at two temperatures via a food-chain approach. The chlorophyte S. obliquus, unsupplemented or supplemented with EPA, was fed to the herbivorous zooplankter Daphnia ambigua, which was in turn offered as prey to Bythotrephes. To determine if EPA supplementation improved growth for the consumers in this study, D. ambigua and Bythotrephes sizes were measured. The predation rates of Bythotrephes over the first 24 h of the experiment
128 were also tracked. Where possible, the effects of EPA enrichment on Bythotrephes' clutch sizes were compared. Finally, FA profiles of Bythotrephes, D. ambigua, and S. obliquus were determined to verify that the manipulation of dietary EPA contents alone was responsible for the various observed effects in my test subjects. I provide evidence that the availability of a single FA, EPA, may well limit the spread of Bythotrephes on the Canadian Shield and beyond by affecting its ability to reproduce. In addition, I find that Daphnia ambigua may be capable of endogenous EPA synthesis (albeit at very low levels) from precursor molecules, and I confirm Fuschino et al.'s (2011) finding that the production of co3 FAs in S. obliquus is inhibited at increased temperature.
MATERIALS AND METHODS
Algal culture and enrichment with EPA
I batch-cultured the chlorophyte S. obliquus from pure starter cultures (Canadian
Phycological Culture Centre, Waterloo, Canada) in autoclaved Bold's Basal Medium
(BBM; modified from Stein 1973; see http://www.phvcol.ca/system/files/BBM for recipe) at both 21°C and 26°C, under 100 (xmol • m"2 • s"1 continuous lighting. Algal cell counts were performed daily using a hemacytometer (Bright-Line™, Cambridge
Instruments, Inc., Moatly, Everington, U.K., model Z359629) and algae were harvested in the log-phase of growth, at which stage cell counts consistently numbered ~107 cells • ml"1. The algae were well suspended in the BBM, then poured into 50 ml centrifuge tubes. I randomly selected half of the tubes from each batch of algae to enrich with EPA
(99% purity; Matreya, Pleasant Gap, U.S.A. via http://biolynx.ca in Canada, product 129 number MT1167) using a modified version of von Elert's (2002) protocol. Algae that were not enriched with EPA were subjected to the same procedures, minus the addition of EPA.
The steps for enrichment were as follows. Algae were concentrated down into a pellet at 1500 RPM for 7 min. From each tube of algae, 30 ml of BBM was removed.
Five ml of Bovine Serum Albumin (BSA; product number A7030; Sigma-Aldrich Co.,
Oakville, Canada) stock solution (3.5 g • L"1), and 400 nl of an EPA-EtOH stock solution
(2.5 mg • ml"1) were added to a clean glass beaker and gently swirled for several seconds.
The EPA-BSA mixture was then added to one tube of algae, which was capped tightly and inverted five times. The resulting suspensions were placed on a rotary shaker set at
100 rpm for 7 hours under approximately 100 |xmol • m"2 • s"1 cool white fluorescent lighting; temperature remained steady at 20°C - 22°C. After the incubation period, the algal cells were concentrated by centrifugation (1500 RPM for 7 min) and the supernatant discarded. To remove excess BSA and EPA-EtOH, the cells were washed twice in 20 ml of sterile BBM. Prior to daphniid feeding algae were re-suspended in FLAMES medium, the soft-water culture medium (Celis-Salgado et al. 2008) used to culture all zooplankton in this study. If a surplus of algae was treated (i.e., more than immediately required for feeding of daphniids), it was re-suspended in 20 ml of sterile BBM and stored at 4°C for a maximum of five days. Following each enrichment episode, the integrity of the algal cells was checked qualitatively by scanning under a compound microscope. Scenedesmus
130 obliquus treated with BSA only7 is designated the 'control' algae while that treated with the EPA-BSA complex is denoted the 'EPA-enriched' algae. EPA was detected in control and EPA-enriched S. obliquus at 0% and 12%, respectively, as proportions of TLs.
Prey cultures
All prey (D. ambigua and Bosmina freyii) were reared in FLAMES medium and housed in Conviron E2/7 growth chambers at either 21°C or 26°C under 100 nmol • m"
2 • s"1 cool white fluorescent lighting and a 14:10 L:D photoperiod. Cultures were fed every other day, transferred to clean FLAMES medium once a week, and covered with
Parafilm® to prevent evaporation and contamination. Daphnia ambigua cultures were initiated in 2 L beakers, and fed a uni-algal diet of S. obliquus ad libitum until one month prior to assays with Bythotrephes. At this time, half of the culture containers were randomly selected and switched to a diet of EPA-enriched S. obliquus. These daphniids are referred to as the 'EPA-enriched' daphniid prey whereas the cultures fed the control S. obliquus are considered the 'control' daphniid prey. EPA was detected in control and
EPA-enriched daphniids at 3% and 6%, respectively, as proportions of TL. Since cultures were fed ad libitum, I assume that variations in any of the parameters examined within a given temperature can be attributed to the differences in food quality. Bosmina freyii cultures (originating from local softwater lakes) were maintained in 1 L straight-sided borosilicate glass containers, and fed EPA-free S. obliquus ad libitum during the study.
7 No significant effects of BSA incubation on altering the FA profiles of S. obliquus have been observed (von Elert 2002). 131 Bythotrephes culture and assay conditions
The maternal generation of Bythotrephes (n = 143) bearing parthenogenic offspring to be used in experiments was collected from Fletcher Lake (45.3°N, 78.8°W) near Dorset, Ontario, Canada, on 12 September 2010 following the field collection protocol of Kim and Yan (2010). Field-collected Bythotrephes were brought back to the nearby Field Laboratory for the Assessment of Multiple Environmental Stressors
(FLAMES, after which the softwater culture medium is named), located at the Ontario
Ministry of the Environment's Dorset Environmental Science Centre in Dorset. The tubes containing Bythotrephes were randomly placed in one of the two growth chambers housing the prey cultures (i.e., set at 21°C or 26°C) and left to acclimate overnight. The following day, animals were transferred to 200 ml capacity glass mason jars previously filled with 175 ml of FLAMES medium incubated to the appropriate temperature.
Unenriched D. ambigua were offered to the Bythotrephes at rates of either 30 or 60 animals • d"1 depending on temperature (21°C or 26°C, respectively); pilot feeding trials indicated that Bythotrephes' predation rate approximately doubles going from 21°C to
26°C (N. Kim, unpublished data) and I wanted to avoid food limitation of Bythotrephes.
Unenriched Bosmina freyii were offered as a supplemental prey species at rates of 10 or
20 B. freyii • d"1, for Bythotrephes reared at 21°C and 26°, respectively.
After four days, adequate numbers of Bythotrephes neonates were attained, and immediately transferred to food treatments (n = 20 neonates • treatment"1). At 21°C, food treatments were the control and EPA-enriched diets, consisting of 30 mid-size D. ambigua (control or EPA-enriched depending on the treatment) and 10 B. freyii (reared 132 on EPA-free S. obliquus). At 26°C, the aforementioned prey densities were doubled. For each Bythotrephes mother, two of her parthenogenic neonates were retained, which were split between the food treatments (control or EPA-enriched daphniid prey) in an effort to minimize variability between treatments from maternal effects. Each day, Bythotrephes were visually assessed for survival, and then transferred to fresh FLAMES medium with new prey. Animals that were immobile for 10 or more seconds were defined as dead.
Contents of the jars previously housing Bythotrephes for the first 24 h of the experiment were examined thoroughly under a Leica MZ 12(5) dissecting microscope to determine approximate predation rates of neonates.
I terminated this study without waiting for natural mortality of the experimental animals, as the majority of the Bythotrephes' broodsacs began noticeably shrinking on the eleventh day at 21°C (Figure 5.1). Previous experiments indicate that parthenogenic
Bythotrephes normally reproduces at ten or eleven days of age at this temperature when it is not food-limited (Kim & Yan 2010). Bythotrephes' broodsacs were examined via dissecting microscope to determine numbers of distinct embryos being resorbed (as described and illustrated by Rivier 1998). Due to the premature termination of this assay,
Bythotrephes mortality could not be assessed.
Length measurements
Upon termination of the assay, Bythotrephes were measured (from the top of the head to the anus for body length, and from the anus to the distal tip of the caudal process for tailspine length, with the sum of these two measurements equaling total length) live
133 under a dissecting microscope with the aid of the NIS-Elements D 3.0 software (Nikon
Instruments, Inc., Melville, U.S.A.). Daphniids were also measured by making 'squash' preparations (one drop of water on a glass slide with a coverslip on top). For each experimental treatment, D. ambigua were sub-sampled via pipet from each culture jar, collected in a large Petri dish, gently stirred, and left to settle for a few minutes. From this pool, specimens were randomly selected until in excess of 100 daphniids had been measured. Daphniids were measured from the top of the head just above the eye to the base of the tail spine, and those with eggs or embryos were not included in the analyses.
Fatty acid analyses (see also Appendix E)
Algal, daphniid, and Bythotrephes tissues were prepared for FA analyses as follows. For every batch of S. obliquus fed to the daphniids, I randomly selected 4 tubes
• treatment"1 to concentrate into a pellet and analyze for FAs. Upon cessation of using daphniids as feed for Bythotrephes, remaining D. ambigua (of mixed ages and reproductive status) were transferred into clean FLAMES medium without food for 15 or
24 h, at 26 and 21°C, respectively (based on Fuschino et al.'s (2010) observations for D. magna)', these depuration times were verified in preliminary trials to be sufficient for the gut clearance of the much smaller D. ambigua. Daphniids from three to five culture jars were combined to in order to provide sufficiently large sample masses for lipid analyses.
Daphniids were concentrated onto a 113 [Am Nitex mesh filter, gently rinsed with
FLAMES medium, and transferred to 2 ml plastic cryovials (n = 5 replicates • treatment"1) which were placed in a Forma 8615 ultra-low temperature freezer (Thermo Fisher
134 Scientific Inc., Nepean, Ontario, Canada) at -86°C. Bythotrephes were starved for only a few hours (as a few specimens were near death at the termination of the study), rinsed with FLAMES medium, and then placed individually into cryovials. All samples were frozen at -86°C until the time of FA analyses.
Prior to extraction, tissue samples were completely freeze-dried and weighed on a microbalance. TLs were extracted from S. obliquus, D. ambigua, and Bythotrephes tissue samples by homogenizing in 2 ml of chloroform/methanol (2:1) using a homogenizer with a Teflon grinding pestle. Total lipid was prepared and measured gravimetrically using a modified version of Folch et al.'s (1957) methods (refer to Iverson et al. 2001 for modifications). Methylation of lipid samples was performed using a methanol/sulphuric acid methylation protocol from D. Tocher (University of Stirling, Scotland), which was modified from Christie (2003). Fatty acid methyl esters (FAMEs) were separated and quantified by Agilent 6890 gas chromatography with a 100 m x 0.25 mm x 0.2 (im capillary column (Supelco SP-2560) and flame ionization detector (FID). Helium was used as the carrier gas. Temperature ramping was from 70 to 140°C at 20°C ' min"1, held at 140°C for 5 min, increased to 170°C at 4°C ' min"1, and lastly to 240°C at 2°C " min"1, and held for 10 min for a total run time of 62 min. Individual FAMEs were identified by comparison to known standards.
To compare the FA profiles of wild Bythotrephes with those of the laboratory- reared animals, second and third instar Bythotrephes with unpigmented broodsacs
(Yurista 1992) were collected from nearby Mary Lake (45.3°N, 79.2°W) in Huntsville,
Ontario—a lake that has harbored Bythotrephes populations since 1990 (Yan et al. 1992).
135 Collections were made on 8 July 2010 using an 80 fim conical zooplankton net, and specimens were pipetted in 50 ml tubes filled with 20 jAm filtered lake water to the
Canadian Centre for Inland Waters in Burlington, Ontario, Canada where samples were immediately freeze-dried and analyzed for FA as per the procedure above.
Statistical analyses
Results are expressed as means plus or minus one standard error (SE) unless otherwise indicated. Two-tailed /-tests were applied for comparisons of daphniid and
Bythotrephes sizes, Bythotrephes predation rates, and Bythotrephes clutch sizes (two- tailed p-values are reported unless otherwise stated, a = 0.05). All count data (i.e., predation rate and clutch size data) were square root transformed prior to statistical testing to meet normality assumptions. All values of FAs present within each tissue sample (S. obliquus, D. ambigua, or Bythotrephes) represent proportions, and were first square-root arcsine transformed to meet normality assumptions. The two treatment means within a given temperature were then assessed using /-tests. I also compared the proportions of FAs in S. obliquus at 21°C and 26°C in the same manner. All statistical analyses were performed in JMP 8 (SAS Institute Inc., Cary, NC, 2009).
RESULTS
Impacts of EPA on Bythotrephes and D. ambigua
At 21°C, dietary EPA impacted non-primiparous daphniid size, which in turn affected juvenile Bythotrephes feeding ability and clutch size. D. ambigua reared on
136 EPA-enriched S. obliquus were significantly larger (0.98 ±0.012 mm, n = 192) than those reared on control S. obliquus (0.94 ±0.012 mm, n = 187; p = 0.015). This likely explains why the mean predation rate of juvenile Bythotrephes raised on EPA-enriched D. ambigua (12.5 ± 1.53 ind. • d"1, n - 15 observations) was significantly lower than those feeding on control daphniids (21.5 ± 1.09 ind. • d~!, n = 15 observations; p = 0.0004;
Figure 5.2). Bythotrephes consumed B.freyii at equal rates regardless of food treatment
(8.2 ± 0.56 and 7.9 ± 0.37 B.freyii' d*1 for the control and EPA-enriched food treatments, respectively) at 21°C.
Eleven-day-old Bythotrephes reared at 21°C on the EPA-enriched diet had significantly higher numbers of embryos within their broodsacs (2.3 ± 0.29, n - 9
Bythotrephes) than those maintained on the control diet (1.5 ± 0.16, n = 11 Bythotrephes; p - 0.015; see also Figure 5.3). Eleven-day-old Bythotrephes (n - 14 • treatment"1) fed the EPA-enriched daphniids tended to be larger than those fed the control daphniids in terms of body (2.45 ± 0.067 mm versus 2.36 ± 0.047 mm), tailspine (6.27 ±0.181 mm versus 6.23 ±0.163 mm), and total (8.72 ± 0.225 mm versus 8.59 ±0.192 mm) lengths.
Bythotrephes reared on control versus EPA-enriched daphniids also tended to be heavier
(113.1 jig ± 0.02 jig versus 92.3 ± 0.01 ng DM; 2 replicates of 7 - 8 Bythotrephes). None of these differences were statistically significant however (p-values = 0.281, 0.860, 0.656, and 0.437, respectively).
At 26°C, dietary EPA did not confer any advantages to D. ambigua or
Bythotrephes. There was no impact of EPA supplementation on daphniid size (1.02 ±
0.015 mm for D. ambigua feeding on control S. obliquus, n = 108, and 1.03 ±0.012 mm, 137 n= 158, for D. ambigua reared on EPA-enriched S. obliquus; p = 0.538). Moreover, there was no effect of EPA availability on juvenile Bythotrephes predation rates, which were virtually identical at 45.4 ± 1.01 (n = 14 observations) and 45.5 ± 1.09 (n = 15 observations) Daphnia • d"1 for the animals fed control and EPA-enriched daphniids, respectively. Finally, only 20% and 5% of Bythotrephes individuals fed the control and
EPA-enriched D. ambigua, respectively, reached reproductive maturity, or the third instar stage. No Bythotrephes survived to reproduce at 26°C however—by days 8 and 11, mortality rates were -50% and 100%, respectively.
Fatty acid patterns in Bythotrephes, D. ambigua, and S. obliquus
Bythotrephes longimanus
There were no significant impacts of diet on any of the FAs examined in
Bythotrephes reared in the laboratory at 21°C (Table 5.2). There was a higher relative proportion of EPA measured in the Bythotrephes fed the EPA-enriched daphniids (7.6%
EPA as TL) compared with those fed control daphniids (4.8% EPA as TL), but this difference was not statistically significant (p - 0.500, n = 2 replicates of 7 - 8 animals).
The dominant FA in all laboratory-reared Bythotrephes was the SAFA, 16:0 (palmitic acid), at -20% of TL—followed by ARA, 18:1 co9 (oleic acid), 18:3co3 (a-linolenic acid,
ALA), and 18:0 (stearic acid), each contributing ~10% to the TL pool. In contrast, the most abundant FA measured in wild-caught Mary Lake and Swedish Bythotrephes was
EPA at -23%, followed by 16:0, then ARA or 18:lto9. The SAFA 18:0 was approximately twice as high in all laboratory-reared and Mary Lake Bythotrephes than in
138 Swedish Bythotrephes, however. Although I did not statistically compare relative proportions of the laboratory-reared and wild-caught Bythotrephes, there was overall less
18:2G)6 (linoleic acid, LIN), and ALA in the wild-caught animals. There were no significant effects of diet on the FA functional groups of laboratory-reared Bythotrephes
(Table 5.3). It is clear, however, that wild-caught animals generally had a higher
X&)3:£(d6, as well as less £SAFA and more £MUFA. Swedish Bythotrephes had the highest ]Tco3:Xco6, followed by Mary Lake Bythotrephes, and all laboratory-reared specimens (with respective ratios of 2.6, 1.6, and 1.2).
Daphnia ambigua
In general, the FA profiles of D. ambigua resembled that of their algal diets, with
ALA observed to be the dominant FA across all temperatures and treatments, followed by
16:0, 18:la)9, and LIN (Table 5.4). Not surprisingly, there were significantly higher proportions of EPA in D. ambigua fed the EPA-enriched S. obliquus than in daphniids fed control S. obliquus at both 21°C (6.1% versus 3.0% EPA as TL; p < 0.0001), and
26°C (4.9% versus 1.8% EPA as TL; p < 0.0001). At both experimental temperatures, small amounts of EPA (~2% - 3%) were also detected in daphniids that were fed control
S. obliquus, suggesting that D. ambigua likely possesses some capacity for de novo synthesis of this FA (as has been seen with D. pulex fed green algae with undetectable levels of EPA; Schlechtriem et al. 2006). In addition, ARA had been detected in only trace amounts in S. obliquus (0% - 0.16% of TL) but was found to make up 5.5% - 9.5% of TL in D. ambigua, suggesting it is preferentially accumulated. At 21°C, there was
139 significantly higher £cfl3 (p = 0.002) and concurrently lower ]Tco6 (p < 0.001) in the
EPA-enriched daphniids, leading to a significantly higher £g)3:£(d6 in the EPA-enriched versus the control daphniids (1.7 compared with ll;/7 < 0 .001; Table 5.5). There was also significantly less £PUFA in the EPA-enriched daphniids (p - 0.021), which is due to the lower amount of £co3 + £co6 relative to £SAFA. At 26°C, the only difference among treatments in the D. ambigua FA functional groups was in the proportion of £(o6 FAs, which was significantly lower in the EPA-enriched daphniids [p < 0.001), likely due to the slight increase in Xco3s, which may be attributed to the higher amount of EPA.
Scenedesmus obliquus
Control S. obliquus was associated with little to no EPA at both 21°C and 26°C
(Table 5.6). My modified version of von Elert's (2002) EPA enrichment procedure proved effective at both temperatures, with significantly higher proportions of EPA detected as TLs in EPA-enriched S. obliquus relative to control S. obliquus (12% compared with 0% at 21°C,p < 0.001; 8.9% compared with 0.1% at 26°C,p < 0.001).
The relative proportions of EPA attained in the TL pool of EPA-enriched S. obliquus
(12% and 9%, at 21°C and 26°C, respectively) appear to be realistic, although they slightly exceed the ranges of EPA as TL measured in the natural seston of water bodies in
Bythotrephes native range (e.g., up to 4% as TL in a Russian reservoir, Bychek &
Gushina 2001; 3% in oligotrophic subalpine Swedish lakes, Persson & Vrede 2006).
ALA was by far the most abundant FA in all S. obliquus samples, regardless of culture temperature or enrichment status. Other dominant FAs included 16:0, LIN, 18:1 o>9, and
140 18:4a)3 (stearidonic acid, SDA). As seen commonly with other species of green algae
(Ahlgren al. 1990), the dominant SAFA in all S. obliquus reared in this study was 16:0.
There were no differences in the proportions of FAs as TL in S. obliquus grown at 26°C, except for EPA, which was present in the enriched algae but detected in only trace amounts in the control algae.
Culture temperature influenced the proportion of FAs in control S. obliquus {n = 5 replicates • temperature"1), as S. obliquus grown at 21°C had significantly more ALA as
TL (p - 0.019) than S. obliquus grown at 26°C. Conversely, S. obliquus grown at 26°C had significantly higher proportions of LIN (p < 0.001) and GLA (p = 0.030) as TL than
S. obliquus reared at 21°C.
Generally, £PUFAs were the dominant FA functional group of S. obliquus, followed by £SAFA and ]TMUFA (Table 5.7). At 21°C, the proportion of £co3 FAs was lower (p = 0.050) in control than in EPA-enriched S. obliquus (53.5% compared with
58.6%), which is expected given that the latter group contains -12% EPA as TL. In EPA- enriched S. obliquus, £SAFA was lower, concomitant with the increase in £PUFAs, while the reverse was seen for control S. obliquus. At 26°C, there were no significant differences in the FA functional groups between control and EPA-enriched S. obliquus.
I did, however, observe a significant effect of culture temperature on the proportions of £&)3 FAs {p = 0.021) in control S. obliquus, attributable mainly to the differences in ALA, LIN, and GLA noted previously. There were significantly lower mean proportions of £ £MUFAs, or £PUFAs in S. obliquus. DISCUSSION At 21 °C, Bythotrephes reared on EPA-enriched daphniids had higher proportions of EPA as TL than Bythotrephes fed control daphniids (7.6% compared with 4.8%, respectively) but the lack of a statistically significant result may be due to: i) inadequate statistical power, as I had only 2 replicates of Bythotrephes for FA analyses, and ii) the observation that Bythotrephes offered EPA-enriched daphniids were consuming less prey throughout the experiment than those fed control daphniids, at least as juveniles. After all, control and EPA-enriched daphniids contained 3.0% and 6.1% EPA as proportions of TL, respectively, and this difference was highly statistically significant. Despite this, EPA availability appeared to impact Bythotrephes parthenogenic clutch sizes. For Bythotrephes kept under optimal food conditions in the past, clutch sizes averaged 2.2 offspring (n = 6; Chapter 3). In this experiment the mean clutch size of the EPA-enriched Bythotrephes averaged 2.3 offspring (n = 9; on par with previous results), whereas that of the control Bythotrephes (1.5 offspring, n = 11) was closer to what was observed when Bythotrephes lacked sufficient food quantity (1.7 offspring; n = 3; Chapter 4). A clutch size of 2 - 4 offspring is typical for Bythotrephes in my study region in late summer (Young 2008). At 21°C, EPA limitation also appears to impact Bythotrephes' body size, but further study is required to confirm this. 142 At 21°C, EPA confers benefits to D. ambigua by way of increased body size. In a controlled study, von Elert (2002) demonstrated improved growth of juvenile D. galeata when reared on EPA-enriched S. obliquus (as well as S. obliquus supplemented with either ALA or DHA; D. galeata appears to have at least some ability to convert these FAs, as well as Ci6-PUFAs, to EPA). Because larger daphniids also tend to be faster swimmers (Dodson & Ramcharan 1991), increases in daphniid size and presumably swimming speed most likely account for juvenile Bythotrephes' reduced predation rates on the EPA-enriched daphniids. In an earlier trial of this experiment that was performed in the same manner as the present test but with lower prey quantity (25 D. ambigua • d"1 and no bosminids), Bythotrephes fed the EPA-enriched daphniids succumbed earlier to higher mortality than those reared on the control daphniids (N. Kim unpublished data). These results suggest that, at sufficiently low prey densities, Bythotrephes may actually be hindered by the presence of strictly high quality (EPA-rich) prey as opposed to lower quality (EPA-poor) prey, if they are unable to capture them in adequate quantities. Well- nourished, larger-bodied daphniid species might then be even less vulnerable to Bythotrephes, given that the D. ambigua used here are amongst the smallest of daphniids. Additional benefits conferred to larger-bodied crustacean zooplankton—both intra- and inter-specifically—include enhanced feeding rates (for cladocerans), lower starvation thresholds, and higher reproductive potential (Brooks & Dodson 1965; Hart & Bychek 2011); advantages that likely ensue when there is increased EPA availability. The most likely explanation for Bythotrephes' resorption of parthenogenic embyros at 21°C is that laboratory-reared animals were generally lacking EPA. After all, 143 the relative EPA levels in the laboratory-reared Bythotrephes were much lower than any of the published values for wild Bythotrephes in Europe, which range from ~11% - 23% as TL (Bychek & Gushina 2001; Persson & Vrede 2006; this study). Rivier (1998) describes the phenomenon of embryo loss and resorption for Bythotrephes populations in Russia's Rybinsk Reservoir. Late summer embryo loss was observed in 36% of all fecund females, with mean inferred clutch sizes of 3; approximately half of the embryos were resorbed in the specimens examined, with loss and resorption taking place at the stage of cephalic segment delimitation and the beginning of swimming antennae development for reasons that are unclear but may be due to eutrophication and chemical contamination (Rivier 1998). A Xco3:]T(jo6 of 1.2 is probably also suboptimal for the successful reproduction of Bythotrephes at 21°C. Wild-caught Bythotrephes from subalpine oligotrophic lakes had the highest ]T FA precursor's desaturation activity (reviewed in Ahlgren et al. 2009). Because ARA was not deficient in the laboratory-reared Bythotrephes (when compared with wild-caught animals) it is possible that endogenous conversions of the shorter chain ALA into EPA— if it even occurs in Bythotrephes—was inhibited. As with D. ambigua, ARA was accumulated in Bythotrephes, which indicates it as an important EFA, but the relative 144 proportions of ARA in laboratory-reared versus wild-caught Bythotrephes were not largely different, so it was most likely not limiting. The physiological importance of ARA in zooplankton remains to be clarified (Kainz et al. 2004). An alternate explanation for the lack of reproduction by Bythotrephes at 21°C is insufficient food quantity. I deliberately biased the prey density to be low, but did not anticipate that Bythotrephes would exhibit differential predation rates on the control versus EPA-enriched prey. The results of food quantity experiments (Chapter 4) indicate that Bythotrephes will be compromised when daily consumption rates are between 105 |xg DM d"1 and 175 fig DM' d"1 when fed a mixed cladoceran prey assemblage reared on chlorophytes along with Artemia franciscana nauplii. In the present experiment, Bythotrephes were offered a relatively low level of food (~230 jug • DM • d ' ), of which I estimate—based on juvenile Bythotrephes predation rates—consumption was -168 Hg • DM • d"1 in the control treatment and ~98 ng • DM • d"1 in the EPA-enriched treatment. These results highlight Bythotrephes'' need to access a diverse prey assemblage. Past work with Bythotrephes in the laboratory revealed that Artemia nauplii are often selected as prey by captive Bythotrephes (Kim & Yan 2010; Chapter 4). Artemia nauplii are known to contain some EPA (~2%) and high amounts of ALA (-27%) in TL (Furuita et al. 1996). Artemia are clearly not a naturally encountered prey item for Bythotrephes, but other sources of EPA may be present in situ to support reproduction, such as copepod nauplii (which Bythotrephes readily consumes; Vanderploeg et al. 1993) and bosminids, particularly after grazing on EFA-rich phytoplankton. Freshwater copepods are known to 145 contain high amounts of ALA, EPA and DHA, with high £g>3:Xco6 ranging from 4.2 to 5.2 (Brett et al. 2009, and references therein). Unlike daphniids, bosminids and copepods also possess the ability to feed preferentially (Brett 1993), and thus may represent a higher quality food source for Bythotrephes. As bosminids and copepod nauplii are also much smaller and slower than daphniids, Bythotrephes can easily capture them, and probably expends less energy in the process. In a review of Bythotrephes ecology in its native range, Grigorovich and colleagues (1998) mention that Bythotrephes can also feed directly on phytoplankton such as the dinoflagellate Peridinium (which contains high levels of EPA as well as DHA; Ahlgren et al. 1990; Ahlgren et al. 1992) and diatoms. While widely considered an obligate carnivore, Bythotrephes probably ends up consuming small amounts of phytoplankton directly as well as indirectly via the gut contents of its prey. At 26°C, Bythotrephes exhibited very poor survival on the predominantly daphniid diet, with only 20 and 5% of test animals (fed control and EPA-enriched prey, respectively) reaching reproductive maturity, and none surviving to reproduce. Bythotrephes is typically considered a cold stenotherm, with respiratory enzymes deactivated at 23°C (Yurista 1999). Previous laboratory work indicates that Bythotrephes has an upper thermal threshold between 25°C and 28°C (Kim & Yan 2010). I included this temperature treatment because it is ecologically relevant; small lakes in my study region of Muskoka, where Bythotrephes continues to spread, often exceed 26°C at the surface in mid-summer (Appendix F). Furthermore, Kim and Yan (2010) have previously cultured Bythotrephes successfully at 25°C. It is possible that I would have 146 observed greater impacts of EPA availability on both daphniids and Bythotrephes had I selected lower experimental temperatures. At 26°C, a diet of EPA-enriched S. obliquus—at the level offered in the assay— did not benefit D. ambigua in terms of body size or escape ability from Bythotrephes. Besides the overall decline in algal EFAs with increasing temperature, it is possible that sterols (as opposed to EPA), became limiting for the daphniids at the higher temperatures. As well as comprising a structural part of the cell membrane (Goad 1981), cholesterol, for example, serves as a precursor for steroid hormones including ecdysteroids, which are required for moulting by crustaceans (Lachaise et al. 1993). In daphniids, a lack of dietary sterols can manifest as declines in somatic and population growth, numbers of viable offspring, and survival (Martin-Creuzburg et al. 2005). Wacker and Martin- Creuzburg (2007) provide evidence that cholesterol is critical for daphniid somatic growth, whereas PUFAs are more important for reproduction. Finally, at higher temperatures, increasing levels of cholesterol are required to ensure optimum membrane fluidity (Sperfeld & Wacker 2009). I did not measure the sterol contents of D. ambigua or S. obliquus, although S. obliquus is not considered to be sterol-deficient (Martin- Creuzburg et al. 2005). If EPA is required for Bythotrephes reproduction—and possibly, somatic growth—as my study suggests, a lack of this single FA can have several repercussions. EPA is clearly important for cladocerans, but it will likely become more limiting in the future owing to changes in phytoplankton FA composition as a result of climate warming (Fuschino et al. 2011). In general, S. obliquus is a depauperate source of EPA for 147 consumers, whether cultivated at 21°C or 26°C. The molecular precursor to EPA, ALA, was the most abundant FA in S. obliquus, but ALA levels declined with the 5°C increase in culture temperature. Fuschino and colleagues (2011) reported similar patterns when rearing S. obliquus (the same CPCC strain as the one used in my study) at 20°C and 28°C; they also found a decrease in to3 PUFAs (53% compared with 37% in TL) with increasing temperature. This was due mainly to a decrease in ALA and a concomitant increase in SAFAs at 28°C (Fuschino et al. 2011). Likewise, I observed a decrease in co3 PUFAs in S. obliquus grown at 26 and 21°C (54% compared with 45% as TL), with most of that also attributable to a decrease in ALA (45% compared with 37% as TL). EPA may also become limiting due to the widespread eutrophication of natural waters leading to increased cyanobacteria dominance (Downing et al. 2001). Of the phytoplankton groups, diatoms, dinoflagellates, and cryptophytes typically contain the highest amounts of EPA, whereas chlorophytes have only trace amounts, and cyanobacteria produce little to no EPA (Ahlgren et al. 1990; Ahlgren et al. 1992; Gulati & DeMott 1997; Fuschino et al. 2011; this study). In waters receiving increased nutrient inputs, we expect to see more frequent incidences of eutrophication and cyanobacterial blooms, with consequent reductions in algal EPA anticipated. Although TP levels are declining in Ontario and in much of the developed world, control of eutrophication is not yet universal. Currently, the relative biovolume of diatoms is declining in Muskoka, Ontario, while colonial chrysophytes appear to be on the rise, for reasons related to multiple anthropogenically-driven stressors (Paterson et al. 2008). The FA contents of 148 chrysophytes common to my study region are not yet well known, but there is evidence that the commonly occurring chrysophyte Synura petersenii contains no EPA, although 22:6c)3 (docosahexaenoic acid, DHA) is present in relatively notable amounts (Liam T. Quinn, York University, personal communication). In Lake Huron, diatom biovolumes have declined in recent years, and a corresponding drop has been seen in cladoceran abundances, along with steep declines in daphniid and cyclopoid copepod egg production (Barbiero et al. 2011). These bottom-up forces may affect Bythotrephes as well. Bythotrephes' future establishment will likely be limited in those lakes impacted by eutrophication (and thus dominated by EPA-deficient phytoplankton such as cyanobacteria), and/or lacking EPA-retentive prey. It has been repeatedly observed that Bythotrephes abundances decrease or populations disappear altogether in European lakes that become eutrophic (reviewed in Therriault et al. 2002; see references therein). Although other drivers likely contribute (e.g., planktivory; Jeppeson et al. 1996), I propose that EPA limitation may also play a role. This may also in part explain why Bythotrephes tends to be absent from very productive systems in its native Norwegian range, unlike L. kindtii (Hessen et al. 2011), which appears to have much lower EPA requirements (Bychek & Gushina 2001). Bythotrephes establishment will also likely be affected by climate change. Not only does algal food quality tend to decline with increased temperature (Fuschino et al. 2011; this study), Bythotrephes is clearly sensitive to warmer water temperatures but in stratified lakes it is constrained to the upper, warmer layers due to its reliance on light for feeding (Pangle & Peacor 2009; Young et al. 2011). 149 Brown and Branstrator (2011) recently highlighted Bythotrephes' need to establish a viable resting egg bank for the inter-annual persistence of populations. These authors also found that heavier (and presumably, resource-rich) Bythotrephes mothers were able to manufacture resting eggs that had an increased likelihood of hatching the next spring (Brown & Branstrator 2011). For D. pulicaria fed S. obliquus (without and with EPA additions) as well as EPA-rich Cryptomonas, Abrusan et al. (2007) found that resting egg production was highly positively influenced by EPA availability, and that mothers invested much more EPA into resting than subitaneous eggs. Sperfeld and Wacker (2012) also observed a greater investment of PUFAs in eggs relative to somatic tissue, and found increased hatching success of D. magna eggs at 20°C when mothers were supplemented with EPA compared to when they were not. Whether this is true for Bythotrephes should be tested in the future. Owing to the sexually reproductive phase in its life cycle, Bythotrephes is not immune to Allee effects, and must maintain sufficient numbers to persist (Wittman et al. 2011). In conclusion, the availability of EPA—originating from phytoplankton and cascading upward through herbivorous zooplankton—impacts Bythotrephes, and requires further investigation. It is plausible that this single FA could ultimately contribute to the success or failure of new invasions, a hypothesis that should be more closely examined. This may be accomplished with further assays of the type presented here, but with modifications to the diet and under a broader range of treatment conditions (i.e., additional temperatures, dietary EPA concentrations). For Bythotrephes-imaded systems supporting economically viable sport fisheries, resource managers may be interested in 150 examining how this zooplanktivore, with its high EPA demand, may impact the nutritional needs/output of potential or known fish predators. ACKNOWLEDGEMENTS I thank the Canadian Aquatic Invasive Species Network (CAISN), the Natural Sciences and Engineering Council of Canada (NSERC), Environment Canada and the York Faculty of Graduate Studies for funding this project. Field and laboratory support for conducting the experiments were provided by S. Hung and staff at the Ontario Ministry of the Environment's Dorset Environmental Science Centre. Fatty acid analyses were performed by M. Rudy and J. Chao of Environment Canada. I am indebted to M.T. 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Abbreviation Full or common name Structure FA(s) Fatty acid(s) - 8 EFA(s) Essential fatty acid(s) - SAFA(s) Saturated fatty acid(s) - MUFA(s) Monounsaturated fatty acid(s) - PUFA(s) Polyunsaturated fatty acid(s) - FAME(s) Fatty acid methyl ester(s) - LIN Linoleic acid 18:2co6 GLA y-linolenic acid 18:3co6 ALA a-linolenic acid 18:3GO3 SDA Stearidonic acid 18:4co3 ARA Arachidonic acid 20:4w6 EPA Eicosapentaenoic acid 20:5co3 DPA co3-docosapentaenoic acid 22:5co3 DHA Docosahexaenoic acid 22:6co3 X«3 Total co3 fatty acids - Xo>6 Total co6 fatty acid - 8 Those FAs deemed "essential" are those that cannot be synthesized by organisms endogenously. Rather, these FAs must be obtained strictly via consumption. 161 Table 5.2. Mean proportions (as % of FAs) of selected FAs in Bythotrephes reared without and with EPA addition at 21°C, ± 1 SD (n.d. = not detected). Also shown are mean proportions of fatty acids in Bythotrephes retrieved from Mary Lake, Huntsville, Ontario, Canada. There were 2 sample replicates of lab-reared Bythotrephes consisting of 7-8 individuals each, and 5 sample replicates of Mary Lake-caught Bythotrephes, consisting of 10 - 19 individuals each. Also included for reference are data from Persson and Vrede (2006) for Bythotrephes collected from four oligotrophic lakes in northwestern Sweden (n = 2-4 replicates total). There were no significant effects of EPA enrichment on any of the FAs in the laboratory-reared Bythotrephes (a - 0.05). Lab-reared Wild-caught Lab-reared Wild-caught Bythotrephes Bythotrephes - Common Bythotrephes Bythotrephes Fatty acid -EPA- Swedish lakes, name - Control - Mary enriched n = 4 lakes *'b diet (%) Lake" (%) diet (%) (%) 14:0 Myristic acid 2.7 ±0.53 3.2 ± 1.18 2.4 ±0.41 4.4 ±0.7 16:0 Palmitic acid 20.8 ± 2.05 20.8 ± 1.01 17.0 ± 1.78 19.1 ± 1.5 16:1 co7 Palmitoleic acid 1.9 ±0.03 2.4 ±0.41 1.4 ±0.09 2.9 ±0.9 17:0 Margaric acid 1.0 ± 0.16 0.9 ± 0.08 1.7 ± 0.16 0.9 ±0.1 18:0 Stearic acid 10.8 ± 1.28 9.7 ±0.59 8.3 ±2.37 4.7 ±0.7 (Included with 18:1 a)9t Elaidic acid 0.2 ± 0.28 0.4 ± 0.07 1.2 ±0.59 FA below) 18:lco9c Oleic acid 10.2 ±0.57 11.1 ±0.04 10.6 ± 1.36 10.3 ± 1.4 (No common 18:1 co7 name) 5.1 ±0.35 5.7 ±0.24 6.3 ± 0.62 6.0 ±0.5 18:2co6 Linoleic acid 6.5 ±0.69 6.4 ±0.42 3.0 ±0.44 3.6 ±0.6 (LIN) 18:3co3 a-Linolenic acid 10.4 ±0.85 10.3 ±0.62 3.4 ±0.54 5.3 ± 1.7 (ALA) 18:4w3 Stearidonic acid 1.7 ± 0.10 1.7 ± 0.16 1.7 ±0.36 4.1 ± 1.5 20:4oo6 Arachidonic acid 11.5 ± 1.14 10.4 ±0.89 13.0 ± 1.98 9.3 ± 1.4 (ARA) 20:5co3 Eicosapentaenoic 4.8 ± 0.60 7.6 ± 0.38 23.3 ± 3.04 23.0 ± 1.4 (EPA) acid 22:5ce>3 " Wild-caught Bythotrephes were excluded from statistical analyses, as other variables (e.g., temperature, light regime) may affect the differences in FA levels. b Data from Table 4 in Persson & Vrede (2006); percentages of individual FAs do not sum exactly 100% because there was -2% unidentified, and traces (< 1%) of FAs are not included here. 162 Table 5.3. Mean proportions of FA functional groups (as % of FAs) considered in this analysis, and £&>3:£a)6 for 11-day-old Bythotrephes reared at 21°C in the laboratory and fed prey cultured on S. obliquus without and with EPA additions, ± SD. For reference purposes, data are also shown for wild-caught Bythotrephes from Mary Lake.There were 2 sample replicates of lab-reared Bythotrephes consisting of 7 - 8 individuals each, and 5 sample replicates of wild-caught Bythotrephes, consisting of 10 - 19 individuals each. There were no significant differences between treatments for Bythotrephes reared in the laboratory. Lab-reared Wild-caught Wild-caught Lab-reared Bythotrephes - Bythotrephes Bythotrephes - Bythotrephes EPA-enriched Swedish lakes, Control diet diet n = 4 lakesb £co3(%) 25.7 ±0.81 25.2 ±0.20 26.3 ±0.66 34.4 ±2.0 I«6(%) 18.6 ±0.06 17.5 ±0.04 17.0 ±0.53 13.5 ±1.0 2>3:£co6 1.2 ±0.27 1.2 ±0.16 1.6 ±0.04 2.6 ±0.3 ISAFA 37.2 ±0.21 36.6 ± 0.09 26.3 ±0.60 30.8 ±1.8 XMUFA 18.3 ±0.04 20.7 ±0.01 30.4 ±0.61 19.2 ±1.1 XPUFA 44.5 ± 0.36 42.8 ± 0.06 43.4 ± 1.03 47.9 ±1.0 " Wild-caught Bythotrephes were excluded from statistical analyses. bData from Table 4 in Persson & Vrede (2006). 163 Table 5.4. Mean proportions (as % of total FAs) of selected FAs in D. ambigua fed the chlorophyte S. obliquus treated without and with EPA, at 21°C and 26°C, ± SD (n.d. = not detected). There were 5 sample replicates for each treatment. Asterisks (*) indicate significantly different treatment means within a given temperature (a = 0.05). 21°C 26°C Algal diet EPA- EPA- Fatty Control (%) enriched Control (%) enriched acid (%) (%) 14:0 Myristic acid 1.9 ±0.44 1.9 ± 0.19 2.5 ± 0.28 2.1 ±0.22 16:0 Palmitic acid 15.8 ±0.43 15.9 ±0.26 17.3 ±0.32 17.0 ± 0.19 16:loo7 Palmitoleic acid 2.7 ±0.12* 2.5 ± 0.11* 2.6 ±0.12* 2.7 ± 0.11* 17:0 Margaric acid 1.0 ±0.08* 0.9 ± 0.03* 0.8 ± 0.05 0.8 ± 0.05 18:0 Stearic acid 7.3 ± 0.36 7.1 ±0.14 6.1 ±0.69 6.3 ±0.26 18:1co9 Oleic acid 12.5 ±0.79 12.3 ±0.35 11.1 ±0.74* 13.0 ±0.24* (No common 18:10)7 8.6 ±0.30* 9.3 ±0.18* 8.3 ±0.36 8.4 ±0.12 name) 18:2(o6 Linoleic acid 10.6 ±0.65* 8.5 ± 0.20* 11.2 ±0.48* 10.1 ±0.12* (LIN) 18:3G)6 y-Linolenic acid 1.3 ±0.19* 1.0 ± 0.11* 1.3 ± 0.10* 1.1 ±0.10* (GLA) 18:3(i)3 a-Linolenic acid 17.1 ±0.63* 18.9 ±0.45* 21.3 ± 1.66 19.6 ±0.74 (ALA) 18:4a)3 Stearidonic acid 3.2 ±0.39 2.7 ±0.36 3.6 ±0.53* 2.9 ±0.16* (SDA) 20:4(o6 Arachidonic acid 9.5 ±0.61* 6.7 ±0.39* 6.0 ± 0.90 5.5 ±0.37 (ARA) 20:5(i)3 Eicosapentaenoic 3.0 ±0.19* 6.1 ±0.31* 1.8 ±0.23* 4.9 ±0.15* (EPA) acid 22:5o)3 co3- 1.3 ± 1.29 1.0 ±0.39 0.8 ±0.78 0.4 ± 0.49 (DPA) Docosapentaenoic acid 164 Table 5.5. Mean proportions of FA functional groups (as % of total FAs) considered in this analysis, and Xco3:Xco6 for D. ambigua fed the chlorophyte S. obliquus treated without and with EPA, at 21°C and 26°C, ± SD. There were 5 sample replicates in each treatment. Asterisks (*) indicate significantly different treatment means within a given temperature (a = 0.05). 21°C 26°C Algal diet Control EPA-enriched Control EPA-enriched I«3 (%) 25.0 ± 1.21 * 29.0 ±0.57* 27.9 ± 1.57 28.2 ± 0.68 £ XSAFA (%) 28.9 ± 0.93 29.4 ± 0.56 30.5 ± 0.75 29.8 ± 0.43 XMUFA (%) 24.2 ± 0.78 24.9 ±0.51 22.5 ±0.51 24.8 ±0.13 XPUFA(%) 47.0 ± 0.64 * 45.7 ±0.25 * 46.9 ± 1.16 45.4 ±0.37 165 Table 5.6. Mean proportions (as % of total FAs) of selected FAs in S. obliquus without and with EPA addition, at 21°C and 26°C, ± SD (n.d. = not detected). There were 5 sample replicates for each treatment. A single asterisk (*) indicates significantly different treatment means within a given temperature, while double asterisks (**) denote significantly different means for control S. obliquus between temperatures (a = 0.05). 21°C 26°C EPA- EPA- Fatty Common Control enriched Control (%) enriched acid (%) name (%) (%) 16:0 Palmitic acid 17.4 ± 1.65* 14.8 ± 1.13* 19.1 ±2.46 17.2 ± 1.66 16:1 co7 Palmitoleic acid 1.0 ±0.21 0.9 ±0.27 1.0 ± 0.12 0.9 ±0.12 18:0 Stearic acid 1.9 ±0.60 1.9 ±0.73 2.1 ± 1.23 1.6 ±0.20 18:l(o9 Oleic acid 7.5 ±0.97 6.7 ±0.84 7.7 ±2.27 6.8 ± 1.49 (No common 18:1 co7 4.4 ±0.88 4.2 ±0.36 5.1 ±0.64 5.4 ± 0.87 name) 18:2co6 Linoleic acid 8.2 ± 1.07** 7.2 ±0.93 13.8 ± 1.95** 13.1 ± 1.69 (LIN) 18:3co6 y-Linolenic acid 0.8 ±0.13** 0.7 ±0.18 1.3 ±0.36** 1.1 ±0.29 (GLA) 18:3o)3 a-Linolenic acid 45.3 ±3.34** 39.4 ±4.82 37.3 ±4.77** 33.8 ± 1.61 (ALA) 18:4co3 Stearidonic acid 7.3 ± 0.26 6.3 ± 0.67 6.8 ±0.27 6.0 ±0.67 (SDA) 24:0 Lignoceric acid 1.1 ±0.37 1.0 ±0.23 0.7 ± 0.46 0.8 ±0.14 20:5co3 Eicosapentaenoic n.d.* 11.9 ±4.09* 0.1 ±0.09* 8.9 ±3.55* (EPA) acid 166 Table 5.7. Mean proportions of FA functional groups (as % of FAs) considered in this analysis, and Xco3:Xcd6 for S. obliquus cultured at 21°C and 26°C in the laboratory without or with EPA addition, ± SD. There were 5 sample replicates in each treatment. A single asterisk (*) indicates significantly different treatment means within a given temperature, while double asterisks (**) denote significantly different means for control S. obliquus between temperatures (a = 0.05). 21°C 26°C EPA- Control Control EPA-enriched enriched 2>3 (%) 53.5 ±3.57 *' ** 58.6 ±3.53 * 45.1 ±5.16** 49.7 ± 3.66 I«6 (%) 9.0 ± 1.08** 8.0 ±0.95 15.1 ±2.05** 14.3 ± 1.90 X«3:£o)6 2.7 ±0.25** 3.1 ±0.30 1.8 ±0.14** 2.0 ±0.19 ISAFA (%) 24.0 ± 1.99* 20.8 ± 1.10* 25.2 ± 4.27 22.5 ± 2.20 IMUFA (%) 13.6 ± 1.34 12.6 ± 2.13 14.6 ±2.21 13.5 ± 1.86 XPUFA(%) 62.4 ± 3.07 * 66.5 ±2.91 * 60.2 ±6.31 64.0 ± 3.52 167 FIGURES Figure 5.1. Example of laboratory-reared, parthenogenic, 11-day-old Bythotrephes female with two embryos in the early stages of resorption (e) visible through the broodsac. This particular female had been fed EPA-enriched daphniids. The smaller spacing between the articular spines (a - b) on the caudal process, compared to (b - c), indicates poor growth at the first instar stage. 168 •Control •EPA««nriched Temp«r»tiir« Figure 5.2. Mean predation rates of Bythotrephes < 24 hour old on D. ambigua, ± SE. D. ambigua were offered at rates of 30 prey • d 1 or 60 prey • d"1 at 21°C and 26°C, respectively. There were 14 to 15 incubations • treatment"1. Bars not connected by the same letter within each temperature treatment indicate means with statistically significant differences (a = 0.05). 169 Control prey EPA-enriched prty Food treatment Figure 5.3. Mean clutch sizes of Bythotrephes reared at 21°C on day 11 of the assay, ± SE. There were 11 and 9 fecund Bythotrephes in the control and EPA-enriched treatments, respectively. Bars not connected by the same letter indicate means with statistically significant differences (a = 0.05). 170 CHAPTER 6 CONCLUDING REMARKS "When observations and theory collide, scientists turn to carefully designed experiments for resolution. Their motivation is especially high in the case of biological systems, which are typically far too complex to be grasped by observation and theory alone. The best procedure, as in the rest of science, is first to simplify the system, then hold it more or less constant while varying the important parameters one or two at a time to see what happens." - E.O. Wilson, The Future of Life Summary and implications for establishment The first stage of the invasion process is the introduction of a non-native species to a new area, followed by the establishment of populations. For both of these events to occur successfully, the potential invader must be able to grow, survive, and reproduce in the novel environment. Among the dominant regulators of zooplankton biology are temperature, resource availability, and predation (Yan & Pawson 1998). Here, I examined the life history consequences of rearing Bythotrephes in environments with differing temperature and resource availability. Life history theory not only analyzes how variations in certain traits contribute to variation in fitness among individuals, it also provides a link from individual to population levels of biological organization (Stearns 1992). Clearly, many localized variables have the capacity to individually and interactively affect Bythotrephes including temperature, dissolved minerals, nutrition in terms of prey quantity and quality, the nutrient status of a lake, water clarity, pH, 171 parasites, disease, competition, and predation. In both its native and invaded ranges Bythotrephes inhabits large, deep, nutrient-poor lakes (Maclsaac et al. 2000; Weisz & Yan 2010; Wang & Jackson 2011). While this generality may be an artifact of the idea that human-mediated propagule pressure is highest in these lake types (Weisz & Yan 2010), my laboratory studies provide some additional insight on why this is so, given the physiological tolerances of this species. I focused primarily on abiotic factors affecting the growth, survival, and reproduction of parthenogenic females by first simulating an optimal habitat within a laboratory environment (Chapter 2). The methods for field collection and laboratory culture developed here have since been employed by other workers conducting laboratory-based behavioural and genetic experiments on this species (e.g., A. Jokela, Queen's University Biology Department, Kingston, ON, personal communication; P. Turko, Queen's University Biology Department, Kingston, ON, personal communication; A. Jaeger-Miehls, Michigan State University Department of Fisheries and Wildlife, East Lansing, MI, personal communication). Bythotrephes is a poikilotherm. Like all crustaceans, it is dependent on behavioural thermoregulation, its potential for thermal acclimation, and dormant stages to endure unfavourable temperatures (Lagerspetz & Vainio 2006). I found that Bythotrephes exhibits the highest population growth at approximately 21°C, but that it is limited at temperatures > 25°C (Chapters 2, 5). In Michigan, Bythotrephes is largely absent from water bodies with summer epilimnetic temperatures exceeding 26°C (Kerfoot et al. 2011). Small, unstratified lakes in my study region of Muskoka, Ontario, currently experience 17? temperatures in this range; a pattern that will likely continue with climate warming. As such, Bythotrephes establishment will likely be limited to the larger, deeper lakes with thermal refugia, which also happen to receive increased anthropogenic activity. Of lesser significance for Bythotrephes is the amount of aqueous calcium in the surrounding environment, but this represents a case where insignificant experimental results may have significant repercussions in situ (Chapter 3). Bythotrephes has already had devastating impacts on pelagic crustacean zooplankton. This trend will likely continue given that calcium is declining (Jeziorski et al. 2008), crustacean zooplankton have very high calcium demands, and Bythotrephes is unaffected by low calcium. On the other hand, pH has risen in the past few decades (Jeziorski et al. 2008), but again Bythotrephes does not appear to be affected by mildly acidic waters, at least in the short term (Appendix G). Following a recent study implicating prey availability as be the most important determinant of Bythotrephes abundances in the field (Young et al. 2011), I devoted the final two chapters to investigating the roles of food quantity and quality on Bythotrephes life history. I found that at 21°C—a temperature at which Bythotrephes exhibits optimal population growth—a daily predation rate of 105 jig prey as dry mass (DM) is likely insufficient to support the growth, survival, and reproduction of this voracious predator (Chapter 4). This predation rate occurs in the laboratory at a density of 15 prey organisms • 0.4 L"1 • d"1 (equivalent to 38 prey • L~' • d"1). Bythotrephes is able to survive and reproduce rather well when offered an edible prey density of > 30 prey 0.4 L"1 • d"1 (equivalent to 75 prey • L"1 • d"1), however. 17"* At 21 °C I also found that when offered a diet of 30 small daphniid prey of differing quality (in terms of the amounts of the co3 fatty acid eicosapentaenoic acid, EPA) and 10 bosminids, juvenile Bythotrephes were less able to catch the larger and faster EPA-supplemented daphniids (Chapter 5). Despite this, modest supplementation with EPA resulted in significantly larger clutch sizes, suggesting that the availability of this single fatty acid may help support Bythotrephes establishment. The diet provided in this study supported only the ontogenesis of Bythotrephes, as embryos were resorbed. Fatty acid analyses revealed that Bythotrephes were severely EPA-impoverished, especially when compared with their field-dwelling counterparts. This result may also help explain in part why Bythotrephes is often absent from highly productive systems, as algal food quality in terms of EPA would be lacking. There are additional factors that were not examined in detail here, which may also affect Bythotrephes survival and reproductive capacities in a new habitat. Light is an important factor, for example, and Bythotrephes is likely present in clear lakes because it strongly relies on visual cues for feeding. It has been demonstrated elsewhere that Bythotrephes exhibits increased feeding rates under higher light conditions (e.g., Pangle & Peacor 2009; Anneli Jokela, Queen's University, Department of Biology, personal communication), which I have also observed in the lab. Studies on the role of cannibalism for Bythotrephes fitness would also be useful, as it is prevalent in this species. In fact, colonization of marginal habitats may be facilitated by increased rates of cannibalism, as seen in flour beetles (Tribolium castaneum; Via 1999) for example. This may be an adaptive characteristic of Bythotrephes that alleviates the severity of Allee 174 effects when operational. Modeling efforts suggest that Bythotrephes is not immune to Allee effects following its initial introduction to a new habitat (Gertzen et al. 2011; Wittmann et al. 2011). Importance of autecological studies Insights on this species' autecology were gained through this study that would not have been possible with only field-based observations. First, I observed the rather frequent production of multiple broods (Chapters 3,4), which is an aspect of this species' life history currently not accounted for in spread and establishment models. Second, I will address a minor discrepancy in the Bythotrephes literature. It is known that Bythotrephes neonates are born with one pair of lateral spines (barbs), and a new pair is added at each moult. When moulting from one instar to the next, Bythotrephes sheds its exoskeleton at the base of the lateral spines, which leaves the old caudal process (tailspine) attached to the new one (Ischreyt 1930 in Ketelaars et al. 1995; Branstrator 2005). Consequently, the practice of identifying the instars of field-collected individuals by counting the number of barb pairs on the tailspine is widespread. In the field it has been noted that Bythotrephes can reproduce as early as the first and second instar stages (Ketelaars et al. 1995), and that second and third instar body sizes converge during summer (Pothoven et al. 2001; Yan & Pawson 1998; Young 2008). This has been variously attributed to favourable environmental conditions (Ketelaars et al. 1995) as well as prey limitation (Yan & Pawson 1998). In Harp Lake, Young (2008) noted that when prey abundance was the highest, second and third instar Bythotrephes were among the 17S largest, and she suggested that the convergence in body lengths was due to atypically large second instar animals rather than undersized third instars. Although a relatively rare occurrence in the laboratory environment, direct observation of Bythotrephes throughout its life cycle indicates that reproducing first and second instar individuals are indeed of the same age as their third instar counterparts (Appendix H). This phenomenon is widespread in field Bythotrephes during summer. Unfortunately, I cannot confirm whether these "first" or "second instar" Bythotrephes were shedding their exoskeletons but failing to add an additional barb pair—as proposed by Yurista (1992)—or if they were skipping a moult (or two) altogether. Stearns (1992) explained that in the case of two arthropod siblings differing only slightly in size at birth and constrained by their exoskeletons, the larger of the siblings may initiate maturation at an earlier instar than its smaller sibling9 (assuming that maturation is initiated once a requisite size threshold is exceeded; Stearns 1992). Ebert (1994) found that female Daphnia magna that were born smaller or grew more slowly endured more juvenile instars than those that were born larger or grew faster. I thus concur with Young (2008), and hypothesize that Bythotrephes' apparent "skipping" of a moult may be due to increased sizes at birth resulting from increased prey availability and Bythotrephes' higher predation rates in warmer waters during summer. Finally, an informal comparison of Bythotrephes sizes attained over the course of various experiments conducted in Chapters 3, 4, and 5 at 21°C (the main difference among trials being diet; Appendices I, J) suggests an overall positive relationship 9 This translates to slower growth for the larger-at-birth sibling as resources are diverted to reproduction versus later maturation but an ultimately larger size attained for the smaller-at-birth sibling (Stearns 1992). 176 between increasing size and population growth rates. Larger Bythotrephes may also have greater impacts on food webs, especially in the absence of fish predators. Prognosis Bythotrephes clearly maintains a strong presence in North America, but given its many documented negative impacts on native pelagic systems, it is likely best to mitigate secondary spread of invasive populations. Unless timely measures are taken to slow or even halt the spread of Bythotrephes, many more freshwater communities in North America will be impacted. One way to approach this is to educate people and promote increased interest in freshwater issues. The main vector of Bythotrephes' secondary spread is human-mediated (Weisz & Yan 2010) after all (via contaminated gear such as boats, bait buckets, livewells, bilge water, anchor and fishing lines). Recreational boaters should be targeted, and precautions such as limiting the transport of baitfish or subjecting them to a 24 hour holding period (defecation period, since resting eggs remain viable following passage from fish guts) should lessen dispersal by this vector (Kerfoot et al. 2011). Recent rapid advances in communication devices and social media should also be exploited as a way to educate the general public. Eradication measures have largely been dismissed, as there is an underlying assumption that it can only be achieved with the complete removal of an invasive population; however this may be erroneous if abundances are sufficiently low (Liebhold & Bascompte 2003). Control measures should focus on exploiting Allee effects and targeting resting egg banks, particularly in areas like Manitoba where invasions are relatively new. There is also the possibility of eradication via the temporary 177 eutrophication of lakes, which should not affect herbivorous zooplankton populations as much as Bythotrephes. Although Bythotrephes represents a serious threat to pelagic diversity, I remain hopeful that there will be human-driven initiatives to limit or halt its spread. Failing that, it is possible that invading populations will eventually become self-regulating. Given that the viability of Bythotrephes resting eggs does not appear to be as lengthy as that of Daphnia resting eggs that are encased in an ephippium, for example, it is hypothetically possible that Bythotrephes will eventually exhaust all prey resources including itself as it cannibalizes conspecifics, such that native species are capable of resurrecting. Eventually, we may even see a situation akin to that observed in Norway where Bythotrephes is actually associated with increased biodiversity (Noreen Kelly, York University, Biology Department, personal communication), particularly if nai've prey species are able to recognize and adapt to the kairomone signals emitted by the predator. Moreover, if fish predators become more efficient at exploiting this food resource, Bythotrephes numbers could be reduced. Barring that, perhaps the option of introducing sportfish that are known to preferentially prey on Bythotrephes in lakes that are invaded and already heavily impacted by humans can be entertained. Over a decade ago, Schindler (2001) forecasted that aquatic communities would undergo drastic changes owing to many stressors (including the invasions of non- indigenous species), and called for increased research and a national water strategy as "the only hope for preventing a freshwater crisis in Canada". Yan et al. (2008) showed that zooplankton communities of the Canadian Shield are currently undergoing major 178 changes which must be considered from the standpoint of multiple stressors. Thus, protecting the integrity of the country's freshwaters will involve concerted efforts from multiple stakeholders and interest groups now more than ever before. Recent interest in developing improved invasive species policies is heartening. REFERENCES Ebert, D. 1994. 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Canadian Journal of Fisheries and Aquatic Sciences 65: 862-877. ISO Yurista, P.M. 1992. Embryonic and postembryonic development in Bythotrephes cederstroemi. Canadian Journal of Fisheries and Aquatic Sciences 49: 1118-1125. 181 Appendix A. Results of curve-by-curve survival analyses for Bythotrephes reared at five different temperatures (16°C, 20°C, 24°C, 28°C, 32°C). P-values marked with asterisks (*) denote significant differences among pairs. Temperature (°C) Wilcoxon X2 P-value treatment comparison 32-16 24.8642 <0.0001* 32-28 24.3377 <0.0001* 32-24 24.3377 <0.0001* 32-20 24.3377 <0.0001* 28-16 10.5049 0.0012* 28-24 6.4303 0.0112* 28-20 6.4303 0.0112* 24-16 0.4188 0.5175 20-16 0.4188 0.5175 24-20 0.0000 1.0000 18? Appendix B. Results of Tukey-Kramer HSD multiple comparisons of Bythotrephes mean daily feeding rates on different prey species. P-values marked with an asterisk (*) denote significantly different predation rates by Bythotrephes (df= 1 for all comparisons). Pair-wise comparison i»-value D. ambigua - D. pulex <0.0001* D. ambigua - H. gibberum <0.0001* B. freyii - D. pulex <0.0001* Artemia - D. pulex <0.0001* B. freyii - H. gibberum <0.0001* Artemia - H. gibberum <0.0001* D. ambigua - Artemia 0.0130* D. ambigua - B. freyii 0.0137* H. gibberum - D. pulex 0.9442 B. freyii - Artemia 1.0000 181 Appendix C. Mean predation rates (± SE) of second instar Bythotrephes (n = 5 individuals • treatment"1) over 24 h when offered 6, 9, and 12 each of barren, mid-size D. ambigua and D. pulex in 175 ml at 21°C and 26°C. Prey offered were doubled for the higher temperature, as Bythotrephes' predation rates increase with this temperature increase (N. Kim personal observation). Prey offered #Z). Total prey Total biomass Temperature, # D. pulex (D. ambigua, ambigua organisms consumed (|*g °C consumed D. pulex) consumed consumed DM d') 6,6 5.6 ± 0.4 5.0 ± 1.7 10.6 ±0.7 200.6 ±23.7 21 9,9 7.2 ±2.7 4.6 ±3.2 11.8 ±2.3 202.2 ±52.1 12, 12 8.8 ±3.4 5.8 ±2.2 14.6 ± 1.8 253.0 ±33.1 12, 12 10.2 ± 1.4 10.0 ±0.7 20.2 ± 1.7 401.4 ±27.4 26 18, 18 12.8 ±2.4 11.4 ± 1.7 24.2 ±3.8 465.8 ± 69.3 24, 24 19.0 ± 1.6 14.8 ± 1.4 33.8 ± 1.7 621.4 ±42.8 184 Appendix D. Bythotrephes (n = 5 individuals ' treatment"1) exhibits significantly higher (f = 2.73, df= 7.9, one-tailed p = 0.013) predation rates on mid-size (500 - 850 (im) Daphnia pulex in light (100 jimol' m" ' s") compared with dark (5 ^imol' m" 's" ) conditions in the laboratory. Initial prey densities were 50 D. pulex' 175 ml"1. c _ -c H a JC o Q 00 % H 25- io OJ a> -c re v_ C & 20- o & o re -c XJ Dark Light Light conditions 18S Appendix E. The dewar housing the cryogenically frozen samples during transport between laboratories malfunctioned on one occasion, and as a result my Scenedesmus obliquus and Daphnia ambigua samples were subject to thawing for a maximum of 12 hours. I do not believe this had a significant impact on the results of the fatty acid analyses, however. All of the algae and daphniids were thawed, thus any differences between the treatments should be real and not as a result of the thawing. The dewar was still cool to the touch and algae were still bright green in colour (algae will normally turn dull green if thawed for too long). In addition, lipids that are held intact/in place inside of organisms in their normal orientation in membranes and storage organelles are reasonably protected for a few hours after death (Michael Arts, Environment Canada, personal communication). It is only when cells are lysed due to either bacterial action or mechanical action (during sample extraction) that lipolytic enzymes come into contact with labile PUFA and more serious degradation is expected (Michael Arts, Environment Canada, personal communication). 1 8ft Appendix F. Temperature data for Lynch Lake (45.2°N, 79.2°W) in Huntsville, Ontario, Canada. TidbiT v2 UTBI-001 temperature loggers (Onset Computer Corporation, Pocasset, MA, USA) were deployed at the deepest point of the lake (maximum depth = 3.8 m). Upper line indicates temperature at 0.1 m from the surface and the lower line indicates temperature at 3.5 m depth. 30 28 -I o 2® 0 $ 24 - 22 " « 1 20 H 0) *- 18 16 H 14 CD CD 05 CD o> CD CD CD O) CD O) O) CD a> CD OS CD O O O O O o O O o o o o O o o o o O O O O O o O O o o o o O o o o o CM CM CM CM CM £M CM CM CNj CM CM CM £SJ CM CM CM CM T— 00 LO CM 9? CO CO O CO CO o h- 00 o ""t CM h- r-- CM CO 00 CM CM CD CNJ CO CD CD f- r- 00 00 00 CD o> O) Date 187 Appendix G. Percent survival of first and second instar Bythotrephes (n = 6 individuals treatment"1) in a 96 hour pH range-finding bioassay conducted at 22°C. < *• o •— —• ni- • 9— • < 0- o < > ^ A A • A A A 12 24 36 48 60 72 84 96 Time (hours) pH 4.0 pH 4.5 pH 5.0 pH 5.5 C ontrol 188 Appendix H. Mean ages (in days) of first, second, and third instar Bythotrephes reproducing under differing conditions in the laboratory, alongside age ranges of third instar animals reproducing. Sample sizes are reported in parentheses. All Bythotrephes were hatched out in the laboratory and observed daily. Mean age Age range Mean age (d) of Mean age (d) of (d) of first (d) of third Assay conditions second instars third instars instars instars reproducing reproducing reproducing reproducing 16°C, n/a 18(2) 15.1 (7) 11-18 moderate food 19°C, n/a 11(1) 10.9 (8) 7-17 moderate food 21°C, n/a 10(1) 12.0 (5) 11-14 high food 21°C, high food, 13(1) n/a 12.3(3) 12-13 0.1 mgL'Ca 21°C, high food, n/a 13(1) 11.4(5) 11-12 1.0 mg-L"1 Ca 22°?' , * n/a 11(1) 8.4(14) 7-11 moderate food 25°C' n/a 8 0^ 7-8 moderate food 8 (2)* * Two individuals had highly pigmented broodsacs but died just prior to offspring release, thus these values are estimates. 18Q Appendix I. Summary of body, tailspine, and total lengths of third instar Bythotrephes observed throughout this study at 21°C. Values for test conditions 1 - 2 indicate results of Chapter 5 {n = 15 and 14 Bythotrephes, for each respective condition); conditions 3, 4, and 6 are presented in Chapter 4 (« = 4, 7, and 6, respectively); and condition 6 is taken from Chapter 3 (n = 7). Test conditions 1 - Low food (B. freyii + D. ambigua) 2 - Low food (B. freyii + EPA-enriched D. ambigua) 3 - Low food (mixed prey) 4 - Medium food (mixed prey) 5 - High food (mixed prey, A. fronciscano ad libitum) 6 - High food (mixed prey) 3 ' 4 Test condition Corresponding r-values: 1) undefined - embryo resorption, 2) undefined - embryo resorption, 3) 0.01,4)0.09, 5) 0.11,6)0.10. ion Appendix J. Growth* at first and second instar for all third instar Bythotrephes observed throughout this study at 21°C. Values for test conditions 3,4, and 6 are presented in Chapter 4 (n = 4, 7, and 6, respectively); and condition 6 is from Chapter 3 (n = 7). T«st UHldltlOM 3 - Low food (mixed prey) 4 - Medium food (mixed prey) 5 - High food (mixed prey, A. froneiscono ad libitum) 6 - High food (mixed prey) 9 0.8- Test condition Corresponding r-values: 3) 0.01,4) 0.09, 5) 0.11, 6) 0.10. * The lengths of the intercalary segments of Bythotrephes' tailspine are used as proxies for growth. 101